Microbiology BOC Review

Bordetella pertussis

"whooping cough" nasopharyngeal specimen is preferred for the diagnosis of pertussis Bordet-Gengou media (potato, glycerol, and blood) Regan-Lowe medium (horse blood, charcoal agar, cephalexin, and amphotericin B) - colonies are said to resemble !!mercury droplets or pearls!! direct fluorescent antibody (DFA) technique is used to directly detect the organism - specimen is added to a microscopic slide, then fluoroscein-conjugated antibody is added, this gets incubated with the labeled antibody, at which time the antigen and antibody form a complex. Washing removes unbound antibody, and the excited fluorochromes emit visible light Newer technologies developed to detect B. pertussis include the use of monoclonal antibodies to directly detect lipopolysaccharide and filamentous hemagglutinin. Polymerase chain reaction methods that directly detect the organism in nasopharyngeal specimens or from culture are also available.

Specimen Collection/Rejection Guidelines

- Requisition and specimen label must match. - Re-collect specimens that do not meet criteria when possible and whose procedures for collection are not difficult or invasive for the patient. - Document why the specimen was rejected and communicate to the individual who collected the specimen why it was rejected. - Try not to reject specimens that are difficult to collect, such as CSF or surgical biopsies. - Set up cultures when the situation cannot be resolved, but comment concerns on the report and how the results may be affected. In general, reject the following specimens: - Twenty-four-hour sputum collections—prone to contamination and may dilute out the pathogen because of the normal flora - One swab for many requests, such as aerobes, anaerobes, and fungi - Leaking containers—may present a biohazard and be contaminated - Nonsterile or contaminated containers - Culture or agar plates that are overgrown and dry except for specific mycology request - Specimens contaminated with dyes, oils, or chemicals - Formalin in any specimen For anaerobes, reject the following: - Gastric washes - Midstream urine - Feces, except for Clostridium difficile and Clostridium perfringens - Throat, nares, or oropharyngeal specimens - Most swabs, including superficial skin swabs

Enterococcus spp.

A gamma-hemolytic Streptococcus that blackens bile esculin agar but does not grow in 6.5% NaCl broth is most likely: E. faecalis causes almost 80% to 90% of human enterococcal infections, followed by E. faecium, which is implicated in 5% to 10% of enterococcal infections. In humans, the enterococci are found as normal flora of the gastrointestinal tract and also on the skin and in the oral cavity and genitourinary tract. E. faecalis is the most common species found colonizing the human gastrointestinal tract. Identification Bile-esculin positive Salt tolerant (can grow in 6.5% sodium chloride (NaCl) broth) PYR postive (Only group A streptococci and group D enterococci are PYR-positive) Pathogenicity Enterococci are opportunistic pathogens and are the agents of nosocomial infections, including urinary tract infections, wound infections, and bacteremia. Infections may be polymicrobic in nature. Intra-abdominal abscesses and endocarditis also are associated with the enterococci. Acquired resistance also is becoming more frequent in the Enterococcus, which has shown increased resistance to the aminoglycosides, macrolides, tetracycline, and glycopeptides, especially vancomycin.

Gram stain

A method for the differential staining of bacteria that involves fixing the bacterial cells to a slide and staining with crystal violet and iodine, then washing with alcohol, and counterstaining with safranin. Results in gram-positive bacteria retaining the purple dye and gram-negative organisms having it decolorized so that the red counterstain shows up. Gram-positive bacteria retain the primary stain because of the peptidoglycan and teichoic acid cross-links. Gram-negative bacteria lose the primary stain because of the large amount of lipopolysaccharide in the cell wall. - True spirochetes are coiled bacteria, such as Treponema, which may possess four to 20 coils. Spirochetes do not Gram stain well. - In cultures 48 hours old or longer, gram-positive bacteria may stain as gram- negative bacteria. For this reason, it is important to use 24-hour cultures for the Gram stain. - When Gram staining for certain organisms, such as Legionella and Campylobacter and certain anaerobes, it is recommended to use carbolfuchsin instead of safranin O as the counterstain.

carbapenamases

A new class of bacterial enzymes capable of inactivating Carbapenems, known as Klebsiella pneumoniae Carbapenamases (KPCs).

MALDI-TOF MS

A technique of mass spectrometry in which macromolecules such as proteins are fragmented and ionized, and their time of flight (TOF) provides information on the molecular mass. Stands for: matrix assisted laser desorption ionization time of flight mass spectrometry

Acinetobacter

Acinetobacter species are oxidase-negative, catalase-positive, nonmotile coccobacilli appearing as plump, paired rods or diplococci The organisms grow well on MacConkey and sheep blood agars, with a translucent appearance. Obligate aerobe Nonmotile Oxidase negative Nonhemolytic Grows well on MacConkey agar Resistant to penicillin Acinetobacter lwoffii is the the second most common and this bacterium is asaccharolytic. It grows on MacConkey agar, producing an ammonia-like odor, and is resistant to penicillin. Acinetobacter is associated with nosocomial and opportunistic infections. The bacteria colonize moist areas of human skin and are normally found in the oropharynx and vaginal and gastrointestinal tracts. The organisms also are found in the hospital environment, soil, and water. Infections include urinary and respiratory tract infections, wound infections associated with soil and water contamination, bacteremia, and meningitis. Hospital equipment that has been contaminated by Acinetobacter species includes endotracheal tubes, respiratory care and dialysis equipment, and venous catheters. Using, the oxidase reaction, Neisseria is oxidase positive, whereas Acinetobacter is oxidase negative.

Types of bacteria

Aerobic bacteria require molecular oxygen for cellular respiration and thus require incubation in the presence of atmospheric oxygen. Strict aerobes or obligate aerobes have an absolute oxygen requirement, needing it as the terminal electron acceptor for cellular respiration. An example of a strict aerobe is Pseudomonas. Facultative anaerobes can multiply in the presence or absence of oxygen and are able to use oxygen as the final electron acceptor in cellular respiration. However, they also can acquire energy through fermentation. Facultative bacteria include Staphylococcus and members of the Enterobacteriaceae. Strict anaerobes or obligate anaerobes are unable to multiply in the presence of oxygen and require incubation in an anaerobic environment, such as in an anaerobic jar or glove box. Examples of obligate anaerobes included Clostridium and Bacterioides. capnophilic = 5% to 10% CO2 with small amounts of oxygen

Extended spectrum β-lactamase (ESBL)

Aminopenicillins: ampicillin, amoxicillin, and bacampicillin Spectrum: extended spectrum of gram-positive and gram-negative bacteria, including Escherichia coli, Proteus mirabilis, Haemophilus influenzae, Salmonella, and Shigella. Can penetrate outer membrane of gram-negative bacteria Many members of Enterobacteriaceae produce extended spectrum β-lactamases and other β-lactamases that produce resistance against many β-lactam drugs.

Specimen collection blood

BLOOD transient bacteremia = normal flora bacteria may be introduced into the blood (brushing teeth/S. viridans) intermittent bacteremia = sporadically discharged from extravascular abscesses or infections into the blood continuous bacteremia = constant release of bacteria into the blood (indwelling catheters) It is recommended to collect two to four specimens from separate venipuncture sites at least 1 hour apart. When possible, collect two sets from the right arm and two sets from the left arm. These actions help to increase the probability of isolating the organism. It is imperative to prepare the patient's skin properly before phlebotomy to ensure that no skin contaminants are collected. Typical methods include a 70% to 95% alcohol. This is followed by either a chlorhexidine or iodophor scrub, which is left on for 1 minute. Preparations containing iodine should not be used for those who are allergic to iodine. Next, a second alcohol rinse is applied. A second method uses a wash with green soap followed by a sterile water rinse. Tincture of iodine is applied and allowed to dry. The final step is the application of 70% alcohol. At least 20 ml of blood should be collected for each adult set of blood cultures and 1 to 10 ml per set for pediatric blood culture sets. An aerobic and anaerobic bottle should be collected. No more than three sets should be collected in any 24-hour period.

Common pathogens of CSF

Bacteria that possess antiphagocytic capsules are some of the most frequent causes of meningitis. These include Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis.

catalase test

Bacteria that produce catalase include Staphylococcus species, Listeria monocytogenes, Bacillus species, and Bacteroides fragilis. The test is important in the differentiation of gram-positive cocci: Staphylococcus are catalase positive, and Streptococcus are catalase negative. Most bacteria are catalase positive. Uses 3.0% hydrogen peroxide (H2O2)

Mycoplasma spp.

Because it doesn't have a cell wall, this little bug doesn't react to Gram stain. - therefore resistant to all antibiotics that inhibit cell wall synthesis, such as the β-lactams IDENTIFICATION chest X-ray film = first shows patchy infiltration of the hilar areas of the lungs Gram stain = mononuclear inflammatory cells with normal oropharyngeal flora Mycoplasmas may be identified through culture, antigen detection, serology, or polymerase chain reaction. For culture methods, blood, sputum, synovial fluid, vaginal discharge, urethral swabs, tissue washings, or amniotic fluid can be examined, based on the site of infection. Throat swabs may be used to isolate M. pneumoniae because the organism can colonize the throat. A diphasic liquid medium that contains fresh yeast extract, peptone, and horse serum is used. In addition, an agar, such as E-agar or Shepard's A7-B agar, should be inoculated. - On E-agar, colonies of M. pneumoniae appear as a typical "fried egg" appearance Culture has a low sensitivity, and because of the long incubation time, it has limited clinical utility. Serological identification = primary method Cold agglutinins = produced in 1/2 patients, IgM antibodies which agglutinate at 4 C Antibody production against M. pneumoniae can be detected using complement fixation, ELISA, or fluorescent antibody methods. Several of these methods are commercially available and able to detect IgG and IgM antibodies against M. pneumoniae. It is most useful to test acute and convalescent sera and show an increasing titer for diagnosis. Enzyme immunoassay tests are used most frequently to diagnose mycoplasma infection in the United States. Polymerase chain reaction is useful in that it shows a higher degree of sensitivity in detecting the organism.

Haemophilus influenzae meningitis

Before the Haemophilus influenzae type b conjugate vaccinations were available and licensed in the United States, this organism accounted for 45% to 48% of cases of bacterial meningitis. • Most of these cases were found in infants and children less than 6 years of age; the peak incidence was found in infants who were 6 to 12 months of age. Today, H. influenzae type b accounts for approximately 7% of the cases of bacterial meningitis in the United States; most of these cases are found in adults. Worldwide, H. influenzae is still widespread where vaccination is not prevalent.

Broad-spectrum antibiotics

Broad-spectrum antibiotics have action against both gram- positive and gram-negative bacteria. An example of a broad-spectrum antibiotic is tetracycline. A disadvantage of broad-spectrum antibiotics is the inhibition or destruction of the normal flora of the patient.

Specimen collection CSF

CEREBROSPINAL FLUID Collected by lumbar puncture in the third or fourth lumbar vertebra by a physician. A local anesthetic is used, and a needle is inserted into the spinal canal. In most adults the collection consists of three tubes. Because the first two tubes may contain skin contaminants, it is usually recommended that the third tube is used for microbiology. There should be no delay in the processing or workup of CSF because some of the organisms associated with meningitis are fastidious and prone to chilling or drying. These include N. meningitidis and H. influenzae. Delays also must be avoided because of the high mortality rate and rapid proliferation associated with the infection. CSF can be held for up to 6 hours at 37°C for bacteria and fungi, but must be maintained at 4°C for virus detection.

Giemsa stain

Chlamydia, Borrelia, Rickettsia, Trypanosomes, Plasmodium

beta-hemolytic streptococci

Complete hemolysis of red blood cells; clearing or colorless zone around colony. S. pyogenes and S. agalactiae S. dysgalactiae S. equi S. anginosus

Corynebacterium spp.

Corynebacterium organisms are slender, pleomorphic, club-shaped, nonbranching gram- positive bacilli, which are aerobes or facultative anaerobes Corynebacterium bacteria are nonspore formers, nonmotile, and positive for both catalase and oxidase reactions. - appear as Chinese letters - narrow zone of β-hemolysis, but can be nonhemolytic too - C. diphtheriae produces gray to black colonies on potassium tellurite medium The most virulent species is Corynebacterium diphtheriae, the etiologic agent of diphtheria, a severe and acute infection. Infection is transmitted through direct contact with contaminated respiratory droplets, such as through coughing or sneezing. Virulence factors diphtheria exotoxin = attacks the mucous membranes of the respiratory tract Nasopharyngeal swabs of the inflamed areas or a vigorously swabbed culture of the pseudomembrane is collected to diagnose diphtheria. Swabs of skin lesions can be used to diagnose cutaneous diphtheria. Dacron swabs should be used for specimen collection. Because nontoxigenic strains of C. diphtheriae may exist, it is necessary to demonstrate the exotoxin. Modified Elek test = In this procedure a filter paper strip impregnated with diphtheria antitoxin is placed in a medium containing rabbit serum, potassium tellurite, and prepared agar. • The medium is allowed to solidify, and the antitoxin diffuses through the medium. • The plate is inoculated with a toxigenic strain of C. diphtheriae, which is made at a right angle to the strip of antitoxin. • A negative (nontoxigenic) control is streaked in the same manner. • The unknown culture is streaked parallel to the positive and negative controls. • After incubation at 35°C for 24 to 48 hours, the plate is observed for precipitin lines. If the diphtheria toxin is present, precipitin lines form at a 45° angle between the inoculum and the antitoxin strip. C. diphtheria infection is treated by giving !!equine antitoxin!! Penicillin or erythromycin is given as supportive respiratory and cardiac care.

Decarboxylation reactions

Detect the ability of the organism to enzymatically remove the carboxyl group from a specific amino acid. Generally, the amino acids lysine, ornithine, and arginine are tested. The decarboxylase reactions are useful in the differentiation of Klebsiella, Enterobacter, and Pantoea and also to speciate within the genus Enterobacter.

Clostridium difficile (Direct detection and molecular methods)

Diagnosis of C. difficile is performed by testing liquid or semisolid fecal specimens, using culture techniques and demonstration of toxins. - it produces toxin A and toxin B Methodologies to demonstrate the toxin include enzyme immunoassay, tissue culture, and latex agglutination. Cell culture methods are known as the gold standard for toxin identification because of their high sensitivity and high specificity. Cytotoxicity testing on cell cultures is used to identify toxin B cytotoxin. Specimens also can be inoculated on either cycloserine cefoxitin fructose (CCF) agar or Clostridium difficile selective agar (CDSA), which are both selective agars for C. difficile. CDSA contains amino acids that when utilized by C. difficile cause an increase in pH, resulting in a color change of the agar from rose to yellow. - C. difficile will appear as yellow, umbonate colonies with a ground-glass appearance after 48 to 72 hours of anaerobic incubation. The organism produces gelatinase but is negative for lecithinase, lipase, and indole. It ferments glucose and fructose but not lactose, maltose, or xylose. Spores are oval and subterminal. The organism is motile and fluoresces under ultraviolet light.

PYR test

Differentiation of Streptococcus and Enterococcus. Group D streptococcus includes enterococci and non-enterococci; enterococci such as E. faecalis are positive for PYR hydrolysis, whereas non-enterococci are negative. Group A β-hemolytic streptococcus (Streptococcus pyogenes) gives a positive PYR hydrolysis reaction, whereas group B streptococcus (Streptococcus agalactiae), which can also give β-hemolysis, is negative for PYR hydrolysis.

Selection of Antimicrobial Agents

Does the antimicrobial agent have strong lethal effects and activity against the microorganism? Is the agent least toxic toward normal flora of the host? Narrow-spectrum antibiotics should be used when possible. What is the age of the patient? Certain agents are toxic to very young patients and may affect geriatric patients in an atypical manner. Can effective blood or tissue levels be achieved? -For example, a large polar molecular, such as vancomycin, is highly water soluble, so its major site of action is in the blood. It is not distributed very well in several other tissues. The sulfonamides, such as Bactrim and rifampin, are nonpolar; they are distributed well and can attain high concentrations in tissues other than blood. Does the antibiotic cross the placenta, which may be toxic to the fetus? Does the antibiotic penetrate the blood-brain barrier, which is necessary in central nervous system infections?

Correlation with other laboratory results - CSF Infection

Fever (body temperature >40°C) or hypothermia (body temperature <36°C) White blood cell count either <4,000/ml or >20,000/ml; hypotension Bacterial meningitis - CSF glucose level DECREASED (reference range, 40-70 mg/dL) is less than 40 mg/dL in 60% of patients. - CSF protein level (reference range, 20-50 mg/dL) is usually ELEVATED - Neutrophils (x10^6/L) 100-10,000 (but may be normal) while Lymphocytes are usually < 100 Viral meningitis - CSF glucose level usually normal - CSF protein level are also usually elevated, though they can be within the reference range. - Neutrophils (x10^6/L) usually < 100 while Lymphocytes can be 10-1000 (but may be normal)

Candida spp

Found normally in the human alimentary canal and gastrointestinal tract and on the mucocutaneous membranes. White-, cream-, or tan-colored colonies with a pasty, creamy appearance. C. albicans is the only yeast that produces a germ tube Urease negative and negative for nitrate reduction and does not produce a capsule. These organisms show round to oval cells, with multilayer budding. Candidiasis can involve almost any organ in the body, and infections range from mild to severe. The most frequently isolated yeast is Candida albicans, which normally is found in small amounts in the gastrointestinal tract and on mucocutaneous areas. C. albicans has a low degree of pathogenicity but causes the most severe infections in the immunosuppressed or debilitated host. Candidiasis may involve the mucous membranes of the mouth (thrush) or vagina (vulvovaginitis). Cutaneous infections, including diaper rash, nail infections (onychomycosis), and cuticle infections (paronychomycosis), also are common Candida infections. Systemic infections include endocarditis, meningitis, urinary tract infections (UTIs), pulmonary infections, and fungemia. Prolonged antibiotic therapy with broad-spectrum antibiotics, such as tetracycline, can suppress the host's normal flora and thus allow the "normal flora" Candida to overgrow into large numbers. Superficial infections are treated with a variety of creams, such as nystatin. Systemic infections are treated with oral ketoconazole or fluconazole.

BACTEC 9240 and 9120

Fully automated and use fluorescence to measure released CO2 or consumed oxygen. There is a gaspermeable CO2 sensor on the bottom of each vial. When released from bacterial metabolism, the CO2 dissolves in water and forms hydrogen ions (H+), which causes the pH to decrease, initiating a fluorescent signal. There is continuous monitoring with repeated measurements every 10 minutes in the 9000 series, and detection is external to the bottle, which decreases the risk of contamination. Thus, bacterial growth is detected as a consequence of production of carbon dioxide or utilization of oxygen.

β-Lactam Antibiotics

Function by inhibition of cell wall synthesis. Penicillins = from the mold Penicillium notatum; gram-positive spectrum other than Staphylococcus; Most Staphylococcus species are resistant. Cephalosporins = from the fungus Acremonium; structurally similar to penicillin but are better able to withstand the action of β-lactamase and are more modifiable; Carbapenems = Examples include imipenem, meropenem, doripenem, and ertapenem. Spectrum: activity similar to third-generation cephalosporins, with slightly greater activity toward Enterobacteriaceae, P. aeruginosa, and anaerobes. They are also active against gram-positive cocci but are not effective for MRSA or vancomycin- resistant enterococcus (VRE). Monobactams (Aztreonam) = Originally made from Chromobacterium violaceum (now produced from a synthetic source) Spectrum: P. aeruginosa and other gram-negative bacteria; low activity against gram-positive bacteria and anaerobes

Specimen collection GI tract

GASTROINTESTINAL TRACT Diagnose the cause of gastroenteritis. The clinical symptoms of gastroenteritis include nausea, vomiting, and diarrhea. The nausea and vomiting usually result from the ingestion of preformed toxins and not from bacterial invasion. There also may be fever. Ingestion of contaminated water may be related to travel to areas where particular bacteria or viruses are endemic. Foreign travel, such as to developing countries with poor water treatment and sanitation methods, also may be associated with specific bacterial pathogens. Recent antibiotic use and hospitalization also are important clues to determine the cause of gastroenteritis. Finally, knowledge of similar symptoms with family members and contacts at school or work may also provide clues to the cause and type of infection. Suitable cultures for gastroenteritis include freshly passed stools, washes, or feces collected during endoscopy. gastric aspirates = acid-fast bacilli (children/infants) gastric biopsies = Helicobacter pylori infection For ova and parasites, three specimens are collected every other day for outpatients, while three specimens are collected on consecutive days for inpatients. When a delay is anticipated, the specimen is preserved in polyvinyl alcohol for the examination of ova and parasites and transported within 24 hours at room temperature. Stool specimens are collected into a sterile wide-mouth container with a screw cap and transported within 24 hours and held at 4°C. Rectal swabs are placed in enteric transport media and also transported within 24 hours at 4°C.

Specimen collection genitals

GENITAL TRACT It is imperative to inoculate the media and incubate the plates in increased CO2 soon after specimen collection to enhance the chances of recovery. Transport media such as JEMBEC (BD Diagnostics) alternatively may be used to provide proper nutrient and atmospheric requirements. For the diagnosis of male urogenital infections, the urethral discharge is usually collected. Prostatic fluid also may be submitted to identify specific pathogens. In female patients, specimens of the uterine cervix, urethra, or cervix may be collected. Because of the abundance of normal flora in the female genital tract, it is important to use a plate selective for N. gonorrhoeae such as Martin-Lewis or modified Thayer-Martin. Such media inhibit the normal flora and allow a better recovery of the pathogen. Anaerobic and aerobic media should be set up for female specimens from the endometrium, cul-de- sac, or Bartholin cysts.

Streptococcus pneumoniae

Gram-positive diplococci are lancet or bullet shaped and form chains; requires 5% to 10% CO2; small, round, glistening, dome-shaped colonies that are transparent, with an entire edge. The colonies may run together. Mucoid colonies indicate the presence of a capsule; wide zone of α-hemolysis Identification S. pneumoniae is identified using the optochin test (P disk). A zone of inhibition of at least 14 mm around a 6 μm optochin disk indicates a positive susceptibility test, which identifies S. pneumoniae. Bile esculin test = will lyse S. pneumoniae so no growth in tube Quellung reaction, or capsular swelling test = identification directly in body fluids (CSF!). The bacteria must be present in large numbers for detection to be accurate. Equal amounts of the specimen are mixed with S. pneumoniae-specific capsular antisera. If the reaction is positive, the antisera will bind to the capsular antigen, making the capsule swell, or become more prominent. S. pneumoniae is transmitted from person to person contact through respiratory droplets of asymptomatic carriers or from individuals who are infected with the organism. Pathogenicity Diseases attributed to S. pneumoniae include pneumonia, bacteremia, sinusitis, otitis media, and meningitis. Most common cause of bacterial community-acquired pneumonia. S. pneumoniae is also a leading cause of otitis media in infants and small children, accounting for numerous middle ear infections. It is also an important cause of bacteremia, septicemia, endocarditis, meningitis, and pericarditis.

S. pyogenes

Group A Streptococcus causes bacterial pharyngitis, skin infections, and other invasive diseases, also rheumatic heart disease and acute glomerulonephritis Morphology = pinpoint, translucent, opalescent, or clear in appearance and white to gray in color. The colonies are surrounded by a wide zone of β- hemolysis. Group A streptococcal colonies may appear either smooth and glossy or round and mucoid, which indicates the presence of M (emm) protein. Presumptive identification of S. pyogenes is through the bacitracin test and the pyrrolidonyl arylamidase (PYR) test. S. pyogenes is susceptible to 0.02 to 0.04 unit of bacitracin and is PYR positive. SXT resistant and CAMP negative Virulence factors = M protein holds antiphagocytic properties. Streptolysin O: Toxic to red and white blood cells; subsurface hemolysin; suppression of neutrophils; induces an antibody response known as antistreptolysin O. Streptolysin S: Oxygen-stable, surface hemolysin; antiphagocytic; toxic to various human cell types. Superantigens: invasion of soft tissue and necrotizing fasciitis fever Streptokinase: Fibrinolysin that lyses blood clots, prevents fibrin barrier, and allows spread of infection

Helicobacter pylori

Has four to six polar flagella very strong urease activity H. pylori is microaerophilic, preferring 85% nitrogen, 10% CO2, and 5% oxygen. It grows optimally at 35°C to 37°C and prefers increased humidity. It is positive for catalase and oxidase. The gold standard to identify H. pylori is through histological staining and culture of biopsies obtained from the stomach or duodenum. - The tissue is ground and plated onto nonselective agar, such as 5% sheep blood agar, and incubated at 37°C in a microaerophilic environment and increased humidity. - Typical growth shows small gray, translucent colonies that are slightly β-hemolytic. Identification is confirmed through biochemical reactions, especially strong urease production. urease breath test, which detects urease in the individual's breath enzyme linked immunosorbent assays to detect Helicobacter antigen in the stool and serological methods to detect IgG antibody in the serum.

inducible clindamycin resistance

Health care-associated infections (HAI) from MRSA are also usually resistant to erythromycin and clindamycin, whereas community-acquired (CA) MRSA infections are usually susceptible to clindamycin. The erm gene activates the resistance to clindamycin. The D test can be used to determine inducible resistance to clindamycin. In this method, the suspension of the organism is streaked onto Mueller-Hinton agar. The erythromycin disk is placed so that its edge is 15 mm to 26 mm from the edge of the clindamycin disk. In the presence of inducible clindamycin resistance, there is a flattened zone observed between the two discs. The zone around the clindamycin disk resembles the letter "D," a pattern that occurs with antagonistic antibiotics. This indicates that the erm gene has been triggered for clindamycin in the organism.

16S ribosomal RNA sequencing

Identification of Streptococcus and Enterococcus, Pseudomonas, Aggregatibacter actinomycetemcomitans, anaerobic gram-positive cocci, and C. trachomatis. The ~1500 bp 16S rRNA gene comprises nine variable regions interspersed throughout the highly conserved 16S sequence. Sequencing the entire gene was originally accomplished by Sanger sequencing. Currently, however, the vast majority of studies sequence only part of the gene, because the widely used Illumina sequencing platform (higher throughput, lower cost, reduced effort compared with Sanger) produces short sequences ( ≤ 300 bases).

urease reaction

Identification of the genera Proteus and Morganella, which are rapid urease producers, as well as in the identification of slower urease producers, such as Klebsiella and some members of the genus Enterobacter. Urease degrades the substrate urea into alkaline end products, which are detected using a pH indicator.

XV factors

If growth occurs around only the X strip, the organism requires only X factor for growth. If growth occurs around only the V strip, the organism requires only V factor for growth. If growth occurs between only the X and V strips, the organism requires both X and V factors. H. influenzae = Needs X and V H. parainfluenzae = Needs V

Candida dublinensis

Important cause of oral candidiasis in those with the human immunodeficiency virus (HIV). It also is associated with infections of the blood and urine and infections in other sites in those who are immunocompromised. It resembles C. albicans because it can produce a germ tube and chlamydioconidia. C. dublinensis can be differentiated from C. albicans by its inability both to grow at 45°C and to assimilate xylose. The organism is believed to be more virulent than C. albicans.

Specimen Transport and Processing

In general, a 2-hour limit between collection and reception into the laboratory is required. If a delay is anticipated, a transport medium such as Stuart's or Amies should be used to increase the viability of the pathogen. The Stuart's system consists of swabs in a test tube with transport media that can be activated by crushing an ampule. The buffered semisolid agar, which contains sodium thioglycollate, can maintain bacteria for up to 72 hours. Cary-Blair medium is designed for the transport of stool specimens and is recommended for the transport of enteric pathogens. The specimen should be examined for adherence to collection guidelines on receipt in the laboratory. The specimen requisition and label must match.

E. coli

Indole postive - closely related through DNA homology to Shigella but Shigella is indole negative TSI: Acid slant over acid deep H2S: NegativeMacConkey: Pink-red colonies Voges-Proskauer (VP): Negative Citrate: Negative Motility: Positive (most) Deaminase (phenylalanine): Negative Urease: Negative E. coli is normal flora of the human lower gastrointestinal tract and is therefore found in the normal stool. E. coli is identified by the presence of pink-red colonies on MacConkey agar or by its characteristic green metallic sheen on EMB It can be presumptively identified if lactose-positive, oxidase-negative, and indole-positive gram-negative bacilli are isolated. Urinary tract infections are frequently attributed to E. coli and include cystitis, pyelitis, and pyelonephritis. - about 90% of UTI's are bc of E. coli in nonhospitalized patients; 25% of all nosocomial UTI Other types of infections include appendicitis, peritonitis, gallbladder infections, pneumonia, endocarditis, neonatal meningitis, wound infections, and septicemia. The septicemia may be associated with endotoxic shock from the release of lipopolysaccharide from the gram-negative cell wall. Specific serotypes of E. coli cause diarrhea and gastroenteritis, often with serious consequences.

motility tests

Involve the use of semisolid media, which typically contain 0.4% agar. A motility medium is often incorporated into a test battery, as with SIM medium and motility indole ornithine (MIO) medium. Motility reactions are very useful in the identification of Shigella and Klebsiella, the only nonmotile members of Enterobacteriaceae. The reaction also is useful in the identification of Yersinia species, which are nonmotile at 37°C but are motile when incubated at 22°C.

Clostridium perfringens

It is a cause of myonecrosis (gas gangrene), food poisoning, post-abortion sepsis, intra-abdominal and pleuropulmonary infections, enterocolitis, and antibiotic- associated diarrhea. The organism is known to produce several toxins, including α-toxin, lecithinase (results in cell destruction), hemolysins, cardiotoxin, collagenase, fibrinolysin, DNase, ribonuclease (RNase), enterotoxin, and proteolytic enzymes. - Myonecrosis is characterized by severe muscle destruction and the production of gas pockets in the tissue The organism is very saccharolytic, and as carbohydrate fermentation occurs, gas is released, which accumulates in the tissue. TREATMENT Treatment of gas gangrene includes surgical debridement or amputation of the necrotic tissue. The use of a hyperbaric oxygen chamber, a chamber of pure oxygen under pressure, can prevent the spread of gas gangrene. Antibiotics, including penicillin, vancomycin, and clindamycin, also are used as a part of the treatment regime. C. perfringens also is a common cause of food poisoning which is a mild and self-limiting condition lasting 2 to 3 days. Symptoms occur 7 to 15 hours after ingestion and include foul-smelling stools and diarrhea, but usually no vomiting occurs. IDENTIFICATION large gram-positive bacilli BOXCAR formation double zone of hemolysis on anaerobic blood agar!! very saccharolytic lipase negative nonmotile lecithinase positive = Nagler plate reverse CAMP test = positive identification with the formation of an arrowhead

Candida glabrata

It is normal flora of the gastrointestinal tract, skin, and upper respiratory tract and an opportunistic pathogen. C. glabrata is an important cause of fungal urinary tract infections and should be suspected when small, glossy yeast colonies are isolated on blood agar. The organism has been isolated from cases of fungemia and kidney, lung, genitourinary, and central nervous system infections involving immunosuppressed, diabetic, and cancer patients. C. glabrata assimilates both glucose and trehalose, a characteristic that is useful in its identification.

Listeria monocytogenes

L. monocytogenes is a tiny, gram-positive, motile diphtheroid and a facultative anaerobe. The organism is most frequently isolated from blood cultures or cerebrospinal fluid. It grows on blood agar, producing smooth, clear to gray colonies, 1 mm to 2 mm in diameter, with a narrow band of β-hemolysis. The organism grows best at 25°C to 35°C, slowly at 4°C, and poorly at 42°C. The organism possesses unique motility characteristics. First, L. monocytogenes produces a characteristic tumbling motility when observed in a wet mount or through a hanging-drop technique. The bacillus flips end over end. When a semisolid agar is used to demonstrate motility, the organisms produce an umbrella-like growth 2 mm to 5 mm below the agar surface. Others have described this pattern as an INVERTED Christmas tree. The organism has optimal motility at 25°C, and these unique characteristics are best observed at this temperature when compared with those observed at 35°C. The major source of infection is contaminated food, and those at highest risk include pregnant females and their fetuses, newborns, the elderly, and the immunosuppressed. In the fetus and newborn, the most common manifestation of listeriosis is meningitis. In early onset listeriosis the fetus becomes infected either in utero or during the first days following birth. - These affected newborns have septicemia, pneumonia, brain abscess, or meningoencephalitis. - They also may have lesions in the liver, spleen, or other organs. In severe cases, the infection may result in spontaneous abortion or stillbirth. In late-onset listeriosis, the infant encounters Listeria from the normal flora of the mother during birth; the infection occurs 1 to 2 weeks after birth. Meningitis is typically associated with late-onset listeriosis, as is conjunctivitis and septicemia. In immunosuppressed adults, particularly those who have undergone transplants or treatment for malignancies, Listeria may cause meningitis, endocarditis, septicemia, conjunctivitis, and urethritis. Antibiotic treatments include penicillin, ampicillin, aminoglycosides, erythromycin, and tetracycline.

Bacillus anthracis

Large Gram positive bacilli with "bamboo rod" appearance Nonmotile Produces acid from glucose, sucrose, and maltose Fails to produce acid from xylose, mannitol, lactose, or salicin Lecithinase: Most strains positive Starch hydrolysis: Positive Casein hydrolysis: Positive Anthrax is associated with cattle hides, goat hairs, and other herbivorous animals. Humans acquire the infection through direct contact with infected animal products, wool, or hair. Anthrax is an occupational hazard for those who handle livestock. - Once the disease is established in an area, bacterial spores from dead infected animals can contaminate the soil. The resistant spore form can remain dormant indefinitely in the soil, and the area can remain infected for years. Colonies are medium to large in size, with a diameter of 4 mm to 6 mm, and gray-white in color. Colonies are raised with an irregular curled margin and whirling projections. This has been described as a !!!"Medusa head"!!! appearance when seen under the dissecting microscope. When the colony is lifted with an inoculating loop, it has the consistency of a beaten egg white. B. anthracis is NONHEMOLYTIC on blood agar, an important reaction in differentiating it from other Bacillus species. Special media to isolate B. anthracis include polymyxin, lysozyme, EDTA, thallous acetate (PLET) media, and bicarbonate media. B. anthracis also produces a "string of pearls" when it is streaked on Mueller- Hinton agar and a 10 U penicillin disk is added and a cover slip applied.

Morganella

M. morganii is the only species within the genus Morganella. Morganella organisms give negative reactions for lactose, citrate, H2S, and LDC. The genus is urease and deaminase positive. Indole: Positive Methyl red: Positive VP: Negative H2S: Negative Citrate: Negative Urease: Positive Deaminase (phenylalanine): Positive Growth in KCN: Positive Esculin hydrolysis: Negative Morganella is an opportunistic pathogen, and infections include gastrointestinal disease and urinary tract and wound infections. Infections are most frequently seen in immunosuppressed patients and those undergoing prolonged antibiotic therapy. More serious infections include septicemia and abscesses.

Klebsiella pneumonia

MacConkey: Pink, mucoid colonies TSI: A/A H2S—Negative Gas: Positive Indole: Negative MR: Negative VP: Positive Citrate: Positive Deaminase (phenylalanine): Negative Motility: Negative Lysine: Positive Ornithine: Negative It is carried as normal flora in the upper respiratory and gastrointestinal tracts of approximately 1% to 5% of healthy individuals. Once known as "Friedlander's bacillus," is encapsulated and appears as mucoid colonies that tend to string. Causes primary bacterial pneumonia, which is more frequently found in those individuals with a predisposing lung disease, such as chronic obstructive pulmonary disease, and also in those with other medical conditions, such as diabetes mellitus. - The pneumonia is necrotic and hemorrhagic, which leads to the appearance of "currant jelly-like" sputum. There may also be lung abscesses and other complications in the lung. The organism can also cause meningitis, surgical wound infections, urinary tract infections, bacteremia abscesses, neonatal meningitis, infections of the gastrointestinal tract, and other wound infections. Those at highest risk of Klebsiella infections include those on prolonged antibiotic therapy, patients on ventilators, and those with intravenous lines. Many clinical isolates are resistant to ampicillin, carbenicillin, and ticarcillin. Additionally, the production of extended-spectrum β-lactamases (ESBLs) through plasmids have caused increased resistance to many β-lactam antibiotics, including the third-generation cephalosporins. The organism has a capsule, which enables K. pneumoniae to resist phagocytosis.

carbohydrates utilization test

Media may incorporate one or more carbohydrates and an acid-base indicator. If carbohydrate is utilized, the indicator changes from its neutral color to its acidic color, denoting a positive reaction. For example, to determine glucose fermentation, a media containing glucose and phenol red indicator is used. After inoculation and incubation, the change in color from pink-red to yellow would indicate a positive reaction. Bubbles or breaks in the media would indicate the production of gas. Carbohydrates commonly tested include glucose, lactose, maltose, sucrose, and fructose.

Neisseria meningitidis meningitis

Most cases (over 90%) of Neisseria meningitidis are sporadic infections. Subgroup B and subgroup C each accounts for approximately 32% of the cases in the United States, and group Y accounts for approximately 24% of the cases. • Group Y accounts for most (53%) of endemic cases of meningococcal meningitis. The quadrivalent meningococcal vaccine, which has been licensed in the United States since 2005, protects against serogroups A, C, Y, and W-135. Meningococcal meningitis is most prevalent in neonates and young adults who have not been immunized. - Risk factors also include living in the same household as a patient or carrier, which places those living in college dormitories and military recruits at a high risk when not immunized. Meningococcal meningitis may range from mild symptoms to a severe bacteremia (meningococcemia) and fulminant meningitis. Individuals with terminal complement component deficiencies also are at increased risk of meningococcal meningitis.

Incubation

Most pathogenic bacteria are mesophilic, preferring a growth temperature of 30°C to 45°C. Most incubators are set at 35°C ± 2°C to meet the preferred temperature of most internal human pathogens. Those bacteria growing on the body surface, such as skin pathogens, prefer a lower temperature of 30°C.

Biosafety cabinet and personal protective equipment

OSHA requires that employees must be protected from hazards encountered during work. In the laboratory, PPE includes protective laboratory clothing, disposable gloves, eye protection, and face masks. Gloves should be changed between patients, and hands should be washed immediately after removing gloves. High-efficiency particulate air (HEPA) respirators should be fit tested for each person; those who encounter mycobacteria through contact with either the patient or specimen should have access to a HEPA respirator. Laboratory employees who have contact with body fluids must be offered the hepatitis B vaccinations free of charge. Cabinets are classified as class I, II, or III based on performance characteristics with regard to biological containment. Class I cabinets = open-fronted, negative-pressure, ventilated cabinets. Unsterilized room air enters and circulates within the cabinet, and the exhaust air from the cabinet is filtered by a HEPA filter. Class II = sterilize both the air entering and circulating within the cabinet and the exhaust air. Type II vertical laminar-flow biological cabinets are open-fronted, ventilated cabinets. Type II cabinets have HEPA-filtered, recirculated airflow within the workspace. The exhaust air from the cabinet also is filtered by a HEPA filter. HEPA filters trap particulates and infectious agents but do not trap volatile chemicals or gases. Class III = provide the highest level of safety, and all air entering and leaving the cabinet is sterilized with a HEPA filter. Supply air is drawn through a HEPA filter while exhaust air is filtered through two HEPA filters. The system is entirely enclosed, and all infectious materials are handled with rubber gloves that are sealed to the cabinet. Most hospital microbiology laboratories routinely use class II-A BSCs. Type II-A BSCs are self-contained with 70% of the air recirculated; type II-A BSCs are not required to be vented and are acceptable for low- to moderate-risk agents.

Brucella

Oxidase: Positive Catalase: Positive Nitrate: Positive Indole: Positive Urease: Negative Acid but no gas produced from glucose, sucrose, and mannose Acid not produced from maltose and lactose Starch hydrolysis: Negative Brucella organisms are catalase-positive, oxidase-positive, nonsaccharolytic, strictly aerobic gram-negative bacilli. Brucellosis is a zoonosis and may be the cause of outbreaks in animals, which may lead to severe economic loss from damage to livestock. It occurs throughout the world and is most prevalent in the countries around the Mediterranean Gulf and Persian Gulf, Central and South America, Mexico, and India. Humans acquire Brucella infections through the ingestion of contaminated animal products, including meats and milk; through inhalation of aerosolized particles; or through direct contact through skin abrasions from handling infected animals. B. canis (dogs), B. abortus (cattle) Pathogenicity Brucella species are obligate parasites and can survive intracellularly in the reticuloendothelial system, bone, liver, and central nervous system of humans. • The organism causes undulant fever (brucellosis), which is a chronic and recurring fever that causes rising and falling fevers, following a pattern that is described as a wave. - The acute form of the disease shows flu-like symptoms, including fever, malaise, anorexia, weight loss, and muscle and back pain. The undulant form occurs within one year of the initial symptoms and includes undulant fevers, arthritis, and possibly arthritis and chronic fatigue. • Granulomatous infections occur in the reticuloendothelial system and in the bones; lymphoadenopathy and splenomegaly also may occur. The infections become chronic because the organism resides intracellularly, protected from cell-mediated immunity. Brucella can be isolated from bone marrow, cerebrospinal fluid, and wounds; however, it is most frequently isolated from the blood. Blood cultures are more likely to be positive between the first and third weeks of febrile illness. Blood cultures generally become negative following the fourth week, after the acute symptoms subside, because antibody production has begun. Because of the difficulty in culturing Brucella, the serum agglutination test (SAT), which is a serological test for antibodies in the patient's serum, may be used to confirm the infection. Brucella is a Biosafety Level 3 biohazard, which includes common and unusual pathogens; the World Health Organization (WHO) identifies Brucella as a pathogen that presents a high risk to laboratory workers. To prevent laboratory-acquired infections, a biological class II or higher safety cabinet must be used to minimize the generation of aerosols and protect against splashing.

Pseudomonas aeruginosa

Oxidase: Positive Pyocyanin: Positive Fluorescein (pyoverdin): Positive Oxidizes glucose, fructose, and xylose in oxidative-fermentative (OF) medium Cannot utilize maltose, sucrose, or lactose in OF medium Grows well on MacConkey agar as lactose-negative colonies Citrate: Positive Urease: Variable Indole: Negative Growth at 42°C: Positive Resistant to kanamycin Susceptible to carbenicillin Accounts for approximately 75% to 80% of all nonfermenters isolated from clinical specimens Identification of the organism is usually aided by characteristic pigments, which are pyocyanin, a water-soluble blue pigment, and fluorescein, or pyoverdin, a yellow fluorescing pigment. Together, the pigments produce the notable blue-green color of P. aeruginosa. Another significant characteristic of P. aeruginosa is its fruity odor of overripened grapes, which also has been described as a "corn tortilla-like" odor, resulting from the production of 2-aminoacetophenone. Other important factors include the organism's ability to grow at 42°C, as well as at room temperature and at 35°C to 37°C. The organism is motile with a single, polar flagellum. P. aeruginosa is oxidase positive and oxidizes glucose but not maltose in the OF medium. It is an obligate aerobe but also can use nitrate and arginine as a final electron acceptor when oxygen is not present. P. aeruginosa is an important pathogen, especially in the immunocompromised host; it is the agent of both acute and chronic lung infections, urinary tract infections, and sepsis. Infections typically occur at sites where water or moisture accumulate, such as in the ears, eyes, and indwelling catheters and most often at sites of burns and wounds. P. aeruginosa also has been isolated from contact lens solutions, cosmetics, hot tubs, and fruits and vegetables. Thus, the organism is often associated with nosocomial infections, such as whirlpool-associated dermatitis, wound infections, urinary tract infections, and lower respiratory tract infections following respiratory ventilation in patients with preexisting lung disorders. A mucoid strain of P. aeruginosa occurs in those with cystic fibrosis, causing a severe and chronic lung disease. Individuals with cystic fibrosis activate the alginate gene, resulting in the production of alginate, which surrounds the bacterial cell wall and protects it from phagocytosis. Virulence factors Fimbriae, which enable it to attach to respiratory epithelial cells. Flagella also enable the organism to adhere to host cells, and alginate is associated with inhibition of chemotaxis and phagocytosis. Elastase, which digests elastin of the arterial walls;collagenase, which breaks down collagen; and protease, which degrades protein, are all important enzymes produced by the organism that destroys tissues. The pigment pyocyanin is known to inhibit lymphocytes and cilia, and exotoxin A inhibits protein synthesis, which leads to tissue destruction and an aggravated host immune response. P. aeruginosa also produces hemolysins, which destroy red cells, and lipopolysaccharide, which functions as endotoxin.

Detection of Candida

PNA FISH = qualitative nucleic acid hybridization assay Cornmeal with Tween 80 agar = used for the differentiation of Candida and other yeasts based on the production of chlamydospores, hyphae, pseudohyphae, and arthroconidia. - C. albicans makes chlamydioconidia Chromogenic agars, such as CHROMagerTM (Becton Dickinson) = differentiation of different species Candida albicans can be presumptively identified based on the production of a germ tube.

Specimen sources

Peripheral: Disinfect bottle tops with 70% isopropyl alcohol (alcohol pad); clean puncture site with alcohol followed by chlorhexidine (CHG) and allow to dry. For adults, collect 10-20 cc and 1-3 cc for a child for each blood culture set; divide blood into two blood culture bottles, one for aerobes and one for anaerobes; two or three blood cultures (by separate stick) per septic episode is sufficient. Intravenous catheter tips: Remove aseptically and cut a ~4 cm segment from tip and place in sterile container; transport rapidly to prevent drying out; Semiquantitative culture of catheter tips is usually performed by rolling the tip across an agar plate; the presence of >15 colonies along with the same organism isolated from peripheral blood with clinical signs and symptoms and no other recognized source is consistent with a CR-BSI.

Specimen collection respiratory

RESPIRATORY TRACT Specimens collected from the upper respiratory tract include throat cultures, nasopharyngeal cultures, and specimens from the oral cavity. Throat = The posterior pharynx between the tonsillar pillars should be firmly swabbed while the roof and sides of the mouth and the tongue should be avoided. Throat specimens should be transported within 24 hours and held at room temperature and plated onto blood agar in the laboratory. Nasopharynx = The nasopharynx is cultured with a flexible thin wire swab that has been premoistened with sterile saline. The swab is gently guided into the nares and backward through the nasal septum until the posterior pharynx is reached. The swab is left in contact with the pharynx for 15 to 30 seconds if possible and then gently removed. Ear & sinuses = Middle ear, or otitis media, infections and sinusitis generally have predictable pathogens and thus, are not usually cultured. Sinus secretions collected by direct sinus aspirations or washes or biopsies collected by endoscopy may be submitted for culture. Cultures of the lower respiratory tract are requested to diagnose bronchitis or pneumonia. Specimens that are used to diagnose lower respiratory tract infections include expectorated sputum, endotracheal specimens, translaryngeal aspirates, and bronchoalveolar lavage. Expectorated sputum = frequently collected to diagnose infections and is considered to be the preferred specimen to diagnose pneumonia. It is important to obtain deep cough secretions that will yield productive results. A saline gargle or use of nebulized saline without disinfectants may enhance the collection. Sputum is collected into a sterile wide-mouth container with a screw cap.

coagulase test

S. aureus Staphylococcus aureus produces both bound and free coagulase. Bound coagulase is detected in the coagulase slide test, which is performed by mixing the colony with a drop of rabbit plasma; a positive reaction is shown by the presence of fibrin clots.

Automated methods

Semiautomated and automated systems usually rely on the principles of colorimetry, nephelometry, or fluorometry. - Colorimetry uses a spectrophotometer to measure a color change in the pH indicator or other indicator as an organism metabolizes a particular substrate. - Nephelometry is based on the principle of light scattering and has been used for antibiotic susceptibility testing. As an organism grows, the well becomes turbid. - Fluorometric procedures use biochemical substrates that have a fluorescent component. If the organism has the enzyme to metabolize a substrate, fluorescence is exhibited. MicroScan® System = uses plastic 96-well microtiter trays in which up to 32 substrates can be used to identify Enterobacteriaceae. The panels may be interpreted manually, and each biochemical result is converted into a seven- or eight-digit code number, which is found in a codebook. Alternatively, an automated tray reader can be used, which will detect bacterial growth or color changes as noted by changes in light transmission. BD PhoenixTM = uses the principle of nephelometry. The instrument automatically takes readings every 20 minutes during incubation. The system measures colorimetric changes and changes in fluorescence intensity levels depending on the type of substrate. There are 45 biochemical reactions, which include fluorogenic, fermentative, chromogenic, and carbon source substrates, to identify gram-negative bacilli. Most organisms are identified within 2 to 12 hours; many results are available within 4 hours.

Direct detection and molecular methods meningitis

Spinal fluids must be processed immediately. In most cases, CSF should not be refrigerated and should be held at room temperature or at 37°C. Some fastidious bacteria, such as S. pneumoniae, N. meningitis, and H. influenzae may lose their viability when refrigerated. The CSF is concentrated by centrifuging for at least 15 minutes at 1,500 × g by using a cytospin or a membrane filter. This will increase the yield of microorganisms, which increases the sensitivity of the stain and culture. • A Gram, methylene blue, or acridine orange stain is performed on the concentrated specimen Rapid methods of examination for CSF include Neufeld quellung reaction, which may be used to detect capsular polysaccharide. Bacterial or fungal antigens also can be detected and identified because capsular polysaccharides are released into the surrounding body tissue and CSF as the bacteria multiply. Other methods include counterimmunoelectrophoresis, coagglutination, or latex agglutination to detect H. influenzae type b, S. agalactiae, and N. meningitidis. Polymerase chain reaction and nucleic acid amplification methods also are available to detect microorganisms in CSF.

Enterobacter

TSI: A/A H2S: Negative Indole: Negative MR: Negative VP: Positive Citrate: Positive Motility: Positive Ornithine: Positive Deaminase (phenylalanine): Negative Enterobacter species are motile and ODC positive. Their habitat includes soil, water, and dairy products. They also are normal flora of the gastrointestinal tract of many animals, including humans. Most Enterobacter infections are opportunistic and include urinary and respiratory tract and wound infections. E. cloacae and E. aerogenes are the most frequent clinical isolates. E. cloacae Associated with urinary and respiratory tract and wound infections as well as infections of the blood. Has been isolated from contaminated intravenous fluids and other medical instrumentation E. cancerogenus Has been isolated from several clinical sources, including bone and wound infections. E. cancerogenus is unique in the genus Enterobacter because it is lactose negative but ONPG positive.

Shigella

TSI: Alkaline slant over acid deep (K/A) H2S: Negative Motility: Negative Citrate: Negative Deaminase (phenylalanine): Negative Urease: Negative MacConkey: Clear colonies (lactose negative) Hektoen enteric: Green colonies (lactose negative) Shigella organisms resemble E. coli but are lactose negative and nonmotile. Serogroup D, S. sonnei, can be separated from the other serotypes because it is ODC positive and produces β-galactosidase (ONPG positive). S. dysenteriae is separated from the others by its inability to ferment mannitol. Shigella is found only in humans and other large primates. Shigellosis, or bacillary dysentery, can be caused by all four species of Shigella. The disease can occur as sporadic cases or in outbreaks in humans. While most infections are confined to the gastrointestinal tract, Shigella infections can be disseminated to other sites in the body. The infection is spread via the fecal-oral route or through contaminated food and water. !!!Shigella should be suspected when a nonlactose fermenter, which is nonmotile and H2S negative, is isolated from the stool.!!! Shigella infections typically incubate for 1 to 7 days, and shigellosis begins with fever, abdominal cramping and pain, and diarrhea. The first stage of the infection involves a watery diarrhea, which may last up to 3 days. This is followed by less frequent bowel movements and then, the dysenteric phase, characterized by frequent stools with the presence of red and white blood cells and mucus. Fluid and electrolyte loss result from the effects of an enterotoxin, and those with severe illness may require fluid and electrolyte therapy. Because S. dysenteriae produces both a neurotoxin and an enterotoxin, infections resulting from this species are the most severe. While the enterotoxin invades the large bowel, the neurotoxin may be associated with paralysis and death. S. dysenteriae also produces a Shiga toxin, adding to the severity of infection. S. sonnei is the most prevalent species in the United States and accounts for 65% to 75% of all Shigella infections. Many infections occur in the day care setting, where it is most often spread through the fecal-oral route, in children less than 5 years of age. Currently, the medications of choice are ampicillin and trimethoprim-sulfamethoxazole, although resistance is common. Alternatively, the fluoroquinolones and azithromycin may be given.

Salmonella

TSI: K/A H2S—Positive Motility: Positive MR: Positive VP: Negative Indole: Negative Lysine: Positive Urease: Negative Deaminase (phenylalanine): Negative !!Salmonella should be suspected when lactose-negative, H2S-positive colonies are isolated from a stool culture.!! S. typhi should be suspected if a lactose-negative organism produces a small amount of H2S in the TSI agar at the point of inoculation, appearing as a curved wedge. The organism is citrate negative, ornithine negative, fails to produce gas in the TSI medium, and is generally less biochemically active when compared to other Salmonella. Types of Salmonella infections include the enteric fevers, which result from infection by Salmonella enterica serotype Typhi. - Humans are the only source for this infection, which is acquired through ingestion of fecally contaminated food or water. - The incubation period is approximately 1 to 2 weeks. Fever, headache, and vomiting occur, and the patient appears ill in 2 to 3 weeks. The fever eventually declines, and the patient usually recovers in the fourth week with appropriate therapy. The organism can be isolated from blood cultures early in the illness (weeks 1 and 2), from the urine later in the illness (weeks 3 and 4), and indefinitely from the stool. - Bismuth sulfite and brilliant green agar are suitable for the isolation of Salmonella serotype Typhi. The organism produces characteristic metallic colonies, with a black ring, on bismuth sulfite agar. Most other Enterobacteriaceae cannot grow on these highly selective media. The most common Salmonella infection is gastroenteritis, which can be spread through asymptomatic human carriers, the fecal-oral route, contaminated water or food products, and direct contact with infected pets or animals. Approximately half of all Salmonella infections are relegated to contaminated poultry or poultry products. The organism can enter hen eggs through small cracks in the shell and then contaminate the egg.

Proteus

TSI: K/A H2S—Positive Swarming motility on sheep blood agar: Urease: Rapidly positive (within 4 hours) Blood agar: Growth in waves or swarms KCN growth: Positive MR: Positive Lysine: Negative Arginine: Negative ONPG: Negative Proteus organisms are found in the environment in soil and water and are normal flora of the human gastrointestinal tract. The genus is characterized by its rapid urease activity and its typical swarming motility on blood agar. P. mirabilis is the most frequently isolated species of Proteus in human infection. It is an important cause of urinary tract infections, such as cystitis and pyelonephritis and also has been shown to be a cause of pneumonia and septicemia. P. mirabilis is indole negative and susceptible to both ampicillin and the cephalosporins. P. vulgaris, is associated with similar infections, but it is indole positive. P. mirabilis has intrinsic resistance to tetracycline and nitrofurantoin but is usually susceptible to ampicillin, amoxicillin, the cephalosporins, and other antibiotics. Proteus can be presumptively identified if colonies on sheep blood agar are swarming and oxidase negative.

triple sugar iron (TSI) agar

Test whether an organism can ferment glucose, lactose and/or sucrose, ferment just glucose, or use the peptone and amino acid Alkaline over acid with hydrogen sulfide production which is typical of Salmonella Acid over acid with hydrogen sulfide production which is seen with Citrobacter fruendii Acid over acid with gas and no hydrogen sulfide production, which is typical of the lactose fermenters such as E. coli, Enterobacter, and Klebsiella.

Propionibacterium spp.

The anaerobe Propionibacterium acnes grows in hair follicles, aided by the presence of oil secreted from the sebaceous glands. This organism produces large amounts of propionic acid and maintains the pH of the skin between 3 and 5, creating an antimicrobial effect. - appear as "picket fences" in stained smears. A rare pathogen, P. acnes is more often found as a skin contaminant in blood culture specimens. Proper disinfection of the skin in preparation for blood cultures, lumbar puncture, or aspiration of abscesses is important to reduce contamination from P. acnes and other skin contaminants. It also can cause infections associated with prosthetic devices, such as heart valves and prosthetic joints; bacteremia; and endocarditis.

Pseudomonas spp.

The family Pseudomonadaceae includes nonfermentative gram-negative bacilli that are motile with polar flagella. Pseudomonas can utilize many nutrients, both in the environment and in many types of media. These organisms oxidize glucose and other carbohydrates and are strict aerobes and usually are cytochrome oxidase positive. Infections most commonly are seen in neutropenic patients, burn patients, and those with other underlying medical disorders.

mecA

The mecA gene encodes for a penicillin-binding protein, which is a β-lactamase- resistant transpeptidase. This protein has a low affinity for methicillin and most other β- lactam antibiotics; the β-lactam ring cannot bind well to methicillin.

spot indole test

The spot test can be performed on bacteria grown only on media that contain tryptophan, which include sheep blood agar and chocolate agar. - It cannot be performed on bacterial colonies from MacConkey agar because this medium does not contain tryptophan. This test is important in the identification of Escherichia coli, which is the only lactose-fermenting member of Enterobacteriaceae that is indole positive. A positive indole reaction is also important in differentiation of Proteus species; Proteus mirabilis is indole negative, and Proteus vulgaris is indole positive. Other bacteria that are indole positive include Aeromonas hydrophilia, Pasteurella multicido, and Vibrio species.

Skin flora

The varied environment of the skin results in locally dense or sparse populations, with Gram-positive organisms (e.g., staphylococci, micrococci, diphtheroids) usually predominating. S. epidermidis = major inhabitant of the skin, and in some areas it makes up more than 90 percent of the resident aerobic flora. S. aureus = nose and perineum Micrococci = not as common as staphylococci and diphtheroids; however, they are frequently present on normal skin. Micrococcus luteus, the predominant species, usually accounts for 20-80% of the micrococci isolated from the skin. Diphtheroids (Coryneforms) = Lipophilic diphtheroids are extremely common in the axilla, whereas nonlipophilic strains are found more commonly on glabrous skin. P. acnes is seen eight times more frequently than P. granulosum in acne lesions and is probably involved in acne pathogenesis. Children younger than 10 years are rarely colonized with P. acnes. The appearance of this organism on the skin is probably related to the onset of secretion of sebum (a semi-fluid substance composed of fatty acids and epithelial debris secreted from sebaceous glands) at puberty. Gram-Negative Bacilli = make up a small proportion of the skin flora; seen in moist intertriginous areas, such as the toe webs and axilla, and not on dry skin; Enterobacter, Klebsiella, Escherichia coli, and Proteus spp. are the predominant Gram-negative organisms found on the skin

Salmonella serotypes

There are more than 2,400 serotypes with the Kauffman-White classification based on somatic (O) and flagellar (H) antigens. Subdivided into serotypes (1, 2, 3, etc.) on the basis of the flagellar (H) antigen. The serogroups are designated as A1, A2, B, B2, etc. Unique serotypes are given names that describe the disease or animal from which the organism was isolated. Also, serotypes are named for the specific geographic location where the organism was isolated.

H. influenzae

There are six serotypes of H. influenzae which are serotyped based on the characteristics of the capsule. The serotype can be determined through identification of the distinct capsular antigen through latex agglutination, capsular swelling, or immunofluorescence with type-specific antisera. There are also currently eight identified biotypes based on biochemical reactions, including carbohydrate fermentation, indole, urease, and ornithine decarboxylase (ODC). Encapsulated strains are able to reduce phagocytosis and are very pathogenic, producing rapid, devastating disease in nonvaccinated children and other susceptible individuals. The infection is spread through direct contact with respiratory droplets from a carrier or another infected individual. Thus, household contacts, day care classmates and infants, and young children are at risk for infection. The organism spreads from the nasopharynx to the regional lymph nodes, to the blood, and finally to the meninges More than 90% of the cases of meningitis are caused by serotype b. Epiglottitis is most often seen in young children and is characterized by a red, swollen epiglottis, which may lead to respiratory distress. Because H. influenzae is very fastidious and adversely affected by changes in temperature and drying, specimens should be held at room temperature and never subjected to cold temperatures. Specimens must be inoculated onto enriched media as soon as possible. Specimens are plated on chocolate agar, and a staphyloccus streak also may be performed on sheep blood agar. Plates are incubated at 35°C to 37°C under ambient air or 5% to 10% CO2 with increased humidity. The organism appears in CSF and blood smears as small, pale staining gram-negative coccobacilli, which also may appear pleomorphic or tangled. Rapid direct identification = Neufeld quellung reaction, or capsular swelling test, in which antisera react with the capsular antigens of the organism, making the capsule more prominent H. influenzae isolates should be tested for β-lactamase - if negative then treat with Ampicillin - if positive use chloroamphenicol, amoxicillin-clavulanate, and third-generation cephalosporins Vaccines are produced from purified polyribosyl ribitol phosphate (PRP).

coagulase-negative staphylococci

These bacteria are important causes of blood infections, infections involving prosthetic devices, and urinary tract infections. Most significant species are S. epidermidis and S. saprophyticus Staphylococcus epidermidis white, creamy colonies that are nonhemolytic; Gram-positive cocci in clusters; Positive growth on CNA; Growth, but lack of fermentation of MSA; Coagulase negative; DNase negative; Susceptible to novobiocin Virulence factors = ability to form biofilms through the production of cell surface and extracelluar compounds that promote the bacteria to adhere to plastic surfaces of prosthetic devices. Ps/A, which assists the organism in its ability to adhere to surfaces and also to resist phagocytosis. S. saprophyticus bright white to creamy colonies that are nonhemolytic; coagulase negative, DNase negative. RESISTANT to novobiocin. important cause of urinary tract infections, such as pyelonephritis and cystitis, especially in sexually active young women. It also is the cause of catheter-associated urinary tract infections in men and women Virulence factors = ability to adhere to epithelial cells in the urinary tract and to urethral cells.

Detection of Trichomonas

Trichomonas may be identified by a standard wet mount procedure. Pear-shaped, motile parasite with four anterior flagella, and the organism shows a JERKY motility when observed in fresh preparations APTIMA® Trichomonas vaginalis Assay is a nucleic acid amplification test (NAAT) for the detection of T. vaginalis. Rapid antigen tests and nucleic acid amplification

Specimen collection urine

URINE Diagnose a urinary tract infection (UTI) of the upper or lower tract. Lower UTIs may involve the bladder (cystitis) or the urethra (urethritis). Upper UTIs include infections of the kidney, such as pyelonephritis and glomerulonephritis. In addition, the renal pelvis (pyelitis) or ureters may be infected. Acceptable specimens for diagnosis of a urinary tract infection include a clean- catch midstream specimen, straight catheterized urine, suprapubic aspirate, and urine collected from a cystoscopy or other surgical method. Specimens that generally are not accepted include urine collected from a Foley (indwelling) catheter or a bagged collection of urine. In most cases, the specimen of choice for bacterial culture of urine is the clean- catch midstream specimen. A straight catheterized urine sample may be used for bacterial culture only if the patient cannot void or the catheter has been inserted for another medical reason. Uncultured urine should be refrigerated at 4°C and transported to the microbiology laboratory within 24 hours to minimize bacterial multiplication. Catheterized (Foley and straight) or suprapubic specimens are also held at 4°C but must be transported to the laboratory within 2 hours.

Multiplex molecular methods

Uses two or more primer pairs to amplify different targets in the same reaction mixture. The first primer pair is the control, or general primer, and is directed toward many sequences, and the second primer can be directed at a specific gene. In multiplex testing, many different targets can be used within one reaction. For example, respiratory infections caused by a panel of bacterial and viral respiratory pathogens that produce similar symptoms in the patient can be tested within one assay. For instance, a multiplex system with RT-PCR can detect influenza A, influenza B, respiratory syncytial virus A and B, parainfluenza virus, rhinovirus, enterovirus, and coronavirus.

vanA

Vancomycin Resistance in Enterococcus The VanA phenotype is encoded by the vanA gene and shows inducible high-level resistance to vancomycin and teicoplanin.

Vibrio spp.

Vibrio organisms are natural habitants of sea water, and all species except V. cholerae and V. mimicus require increased sodium chloride (NaCl) for growth (halophilic). Vibrio species have been isolated from the gastrointestinal tract as causes of gastritis and also have been found in blood and wound infections. Thiosulfate citrate bile salts sucrose (TCBS) agar = sucrose, oxgall, sodium cholate, and bromthymol blue and thymol blue indicators alkaline (pH of 8.6), which enhances the growth of Vibrio • A yellow to orange color indicates sucrose fermentation on TCBS, which can be used in the speciation of Vibrio. Sucrose fermentation is positive for V. cholerae but negative for V. parahaemolyticus. ~Vibrio cholerae~ nonhemolytic on sheep blood agar, Voges-Proskauer (VP) negative, and susceptible to polymyxin B and fails to agglutinate chicken red blood cells V. cholerae produces smooth, yellow colonies on TCBS agar because it is a sucrose fermenter. The organism produces a positive string test. Responsible for pandemics Has choleragen which attacks the bowel mucosa, leading to a tremendous outpouring of water and electrolytes and the production of the characteristic "rice-water stool" Cholera is treated with an aggressive replacement of fluids and electrolytes. No vaccines available in the United States. Cholera is prevented through proper sanitation methods, appropriate water treatment, and good personal hygiene. Vibrio parahaemolyticus = wound and diarrheal infections, 50% food-borne disease in Japan, Chesapeake Bay area. V. parahaemolyticus does not ferment sucrose and produces green colonies on TCBS Vibrio vulnificus = wound infections and septicemia from raw, contaminated oysters. V. vulnificus produces a cytolysin, which contributes to its virulence and high mortality rate from septicemia. Most strains produce green colonies on TCBS; the organism is a lactose fermenter and is positive for ONPG

Specimen collection wounds & abscesses

WOUND AND ABSCESSES Suitable specimens include pus aspirates, irrigation fluids, and swabs of purulent drainage from the dermis. It is recommended always to collect specimens for wound culture using a needle and syringe to aspirate the drainage. This avoids the collection of normal flora and also enhances the recovery of anaerobes, which are often associated with wound infections and abscesses. Swabs for superficial wounds should be collected along the edge of the wound after irrigation with sterile saline. Specimens from deep wound infections include any purulent drainage, necrotic tissue, or other tissue suspected of infection. These specimens usually are collected by needle aspiration and cultured for both anaerobes and aerobes. Exogenous infections = animal and human bites, burns, ulcers, and traumatic wounds (gunshot or stabbing). Endogenous infections = indigenous bacterias within the patient and include cellulitis, dental infections, and septic arthritis. Many endogenous infections are nosocomial and occur as a result of contamination during an invasive procedure.

Chlamydia trachomatis

agent of lymphogranuloma venereum, endemic trachoma, nongonococcal urethritis, and infant pneumonitis - Chlamydia attaches to the host cells, entering through minor breaks in the tissue or abrasions at the site of infection. - Trachoma is the leading cause of preventable blindness in the world Specimen collection Specimens that are collected to diagnose chlamydial diseases depend on the site of the infection. Scrapings are preferred over swabs whenever possible, and Dacron and rayon swabs are recommended because cotton is toxic to the organism. Swabs are placed in a suitable transport medium, which contains sucrose and phosphate buffer and antibiotics to inhibit other organisms. Identification cytological methods, cell culture, antigen detection, nucleic acid probes, and nucleic acid amplification tests Cytological tests = require epithelial cells, which have been scraped from the infected areas. The cells are stained with iodine solution or Giemsa stain and examined for the presence of cytoplasmic and perinuclear chlamydial inclusions. Glycogen in the inclusions stains light to dark brown with iodine stain, whereas the inclusions stain purple with Giemsa stain. Cell cultures = McCoy's heteroploid murine cells that have been pretreated with cycloheximide. - In this process, a monolayer of the cell culture is inoculated with the skin scrapings, and a coverslip is applied. - The cell cultures are examined in 48 to 72 hours, at which time the coverslip is removed and stained with iodine and Giemsa and then examined for inclusion bodies. - Alternatively, fluorescein-labeled chlamydial antibody can be added to the coverslip, which is then observed for fluorescent inclusion bodies. Although extremely sensitive, cell culture methods are complex and may be labor intensive. Serological methods = complement fixation, enzyme immunoassay, and immunofluorescence. This is limited bc lot of ppl have antibodies direct fluorescent antibody (DFA) Molecular methods = nucleic acid amplification and polymerase chain reaction, have been shown to have high sensitivity Nucleic acid probes identify the specific 16S ribosomal RNA sequence. Because amplification is not required, these tests are rapid and cost effective; however, sensitivity may be low because of low levels of antigen in the specimen.

Yersinia pestis

agent of plague, which is a zoonosis and transmitted via the flea- rodent life cycle Three types of plague exist: bubonic, septicemic, and pneumonic, of which bubonic plague is the most common. Bubonic plague is a highly fatal zoonosis and has been associated with three pandemics. - The organism is harbored in rats, squirrels, rabbits, chipmunks, and prairie dogs Y. pestis multiplies within the white blood cells, producing a protein and lipoprotein antigen. It then lyses the cell and escapes. Y. pestis is resistant to phagocytosis because of a protective antigen that enters the host's white blood cells. Clinical symptoms include swelling of the cervical, axillary, and inguinal lymph nodes, and it may be hematogenously spread to organs and tissues. Additional effects include disseminated intravascular coagulation, endotoxic shock, and DARK discoloration of extremities. On blood smear = safety pin appearance TSI shows weak acid in slant with little or no change in deep (yellow/red). gram-negative coccobacillus

Candida tropicalis

associated with vaginitis and urinary tract, intestinal, pulmonary, and systemic infections. It is especially virulent in individuals with leukemia.

Intrinsic resistance

bacteria must be resistant to any antibiotic that they themselves produce Examples include the intrinsic resistance of Staphylococcus saprophyticus to novobiocin Proteus, Providencia, Morganella, and Edwardsiella are intrinsically resistant to the polymyxins. The polymyxins cannot bind to the membranes of Proteus, Providencia, Morganella, and Edwardsiella, rendering them ineffective. Another example of intrinsic resistance includes the resistance of Pseudomonas aeruginosa to trimethoprim sulfamethoxazole and to tetracycline, which are pumped out of the bacteria cell by an inherent mechanism.

SELECTION AND INOCULATION OF PRIMARY MEDIA

broth = liquid media agar = gel or semisolid media; most agar is solidified by using either the red algae polysaccharide agar agar; agarose also can be used to solidify agar General isolation media = also known as supportive media, support the growth of most nonfastidious bacteria. Examples include nutrient agar, trypticase soy agar, and nutrient broth. No growth advantage is given to any group of bacteria. Nonselective isolation media = also known as enriched media, contain a nutrient supplement. Examples include sheep blood agar (SBA) and chocolate agar. SBA: trypticase soy agar base + 5% defibrinated sheep RBC Sheep blood agar does not support the growth of Haemophilus influenzae or Haemophilus haemolyticus. Chocolate agar = Sheep agar that's been heated or enzyme treated to hemolyze the red blood cells and release NAD. Also its enriched with IsoVitaleX, which contains dextrose, cysteine, vitamin B12, thiamine, and ferric nitrate and supports the growth of fastidious bacteria, such as N. gonorrhoeae and H. influenzae. Differential media = provide distinct colonial appearances of microorganisms to aid in their identification. Ex: MacConkey agar (gram negatives): If the organism can ferment lactose, the colonies appear pink to red in color. If the organism is unable to ferment lactose, the colonies appear clear. eosin-methylene blue (EMB) agar (gram negatives): Bacteria that can ferment lactose are purple. Gram-negative bacteria that cannot ferment lactose appear clear on the medium Enrichment broths = Inhibit the growth of one organism while enhancing that of another organism by providing nutrients. Examples of these broths include gram negative, selenite, and tetrathionate Gram-negative broth: bile salt; which is toxic to gram-positive bacteria and inhibits normal flora coliforms Selenite: sodium hydrogen selenite, which allows for the isolation of Salmonella and Shigella Tetrathionate: bile salts and sodium thiosulfate, which enhance the isolation of Salmonella and Shigella Thioglycollate broth = differentiation of aerobes and anaerobes. Contains thioglycollic acid as a reducing agent, a small percentage of agar to prevent oxygen from reaching all areas of the broth. Selective media = contain agents that inhibit the growth of all bacteria except those that are sought. Examples include Hektoen enteric (HE) agar, Salmonella-Shigella (SS) agar, and xylose-lysine-deoxycholate (XLD) agar. All these media selectively inhibit gram-positive bacteria and gram-negative coliform bacteria and permit the isolation of stool pathogens. HE agar: Organisms that ferment one or more of the carbohydrates will appear as yellow-orange colonies, whereas nonfermenters appear green or blue in color. Hydrogen sulfide-producing bacteria = black precipitate SS agar: allows for the differentiation of lactose fermenters from nonfermenters, as well as the detection of hydrogen sulfide producers XLD agar: elective for gram-negative enteric pathogens and allows for the detection of xylose fermentation, lysine decarboxylation, and hydrogen sulfide production. Antibiotic media = selective for a specific group of bacteria through the addition of specific antibiotics. Examples include colistin-nalidixic acid (CNA) and modified Thayer-Martin (MTM). CNA = contains sheep blood agar base with the antibiotics colistin and nalidixic acid added. Colistin (polymyxin E) disrupts the cell membrane of gram-negative organisms, and nalidixic acid blocks DNA replication in gram-negative bacteria. Thus CNA allows for the selection of gram-positive bacteria. MTM = chocolate agar base with the antibiotics vancomycin, colistin, nystatin, and trimethoprim lactate added. The medium selectively isolates N. gonorrhoeae.

blaKPC gene

carbapenem resistance The genes blaKPC and blaNDM encode KPC and NDM enzyme production, respectively. KPC (Klebsiella pneumoniaecarbapenemase) NDM (New Dehli metallo-beta-lactamase)

Yersinia enterocolitica

cefsulodin-irgasan-novobiocin (CIN) plate = incubated at room temperature (22°C to 26°C) for 24 hours to enhance its recovery - contains mannitol, peptones, yeast, neutral red indicator, and the inhibitory agents crystal violet, novobiocin, irgasan, and cefsulodin Agent of enterocolitis, inflammation that occurs in a person's digestive tract, and occurs most frequently in infants and children. In adults the bacterium is also an agent for enterocolitis, ileitis, and septicemia. Animals serve as the major route of infection, and humans may acquire the infection through ingestion of contaminated water, pork, beef, or milk products. Fecal-oral transmission also has been implicated as a route of infection. Y. enterocolitica ferments mannitol and appears as RED "bull's-eye" colonies surrounded by a colorless halo. The species is ODC positive and ferments both sucrose and sorbitol. - gram-negative bacillus

Calcofluor white

fluorescent stain used to stain specimens directly for the presence of fungi. The fungal elements appear blue-white when observed with a fluorescent microscope.

Acinetobacter baumannii

gram negative coccobacilli; oxidase-negative, catalase-positive, nonmotile; Obligate aerobe; Nonhemolytic; Grows well on MacConkey agar; Resistant to penicillin A. baumannii produces large, gummy, translucent, gray-to-white, convex, entire colonies on sheep blood agar. A bluish or peach-to-pink tint may be observed on MacConkey agar, and a cornflower blue color is produced on EMB. Growth on MacConkey agar and the negative oxidase test may lead one to identify the organism mistakenly as a member of the Enterobacteriaceae. Can cause infections in the blood, urinary tract, and lungs, and wounds.

Neisseria spp

gram-negative diplococci, resembling tiny coffee or kidney beans in the Gram stain capnophilic = prefer increased carbon dioxide (CO2) very sensitive to temperature changes and should be protected from the cold All Neisseria are cytochrome oxidase positive almost all members of the Neisseria are catalase positive, except for N. elongata. The pathogenic Neisseria are very fastidious; N. gonorrhoeae requires chocolate agar, and N. meningitidis and M. catarrhalis require blood agar as the minimal growth standard. N. gonorrhoeae requires the amino acid cysteine Isolation Media for Neisseria Modified Thayer-Martin and Martin-Lewis are the same but the former uses Nystatin and the latter Anisomycin to inhibit the growth of mold / yeast New York City Neisseria organisms establish disease through attachment to the mucous membranes of the host through pili, hairlike structures on the bacterial cell that enable the bacteria to bind to human cells. ~Neisseria gonorrhoeae~ The Gram stain serves as a presumptive identification in smears prepared from the urethral exudate in male patients and is considered to be highly specific and sensitive in symptomatic males. The specimen should be plated immediately both on a plate selective for N. gonorrhoeae and on chocolate agar. Plates are incubated at 35°C to 37°C in the candle jar CO2 incubator, providing an atmosphere of 3% to 5% CO2. Colonies are typically clear gray to medium gray in color and opaque. Type 1 colonies are small, raised, and moist, whereas type 2 colonies appear small and raised but dry. N. gonorrhoeae produces acid from glucose but no other sugar Penicillin is mostly used, but if resistant (PPNG) use spectinomycin and tetracycline.

S. agalactiae

group B Streptococcus Morphology Colonies are medium in size, flat, and translucent or opaque. Most often, the colonies show narrow-zone β-hemolysis. Identification CAMP positive, Hippurate positive, Bactitracin resistant, SXT resistant, PYRase negative, Bile-esculin hydrolysis negative Group B Streptococcus is normal flora of the GI tract, and vaginal tract. GBS is an important cause of neonatal infections, including pneumonia, meningitis, and sepsis. Infections are generally treated with penicillin and an aminoglycoside.

oxidase test

identifies bacteria that have cytochrome oxidase Cytochrome oxidase in the presence of atmospheric oxygen oxidizes tetramethyl-para- phenylenediamine dihydrochloride (oxidase reagent) to form a colored compound— indophenol. (blue) The cytochrome system is found in the following genera: Neisseria, Aeromonas, Pseudomonas, Campylobacter, and Pasteurella but not in any of the Enterobacteriaceae.

Common agents of endocarditis

ie: inflammation of your heart's inner lining Approximately 80% of infective endocarditis cases are caused by the bacteria streptococci and staphylococci. The third most common bacteria causing this disease is enterococci, and, like staphylococci, is commonly associated with healthcare-associated infective endocarditis. Viridans streptococcus = a significant cause of subacute bacterial endocarditis when established in the heart. S. epidermidis = associated with prosthetic valve endocarditis N. gonorrhoeae = can cause disseminated gonococcal infection (DGI), which leads to endocarditis Group B Streptococcus Staphylococcus aureus Streptococcus pneumoniae Moraxella catarrhalis E. coli, E. cloacae, and E. aerogenes Burkholderia cepacia

Bacteroides fragilis

major normal flora of the bowel and is a significant cause of intra-abdominal abscesses pale-staining pleomorphic bacillus grows on BBE and hydrolyzes esculin grow well in 20% bile Generally, the Bacteroides organisms have been displaced following surgery or trauma to the abdominal cavity or female genital tract. Other types of infections from Bacteroides include lower respiratory tract infections, skin and soft tissue abscesses, decubitus ulcers, chronic wound infections, and bone infections. On anaerobic blood agar, colonies are large, gray, moist, and nonhemolytic with an entire margin and ring-like structures TREAT with rifampin!! Very resistant to strong drugs like vancomycin and kanamycin.

Staphylococcus aureus

medium to large colonies, 1.0 μm to 2.0 μm in diameter after 24 hours of incubation. Colonies show a smooth, butyrous, creamy appearance. The edge is entire, and the colonies may be pigmented white to golden yellow. Most strains of S. aureus exhibit a narrow zone of β-hemolysis, whereas some strains are nonhemolytic. Identification S. aureus is the only staphylococcal species pathogenic to humans that produces coagulase Other tests to identify S. aureus include growth and fermentation of MSA. The organism can grow in 6.5% to 10% NaCl and ferment the carbohydrate alcohol mannitol. S. aureus also gives a positive DNase test reaction, as indicated by a clearing of the dye toluidine blue, which is incorporated into the nutrient agar- based medium. Chromogenic agars also can be used to isolate and identify staphylococcal species. When using HardyCHROMTM Staph aureus agar, S.aureus appears as deep pink-to fuchsia-colored colonies, whereas S. epidermidis may be partially or totally inhibited and S. saphrophyticus appears as turquoise-colored colonies within 24 hours. Transmission S. aureus causes disease by invading tissues and producing toxins. The organisms may be spread from the site of carriage to the site of infection by breaks in the skin, including surgical wounds or skin abrasions. The organism may then be spread through the blood to the lungs, bones, liver, brain, or heart. Many infections are nosocomial, including surgical wound infections and those involving prosthetic devices. Bacteremia and septicemia from S. aureus also occur in patients with cardiovascular disease, diabetes mellitus, and immunosuppressive conditions and in those with prosthetic devices. Many cases of bacteremia are nosocomial, and complications include endocarditis. Virulence mechanisms enterotoxin = food borne disease or gastroenteritis Hemolysin = lyse red blood cells leukocidan = inhibit white blood cells

Campylobacter spp.

microscopic morphology, that is, small, curved or seagull-winged, faintly staining, gram-negative rods can sometimes be detected by direct Gram stain examination of stool oxidase positive and unable to ferment carbohydrates microaerophilic, preferring decreased amounts of oxygen capnophilic, preferring increased levels of carbon dioxide Humans become infected by ingestion of contaminated animal products, direct contact with contaminated animal feces, or though contact with another infected human. The clinical signs of Campylobacter infection include fever, abdominal pain, cramping, and bloody diarrhea. The stool characteristically contains segmented neutrophils and red blood cells. The organism may invade the bloodstream and also cause endocarditis, septic arthritis, or meningitis. Virulence factors for Campylobacter include the production of cytotoxin, cytotonic factor, and an enterotoxin. Becton-Dickinson media = blood agar base with the antibiotics vancomycin, polymyxin, cephalothin, trimethoprim, and amphotericin B added Grow optimally at 42°C to 43°C Growth is generally present within 48 hours, and Campylobacter produces colonies that are gray to pink or yellow to gray in color. darting motility in wet prep

Acridine orange

most useful for visualizing RBC parasites Selectively binds to any nucleic acid and is useful in demonstrating small amounts of bacteria in blood cultures, cerebrospinal fluid, urethral smears, and other exudates. It is very helpful in observing bacteria in those specimens contaminated with tissue and other debris.

Colony morphology

observe at the edge of the colony swarming of Proteus, star-like appearance of yeast Red (some Serratia marcescens), green—Pseudomonas aeruginosa Elevation: flat, convex (dome shaped), raised, umbilicate (depressed center), umbonate (raised center) Characteristic odors, such as Pseudomonas aeruginosa, which has been described as smelling like grapes, tortilla chips, or fruity gum.

Aeromonas spp.

oxidase-positive, fermentative, gram-negative bacilli that are generally motile with polar monotrichous flagella Aeromonas is naturally found in freshwater and seawater and is known to cause disease in cold-blooded animals The most common types of human infections include cellulitis and wound infections, acquired through contact with contaminated water or soil, as well as gastrointestinal disease. Miscellaneous types of infections associated with Aeromonas include septicemia, urinary tract infections, and ear infections. Differentiated from Enterobacteriaceae by a POSITIVE oxidase reaction It can be differentiated from Pseudomonas aeruginosa by its ability to ferment glucose and produce indole. The most common human isolate is A. hydrophilia, which means "water loving" and is the species that most often causes gastrointestinal disease. The organism produces a heat-labile enterotoxin and a heat-stable cytotoxic enterotoxin. A. hydrophilia also produces protease, lipase, and nuclease.

Enterotoxigenic E. coli (ETEC)

penetrate the intestinal epithelium and produce a toxin that causes gastroenteritis. It is commonly known as traveler's diarrhea, weanling diarrhea, or Montezuma's revenge or "turista" and is associated with ingestion of contaminated water or food. ETEC is seen more frequently in developing countries. Enterotoxin production occurs in certain serotypes, which include O6, O8, O15, O25, O27, O63, O78, O148, and O159. ETEC may produce a heat- labile toxin (LT) and/or a heat-stable toxin (ST), which leads indirectly to fluid loss. The intestinal wall is invaded, leading to inflammation and fluid loss. The illness is characterized by nausea, diarrhea without blood, pus or mucus, mild vomiting, chills, and headache. There usually is a low-grade fever or no fever. The incubation period is 1 to 2 days, and the actual illness lasts for 5 to 10 days.

Preparation methods for slides used for stains

saline mount = specimen (or organism) is mixed with saline and observed with the condenser lowered. This method permits one to detect the presence of bacteria in a specimen and also to observe for motility. One also can observe for trophozoite forms of pathogenic stool parasites, such as Giardia lamblia and Trichomonas vaginalis in urine sediment. iodine mount = Lugol's iodine is mixed with the stool specimen. This technique is used to stain stools for parasitic ova whose nuclei appear orange to brown. potassium hydroxide (KOH) preparation = fungal elements can be detected by mixing the tissue with 10% KOH. The KOH dissolves tissue, such as skin, hair, or nails, making the fungal elements more visible. India ink, or nigrosin = produces a black, semi-opaque background, which makes the clear capsule more visible

Francisella tularensis

small, nonmotile, pleomorphic gram-negative bacillus, a disease of rodents, primarily rabbits The bacterium also can be acquired indirectly through insect vectors, primarily biting flies and ticks It is highly infective when grown in culture, and extreme caution must be used in the clinical laboratory to avoid laboratory-acquired infections. Extreme caution should be exhibited when F. tularensis is suspected because it readily can penetrate through small breaks in the skin. It is a Biosafety Level 2 pathogen, which requires that laboratory workers wear gloves and work in a biological safety cabinet. F. tularensis possesses a capsule that enables it to resist phagocytosis and also has invasive properties that allow the bacterium to penetrate intact skin. inhalation = pneumonia splashing or infecting the eye with contaminated hands = oculoglandular disease (conjunctiva) ingesting contaminated food or water = primary lesion is found in the pharynx Identification F. tularensis requires a special medium, blood-cysteine-glucose agar with thiamine, to grow. It takes 2 to 4 days to grow and is a strict aerobe. Colonies are smooth, bluish gray, and slightly mucoid with a narrow α-hemolytic zone. It also grows on modified charcoal-yeast agar and chocolate agar with IsoVitaleXTM. Biochemical reactions used to identify Francisella include catalase-positive, oxidase-negative, and positive fermentation of glucose, maltose, and mannose. Serological techniques are the recommended method for identification. The DFA stain is used to detect antibodies to F. tularensis in the patient's serum. Enzyme linked immunoassay techniques also are available.

Trichrome stain

stain commonly used for fecal specimens It is a rapid, simple procedure, which produces uniformly well-stained smears of the intestinal protozoa, human cells, yeast, and artifact material.

Plesiomonas shigelloides

straight, round, gram-negative bacillus, which is motile with polar lophotrichous flagella (multiple at one end) An important infectious agent in Japan and in the tropics and subtropics as a cause of gastroenteritis. The infection is generally mild with no blood or mucus in the stool. - The organism is carried on various cold-blooded animals and is found in the water and soil. - Infection is acquired through the ingestion of contaminated or unwashed foods. P. shigelloides grows on blood agar and as a nonlactose fermenter on MacConkey agar, and eosin-methylene blue. It can be differentiated from Enterobacteriaceae by a POSITIVE oxidase reaction.

Modified acid fast stain

used to stain *Nocardia*

Acid-fast stains

used to stain mycobacteria that possess thick, waxy cell walls. Once stained, these bacteria resist decolorizing by acid alcohol, and thus the designation acid fast. Ziehl-Neelsen technique = carbolfuchsin is heated to drive the stain into the cell wall of the mycobacterium. Kinyoun carbolfuchsin method = a detergent is used with the carbolfuchsin to enable the stain to penetrate the cell wall.

Enterobacteriaceae

very large and diverse family of bacteria consisting of gram-negative bacilli and coccobacilli Infections may be associated with lapses in personal hygiene through the fecal-oral route, poor sanitation in underdeveloped countries, or colonization of the skin and respiratory tract of hospitalized patients. Humans may acquire these bacteria through ingestion of contaminated food or water, nosocomially through contact with patients or health care personnel or contaminated medical instruments, and endogenously through their own normal flora. Common types of infections attributed to the Enterobacteriaceae include urinary tract, respiratory tract, and wound infections; bacteremia; and gastroenteritis. These bacteria also are known to cause disease in poultry, livestock, fish, and vegetable crops, in addition to being significant human pathogens. All Enterobacteriaceae are glucose fermenters and cytochrome oxidase negative. Most species, except for Erwinia and Pantoea agglomerans reduce nitrates to nitrites. Most members of this family are motile, with peritrichous flagella, and some species possess pili or fimbriae, which serve as structures for attachment. Facultative anaerobes Enterobacteriaceae most often associated with diarrhea are Salmonella, Shigella, specific diarrheagenic E. coli, and Yersinia enterolitica. Identification Any oxidase-negative organism isolated from MacConkey agar can be suspected of being a member of the Enterobacteriaceae family. The organism's ability to ferment lactose can be determined through observation on MacConkey or EMB agar. Most members of the Enterobacteriaceae produce hydrogen and carbon dioxide (CO2) gas during fermentation, which can be observed as cracks in the tubed media. Especially large amounts of gas are produced by members of Klebsiella, Enterobacter, Hafnia, and Serratia. - Of special note is the genus Shigella, which cannot produce gas during fermentation of carbohydrates. The ONPG reaction is valuable in the differentiation of Citrobacter species, which are ONPG positive, from most Salmonella species, which are ONPG negative. - Salmonella arizonae is the only ONPG-positive Salmonella serotype.