Module 4
Which Klebsiella species is indole positive?
K. oxytoca
Schematic diagram for Enterics: K/A H2S+ and PAD Negative
K/A H2S + PAD Negative: Salmonella spp. C. freundii Edwardsiella tarda Lysine decarboxylase -Positive: --Salmonella spp. --E. tarda -Negative --C. freundii --Salmonella Paratyphi A
Tribe Proteeae
Members consist of: -Proteus, Morganella, and Providencia Tribe members are: -widely disseminated in the environment -part of normal intestinal microbiota most clinical isolate in tribe: -Proteus mirabilis Key biochemical reactions: -Positive PAD -nonlactose fermenter
What are the general characteristics of Enterobacteriaceae?
-all ferment glucose -reduce nitrate to nitrite -oxidase negative (EXCEPT PLESIOMONAS) -motile at body temperature (EXCEPT Klebsiella, Shigella, Yersinia) -Morphology: EXCEPT for Klebsiella, Proteus, and some Enterobacter species none have remarkable morphology on routine media (BAP, Choc, and most nonselective media)
Salmonella Virulence Factors
-fimbriae used in initiating intestinal infection -ability to traverse intestinal mucosa -enterotoxin involved in gastroenteritis
Differential tests to identify C. freundii from Salmonella
C. freundii: -colony morphology: on primary selective media may be mistaken for Salmonella -decarboxylate lysine: no -Hydrolyze urea: 70% yes Salmonella: -Colony morphology: on primary selective media, resembles Citrobacter -Decarboxylate Lysine: Yes (most strains) -Hydrolyze urea: no Both produce H2S
Plesiomonas Clinical Manifestations
Causes at least three major clinical types of gastroenteritis -more common watery or secretory diarrhea -subacute or chronic disease that lasts from 14 days to 2 to 3 months -more invasive dysenteric form that resembles colitis most notable extraintestinal infections -meningitis in neonates -septicemia -shock
Salmonella- Nontyphoidal Bacteremia
Characterized by prolonged fever and intermittent bacteremia Two groups at risk for developing infection -young children --fever and gastroenteritis with brief episodes of bacteremia -adults --transient bacteremia during episodes of gastroenteritis or develop symptoms of septicemia without gastroenteritis
My IMViC reaction is 0+0+ (-+-+). I am Lysine Decarboxylate negative (LDC) and Urease negative. My TSI reaction is K/A with H2S production. I may cause UTI, pneumonia and diarrhea. Who am I?
Citrobacter fruendii
Schematic diagram for Enterics: K/A H2S- ; PAD Negative
Citrobacter spp Shigella spp Escherichia coli Yersinia pestis Serratia spp Motility (37 degrees Celsius) -Positive --E. coli --Citrobacter spp. --Serratia spp. -Negative --Shigella sp. --Y. pestis
Salmonella- Enteric Fevers
Clinical Features: -prolonged fever -bacteremia -involvement of the reticuloendothelial system (RES), particularly the liver, spleen, intestines, and mesentery -dissemination to multiple organs Enteric fever caused by Salmonella typhi is called TYPHOID FEVER
Enterobacter Species
Clinically significant isolates -the most commonly encountered species isolated from wounds, urine, blood, CSF --E. colacae --E. aerogenes other species: -E. gergoviae: respiratory samples, rarely blood -E. hormaechei
Members of the family Enterobacteriaceae share which one of the following characteristics? a. produce cytochrome oxidase b. ferment lactose c. produce beta-hemolysis d. reduce nitrate to nitrite
reduce nitrate to nitrite Members of Enterobacteriaceae are oxidase negative, ferment glucose, and reduce nitrate to nitrite
What are some characteristics of the Enterobacteriaceae?
-all members are gram negative bacilli or coccobacilli -all members are cytochrome oxidase negative -all members grow on MacConkey agar
Biochemical reactions of an organism are consistent with Shigella. A suspension is tested in antiserum without resulting agglutination. However, after 15 mins. of boiling, agglutination occurs in group D antisera. The Shigella species is: a. dysenteriae b. flexneri c. boydii d. sonnei
sonnei Boiling removed the capsule so that the antiserum could react with cell wall antigen. Group D Shigella is S sonnei.
MacConkey media for screening suspected cases of hemorrhagic E coli O157:H7 must contain: a. indole b. citrate c. sorbitol d. lactose
sorbitol Sorbitol replaces lactose in MacConkey. E coli 0157:H7 does not ferment sorbitol, whereas other species of E coli are positive for fermentation of sorbitol. This makes the media a good screen for 0157:H7
Is Enterobacteriaceae generally aerobic or anaerobic?
they are facultatively anaerobic Thioglycolate broth: growth throughout the tube
What is the primary function of Hektoen enteric (HE) agar?
to find salmonella and shigella
Providencia Species- P. rettgeri
urinary tract pathogen occasionally nosocomial outbreaks
Salmonella Carrier State
Individuals who recover from infection may harbor the organisms in the gallbladder -gallbladder becomes the site of chronic carriage -organisms are excreted in the feces either continuously or intermittently Carrier state may be terminated by antimicrobial therapy if gallbladder infection is not evident Cholecystectomy has been the only solution to chronic carrier
Yersinia pseudotuberculosis- Clinical Manifestations
Ingested organisms spread to the mesenteric lymph nodes -general infection that is usually self-limiting May include septicemia accompanied by mesenteric lymphadenitis -presentation similar to appendicitis
What is the capsular antigen and is it heat stable or labile?
K antigen is the capsular antigen and it is heat labile Ex: K1 antigen of E. coli, Vi antigen of Salmonella typhi
Schematic diagram for Enterics: K/A H2S+ ; PAD Negative; Lysine decarboxylase Positive
K/A H2S+ PAD Negative Lysine Decarboxylase Positive: -Salmonella spp. -E. tarda Indole -Positive: --E. tarda -Negative: --Salmonella spp.
Salmonella enteria- Differentiation Salmonella subgroups and members
Lysine decarboxylase rules out S. paratyphi Ornithine decarboxylase rules out S. typhi Trehalose fermentation rules out S. choleraesuis (S. Typhi is least reactive)
Bacillary Dysentry
Marked by penetration of intestinal epithelial cells after attachment to mucosal surfaces Local inflammation Shedding of the intestinal lining Formation of ulcers after epithelial penetration
Does Enterobacteriaceae reduce nitrate to nitrites?
yes
Salmonella Antigenic Structures
O and H antigens H antigen: -Antigens occur in phases (Phase I and Phase II) -Phase I antigens are not always present, but if they are they can identify a particular serotype -Phase II are nonspecific -flagellar Capsular antigen Vi similar to K capsule antigens: -prevents phagocytosis -heat labile Vi antigen often blocks O antigen during serologic typing but may be removed by heating (boiling)
What is the somatic antigen and is it heat stable or labile?
O antigen is the somatic antigen and it is heat stable
Shigella Antigenic Structures
O antigens are separated by serologic grouping K antigens -must be removed to type O antigen -Heat labile Lack H antigens -nonmotile
Yersinia enterocolytica- Appendicitis Like Syndrome
Occurs primarily in older children and adults possible symptoms: -severe abdominal concentrated in right lower quadrant -Fever -enlarged mesenteric lymph nodes -inflamed ileum and appendix -Erythema nodosum
Hafnia
One species: H. alvei -linked to gastroenteritis -occasionally isolated from stool cultures -delayed citrate reaction H. alvei biotype 1 -grows in beer wort --has not been isolated clinically
What are the two major types of clinically significant pathogens?
Opportunistic pathogens Primary pathogens
Serratia species
Opportunistic pathogens associated with outbreaks in health care settings -urinary and respiratory tract infections -nursery, cardiac surgery, burn unit outbreaks -contamination of antiseptic solution --resulted in an epidemic of septic arthritis Biochemical reactions Positive o-nitrophenyl-B-D-galactopyranoside (ONPG)-- slow lactose fermenter DNase varies- may help in species identification (ID) highly resistance to antimicrobials
Schematic diagram for Enterics: K/A H2S- and PAD Positive
P. rettgeri M. morganii P. stuartii P. alcalifaciens Citrate -Positive --Providencia sp. -Negative: --Morganella morganii
What is known as being primarily a plant pathogen?
Pantoea agglomerans
Why might it be necessary for both pink (lactose positive) and colorless (lactose negative) colonies from an initial stool culture on MacConkey agar by subcultured and tested for possible pathogen?
Pathogens can be either lactose positive or lactose negative
Yersinia pseudotuberculosis
Primary pathogen of rodents (especially guinea pigs), farm animals, birds Human infection rare -associated with close contact with infected animals or their fecal material or ingestion of contaminated drink and foodstuff
Serratia marcescens
Prodigiosin: pink to red pigment when growing at room temperature usually most clinically relevant nosocomial infections of the urinary tract, respiratory tract, bacteremia
I cause wound, urinary, ear, and bacteremic infections. My TSI reaction is K/A, H2S production. I am indole negative, ornithine decarboxylase positive, and PAD positive. Who am I?
Proteus mirabilis
Schematic diagram for Enterics: K/A H2S+
Proteus mirabilis Salmonella sp. Citrobacter freundii Edwardsiella tarda PAD -Positive (+): --P. mirabilis -Negative (-): --Salmonella spp. --C. freundii --Edwardsiella tarda
The stock cultures needed for quality control testing of phenylalanine deamination activity are: a. Escherichia coli / Klebsiella pneumoniae b. Salmonella typhimurium / Escherichia coli c. Escherichia coli / Pseudomonas aeruginosa d. Proteus mirabilis / Escherichia coli
Proteus mirabilis / Escherichia coli Of the organisms listed only Proteus mirabilis is phenylalanine deaminase positive
Key Characteristics of Specific Shigella Species
S. dysenteriae (Group A) -most serious infection (developing countries) S. flexneri (group B) -second most common isolate in the United States -associated in men who have sex with men and in young adults ~25 years old S. boydii (Group C) -more common in developing countries S. sonnei (Group D) -most common isolate in the United States --short, self-limiting; patients have fever and watery diarrhea
Which species of Shigella is associated with men who have sex with other men and in young adults ~25 years old?
S. flexneri (group B)
Is Enterobacteriaceae motile?
All motile at BODY TEMPERATURE EXCEPT Klebsiella, Shigella, and Yersinia
Enteroadherent E. coli
Associated with two kinds of human disease: -diarrheal syndromes -UTIs Two types of EAEC: -EAEC: enteroaggregative E. coli (EAEC) -DAEC: diffusely adherent E. coli (DAEC) -may be associated with UTIs and diarrheal disease
What is the selective agent of Hektoen enteric (HE) agar?
Bile salts inhibits gram-positive
What are the selective agents of Salmonella and Shigella (SS) agar?
Brilliant green and bile salts inhibits gram-positive
What is the pH indicator of Hektoen enteric (HE) agar?
Bromethymol Blue
Media for the Isolation of Yersinia Species
CIN agar and Yersinia-selective agar (YSA) help isolate Yersinia species -Now CIN II, which can also isolate Aeromonas
Shigella Colony Morphology
Clear, nonlactose-fermenting colonies S. sonnei: 24 hours lactose negative; 48 hours lactose Positive
Isolation and Identification
Consider site of origin Generally enteric opportunistic organisms isolated from sites that are normally sterile are highly significant Complete identification should be directed only toward true intestinal pathogens
Providencia Species
Five species- two major pathogens: -P. rettgeri -P. stuartii both have high resistance to antimicrobials
Cronobacter sakazakii
Formerly Enterobacter sakazakii Produces yellow pigment Documented pathogen causing meningitis and bacteremia in neonates from powdered infant formula also has been found: brain abscesses, respiratory, and wound infections
What are the primary pathogens of Enterobacteriaceae?
Salmonella sp Shigella sp Yersinia sp --generally from ingesting contaminated food and water NOT NORMAL FLORA
I am a stool pathogen that is transmitted through undercooked or uncooked poultry, milk, and eggs. I cause gastroenteritis and typhoid fever. My IMViC reaction is 0+0+ (-+-+) and my TSI reaction is K/A H2S production. I am urease negative. Who am I?
Salmonella typhi
Salmonella- Typhoid Fever Transmission and Epidemiology
Transmission: -ingestion of contaminated food originating from infected individuals or carriers -no known animal reservoir Epidemiology highlights: -tropical and subtropical areas where international travelers are more likely to acquire the infection -improper disposal of sewage, poor sanitation, and lack of a modern potable water system contribute to typhoid fever cases and outbreaks
Morganella Species- M. morganii
UTI infections -documented cause Possible agent of diarrheal disease -has not been fully examined to date identified as a cause of neonatal sepsis nonswarmer motile
Yersinia pestis
Y. pestis -Causative agent of PLAGUE --Bubonic, septicemic, and pneumonic forms Transmitted through bite of INFECTED FLEAS -pneumonic plague through respiratory droplets Gram-negative coccobacilli resembling a SAFETY PIN appearance (bipolar staining) -preferential growth at 25 to 30 degrees Celsius Class A bioterrorism agent
Is Plesiomonas oxidase positive or negative?
oxidase positive
All of the following are categories of diarrhea-causing Escherichia coli except a. ETEC b. EMEC c. EIEC d. EPEC
EMEC
Which of the following tests best differentiates Salmonella and Citrobacter species?
KCN, malonate, beta-galactosidase, lysine decarboxylase
The organism is nonmotile, LDC +, ODC -, ADH - which would most likely indicate that the isolate is:
Klebsiella pneumoniae
A clean catch urine sample from a nursing home patient is cultured using a .001mL loop. It grows 67 colonies of a lactose fermenter that has the following biochemical reactions: TSI: Acid/Acid Oxidase: negative Motility: positive Indole: negative Citrate: positive VP: positive Lysine decarboxylase: negative Ornithine decarboxylase: positive Urea: negative What should the microbiologist report?
67,000 CFU/mL Enterobacter cloacae
A surgical wound specimen produces pink colonies on MacConkey agar. There is no growth on CNA and abundant flat, spreading gray mucoid colonies on blood agar. The next four questions are talking about this case study above. This specimen contains:
A gram negative bacillus that ferments lactose
Enterohemorrhagic E. coli (EHEC) is:
Also known as verocytoxic E. coli May be associated with hemolytic uremic syndrome May often serotype as 0157:H7
Biochemical reactions of an organism are consistent with Salmonella. A suspension is tested in polyvalent antiserum A through G and Vi antiserum. There is agglutination in the Vi antiserum only. What should be done next?
Boil suspension of the organism for 10 minutes to inactivate the Vi antigen
Which of the following agars is not selective for Enterobacteriaceae? a. Buffered charcoal yeast extract b. MacConkey (MAC) c. Hektoen enteric d. Xylose-lysine deoxycholate
Buffered charcoal yeast extract
________ media is used to isolate Yersinia enterocolitica.
CIN Cefsulodin-irgasin-novobiocin
What is the name of the organism that has been a documented cause of nursery outbreaks of neonatal meningitis and brain abscesses? a. Citrobacter freundii b. C. asteroides c. C. braakii d. C. koseri
C. koseri
You have a TSI reaction that is Alkaline over Acid. What colony morphology would you suspect on MacConkey agar? A) Mucoid B) Red pigment C) Swarmer D) Clear
Clear
On Hektoen Enteric (HE) agar, Shigella appears as:
Clear or colorless colonies.
A microbiologist is reading stool culture plates. She is looking for enteric pathogens on the MAC plate. What do they look like? a. Clear, colorless colonies b. Large, bright pink colonies c. Small, mucoid, green colonies d. Small, orange colonies
Clear, colorless colonies
All of the following are clinically significant isolates of the genus Enterobacter, except a. Enterobacter cloacae b. E Aerogenes c. E gergoviae d. E Hartmanni
E Hartmanni
The IMVC reactions are - - + + which would eliminate __________ as a possible organism.
E. coli
What genus has the following characteristics:urea negativepositive for lysine decarboxylasehydrogen sulfide and indoledoes not grow on Simmon's citrate? a. Providencia b. Edwardsiella c. Escherichia d. Morganella
Edwardsiella
A fecal specimen, inoculated to xylose lysine deoxycholate (XLD) and Hektoen enteric (HE) produced colonies with black centers. Additional testing results are as follows: Biochemical screen: - Glucose: positive - H2S: positive - Lysine decarboxylase: positive - Urea: negative - ONPG: negative - Indole: positive Serological test: - Polyvalent: no agglutination - Group A: no agglutination - Group Bl: no agglutination - Group C: no agglutination - Group D: no agglutination - Group Vi: no agglutination The most probable identification is:
Edwardsiella tarda
A microbiologist is reading a stool culture and notices a mucoid pink colony on MAC agar. What is most likely organism? a. Klebsiella pneumoniae b. Escherichia coli c. Proteus vulgaris d. Yersinia enterocolitica
Klebsiella pneumoniae
What substance produced by the Salmonella spp, which cause gastroenteritis, has been implicated as a significant virulence factor? a. Hyaluronidase b. Protease c. Enterotoxin d. Endotoxin
Enterotoxin
The most important cause of UTIs is a. Klebsiella pneumoniae b. Escherichia coli c. Hafnium alvei d. Morganella morganii
Escherichia coli
What organism presents as a pink colony with a halo on MAC agar and a green metallic sheen on eosin methylene blue (EMB) agar? a. Salmonella enterica b. Proteus Mirabilis c. Escherichia coli d. Klebsiella pneumonia
Escherichia coli
Which of the following organisms can grow in the small bowel and cause diarrhea in children, traveler's diarrhea, or a severe cholera-like syndrome through the production of enterotoxins?
Escherichia coli
Infection of the urinary tract is most frequently associated with: a. Staphylococcus aureus b. Escherichia coli c. Enterococcus faecalis d. Serratia marcescens
Escherichia coli >80% of uncomplicated UTIs are caused by E coli
What are the organisms of interest for clinical isolates in acute-care settings?
Escherichia coli Klebsiella pneumonia Proteus mirabilis
An organism gave the following reactions: TSI: acid slant/acid butt; no H2S gas produced Indole: positive Motility: positive Citrate: negative Lysine decarboxylase: positive Urea: negative VP: negative This organism most likely is: a. Klebsiella pneumoniae b. Shigella dysenteriae c. Escherichia coli d. Enterobacteria cloacae
Escherichia coli These biochemicals are characteristic for E coli. Klebsiella pneumoniae is indole negative and nonmotile. Shigella dysenteriae is nonmotile and Enterobacter cloacae is indole negative.
The TSI is yellow over yellow with cracks but no blackening. This would be interpreted as:
Ferments glucose with gas production, ferments lactose and/ or sucrose
What are the primary antigens used in serologic grouping of salmonellae? a. Somatic A antigen and flagellar O antigens b. Capsular A antigen and somatic O antigens c. Flagellar H antigen and capsular A antigen d. Flagellar H antigen and somatic O antigen
Flagellar H antigen and somatic O antigen
All of the following organisms are commonly associated with urinary tract infections except a A) VP-negative, citrate-negative, indole-positive, lactose fermenter. B) H2S-positive, urea-negative, lysine-positive, non-lactose fermenter. C) non-motile, VP-positive, indole-negative, lactose fermenter. D) H2S-positive, urea-positive, indole-negative, non-lactose fermenter.
H2S-positive, urea-negative, lysine-positive, non-lactose fermenter.
Components of media for gram negative rods have specific properties enables media to be selective or differential. Eosin and Methylene Blue act as: A) Inhibitor B) pH indicator C) Fermentation D) Hydrogen Sulfide Detector
Inhibitor
A sputum culture from an alcoholic seen in the ER grows gray, mucoid, stringy colonies on SBA. The isolate grows readily on MacConkey agar and forms mucoid, dark pink colonies. The colonies yield the following test results: ONPG - positive indole - negative glucose - positive oxidase - negative citrate - positive VP - positive The organism is most likely: a. Edwardsiella tarda b. Klebsiella pneumoniae c. Escherichia coli d. Proteus vulgaris
Klebsiella pneumoniae Klebsiella is the only distractor that is VP positive, and the other biochemical reactions are typical for K pneumoniae
A microbiologist is reading stool culture plates. She sees an organism that has a large, mucoid pink colony on MAC. What is a good presumptive identification of this organism? a. Enteric pathogen b. Escherichia/Citrobacter-like organism c. Klebsiella/Enterobacter-like organism d. Haemophilus influenza
Klebsiella/Enterobacter-like organism
What enteric organism may produce a blue-violet pigment on non-blood-containing media? a. Kluyvera b. Shigella c. Ewingella d. Cedecea
Kluyvera
What organism is the genus Providencia is incriminated in nosocomial outbreaks in burn units and has been isolated from urine cultures? a. Providencia stuartii b. P. heimbachae c. P. rustigianii d. P. alcalifaciens
Providencia stuartii
What organism possesses the Vi antigen? a. Salmonella typhi b. S. sonnei c. Shigella sonnei d. Citrobacter freundii
Salmonella typhi
Does Shigella produce H2S or gas?
No
Is the H antigen found in the cell wall?
No
Which of the following antigens remains after boiling a suspension of a member of the Enterobacteriaceae? A) K B) H C) O D) Vi
O
What bacterium produces swarming colonies on nonselective media, such as sheep blood agar (SBA)? a. Enterobacter aerogenes b. Klebsiella pneumoniae c. P. mirabilis d. Morganella morganii
P. mirabilis
a 64-year-old male with lymphoma has a positive blood culture at 18 hours incubation. The organism is a nonlactose fermenting gram-negative bacillus on MacConkey agar. Further testing gives the following reactions: Oxidase: negative TSI: alkaline/acid, no hydrogen sulfide Motility: positive Indole: positive Citrate: positive Ornithine decarboxylase: negative Urea: positive Phenylalanine deaminase: positive VP: negative The genus is:
Providencia
Rapid urease activity is characteristic of the genus _________.
Proteus
Swarming colonies on sheep blood agar would lead one to suspect:
Proteus
The primary pathogens of the Enterobacteriaceae include all of the following except a. Salmonella enterica b. Proteus mirabilis c. Shigella spp d. Yersinia spp
Proteus mirabilis
>100,000 CPU/mL of a gram-negative bacilli were isolated on MacConkey from a urine specimen. Biochemical results are as follows: Glucose: acid, gas produced Indole: negative Urea: positive TDA: positive H2S: positive The organism is most likely: a. Morganella morganii b. Proteus mirabilis c. Proteus vulgaris d. Providencia stuartii
Proteus mirabilis Morganella and Providencia do not produce H2S; the indole reaction differentiates P mirabilis and P vulgaris
The production of deaminase is found in which of the following groups of bacteria?
Proteus, Providencia, and Morganella
The most frequently implicated species of Shigella in the United States is:
S. sonnei
What organisms produce H2S or gas?
Salmonella Proteus Citrobacter
A DNase-positive member of the family Enterobacteriaceae is suggestive of a. Enterobacter b. Serratia c. Proteus d. Escherichia
Serratia
Phenylalanine deaminase is produced by all of the following organisms except A) Providencia stuartii. B) Morganella morganii. C) Proteus vulgaris. D) Serratia marcescens.
Serratia marcescens
__________ ____________ produces DNAse, lipase, gelatinase and sometimes a red pigment.
Serratia marcescens
The stock cultures needed for quality control testing of deoxyribonuclease (DNase) production are: a. Salmonella typhimurium / Escherichia coli b. Escherichia coli / Pseudomonas aeruginosa c. Proteus mirabilis / Escherichia coli d. Serratia marcescens / Escherichia coli
Serratia marcescens / Escherichia coli Of the organisms listed only Serratia marcescens is DNase positive
Bacillary dysentery caused by this organism is marked by penetration of intestinal epithelial cells following attachment of the organisms to mucosal surfaces, local inflammation, shedding of the intestinal lining, and formation of ulcers that follow the epithelial penetration. What is the organism? a. Enterobacter b. Citrobacter c. Shigella d. Salmonella
Shigella
Which one of the following genera is among the least biochemically reactive members of the Enterobacteriaceae? a. Proteus b. Pseudomonas c. Citrobacter d. Shigella
Shigella Shigella is lactose negative, most species do not produce gas, are VP, urea, lysine decarboxylase and citrate negative, and they are nonmotile
A stool culture from an 11-month-old male suffering from bloody diarrhea gave the following results on differential enteric media:MacConkey agar: clear coloniesXylose Lysine Deoxycholate agar: clear coloniesHecktoen Enteric agar: clear coloniesWhich of the following tests must be performed to confirm the final identification of this enteric pathogen? A) No further testing is required B) Latex agglutination for E. coli O157:H7 C) Salmonella serotyping D) Shigella serotyping
Shigella serotyping
An isolate from a stool culture gives the following growth characteristics and biochemical reactions: MacConkey agar: colorless colonies Hektoen agar: yellow-orange colonies TSI: acid slant/acid butt, no gas, no H2S Urea: positive These screening reactions are consistent with which of the following enteric pathogens? a. Yersinia enterocolitica b. Shigella sonnei c. Vibrio parahaemolyticus d. Campylobacter jejuni
Shigella sonnei Shigella has colorless colonies on both MacConkey and Hektoen agars. Yersinia is lactose negative, but Hektoen agar [has both lactose and sucrose] produces yellow colonies from the fermentation of sucrose. V parahaemolyticus needs at least 1% NaCl to grow and Campylobacter does not grow on MacConkey or Hektoen agars.
Which of the following properties or reactions consistently differentiates gram-negative rods of the Enterobacteriaceae from the non-fermenting gram-negative rods? A) Oxidase reaction B) Growth on MacConkey C) Presence of LPS D) TSI reaction of K/A or A/A
TSI reaction of K/A or A/A
Why is it that most cases of salmonella food poisoning are not treated with antibiotics? a. The organisms are very resistant to antibiotics b. Antibiotics cannot reach the organisms, because they are deeply embedded in the intestinal walls c. Antibiotics would only kill the bacteria, then release the endotoxins into the blood, making things worse d. The disease is self limiting and will subside on it's own
The disease is self limiting and will subside on it's own
Why can direct microscopy not be used to provide a presumptive identification of the enteric bacteria? a. Some members of the genus gram stain weakly b. The microscopic characteristics are indistinguishable from other gram-negative bacteria c. Some members of the genus do not decolorize well and appear gram-positive d. Some members have very thick capsules and these do not allow the penetration of the gram stain
The microscopic characteristics are indistinguishable from other gram-negative bacteria
What disease produced by a Salmonella spp is a severe form of enteric fever? a. Pontiac fever b. Typhoid fever c. Undulant fever d. Malignant fever
Typhoid fever
Are members of the Klebsielleae family VP positive or negative?
VP positive
Which of the following reactions is associated with the gram-negative rod best known for its capsule production? This organism is the most commonly recovered isolate from respiratory tract samples of hospitalized patients. A) PAD positive B) Positive motility C) VP positive D) H2S production
VP positive
One of the enterotoxins produced by enterotoxigenic Escherichia coli in traveler's diarrhea is similar to a toxin produced by:
Vibrio cholerae
In the ____________ __________ pathway for glucose fermentation, acetoin is detected.
Voges Proskauer
What type of media contains lactose, sucrose, Xylose and lysine. A) HE B) MAC C) XLD D) EMB
XLD
A 10-year-old boy was admitted to the emergency room with lower right quadrant pain and tenderness. The following laboratory results were obtained: Patient value Normal range % segmented neutrophils 75% 16% - 60% WBC count 200 x 10^3/uL (200 x 10^9/L) 13.0 x 10^3/uL (13.0 x 10^9/L) The admitting diagnosis was appendicitis. During surgery the appendix appeared normal; an enlarged node was removed and cultured. Small gram-negative bacilli were isolated from the room temperature plate. The organism most likely is:
Yersinia enterocolitica
Which one of the following gram-negative bacilli ferments glucose a. Alcaligenes faecalis b. Pseudomonas cepacia c. Acinetobacter lwoffii d. Yersinia enterocolitica
Yersinia enterocolitica Alcaligenes, Pseudomonas and Acinetobacter are all nonfermenters; Yersinia is a member of the Enterobacteriaceae and, by definition, ferments glucose
What bacterium is causative agent of the plague? a. Salmonella enteritidis b. Citrobacter freundii c. Shigella sonnei d. Yersinia pestis
Yersinia pestis
___________ ___________ causes plague, which is transmitted through fleas and rodents.
Yersinia pestis
Which genera of the Enterobacteriaceae are considered enteric pathogens? A) Enterobacter, Citrobacter, and Morganella B) Yersinia, Salmonella, and Shigella C) E. coli, Citrobacter, and Klebsiella D) Enterobacter, Providencia, and Proteus
Yersinia, Salmonella, and Shigella
The lipopolysaccharide associated with the Enterobacteriaceae is A) protective against antimicrobial agents. B) an endotoxin. C) used in conjugation. D) part of the plasma membrane.
an endotoxin
A gram-negative bacillus has been isolated from feces, and the confirmed biochemical reactions fit those of Shigella. The organism does not agglutinate in Shigella antisera. What should be done next? a. test the organism with a new lot of antisera b. test with Vi antigen c. repeat the biochemical test d. boil the organism and retest with the antisera
boiling the organism and retest with the antisera Some Shigella produce capsular antigen that mask the cell wall and boiling removes the capsule
An 8-year-old female was admitted with a complaint of right lower quadrant pain. She was taken to surgery for removal of her appendix. The appendix appeared normal, but lymph nodes in the peritoneal cavity were inflamed. Cultures of the nodes were taken and sent to the microbiology department. The organism that grew appeared as clear colonies on MAC and provided the following reactions:TSI A/A 37∘C SIM —/—/— 25∘C SIM —/—/+MR/VP +/— PAD — Urea +Citrate — ONPG + Arginine —Lysine — Ornithine +This is organism benefited from cold enrichment. Humans usually acquire the organism through contact with animals or by ingesting contaminated food or water. A) bulls-eye colonies on CIN agar. B) lipase. C) DNase. D) clear colonies on XLD agar.
bulls-eye colonies on CIN agar
Escherichia coli strains have all of the following properties except a. indole production b. fermentation of glucose and lactose c. methyl red (MR) positive, Voges-Proskauer (VP) negative d. can use citrate as a sole carbon source
can use citrate as a sole carbon source
Which animals are a common source of Salmonella serotypes? a. dogs b. cold-blooded animals (fish) c. cats d. horses
cold-blooded animals (fish)
In the carrier state, where are pathogenic salmonella carried? a. gallbladder b. liver c. intestines d. stomach
gallbladder
Is Proteus mirabilis indole positive or negative?
indole negative
All of the following are biochemical features of Salmonella, except a. lactose positive b. indole negative c. hydrogen sulfide positive d. VP negative
lactose positive
A 25-year-old female complained of burning on urination. The urine culture grew a clear colony on MacConkey agar. Biochemical testing revealed the following:TSI A/A H2S positive SIM +/+/+ Citrate — Urea + MR/VP +/— ONPG —PAD + Arginine — Lysine — Ornithine —This organism will also A) need to be serotyped. B) exhibit multiple antibiotic resistance. C) swarm. D) appear as a mucoid colony.
swarm indole + proteus vulgaris indole - proteus mirabillis
All of the following are characteristics of Shigella spp, except a. nonmotile b. urea positive c. hydrogen sulfide negative d. lysine decarboxylase negative
urea positive
Enterobacter Species Characteristics
-Citrate positive -Potassium cyanide broth positive -MR negative -VP positive -usually produce ornithine decarboxylase --Lysine decarboxylase produce by most (not E. cloacae or E. gergoviae) infection sites -wound -urine -blood -CSF
E. coli Biochemical Reactions
-Fermentation of glucose, lactose, trehalose, and xylose -indole production from tryptophan -Glucose fermentation via mixed acid pathway --MR positive and VP negative -Does not produce H2S, DNase, urease, or phenylalanine deaminase (PAD) -IMViC pattern: ++-- -cannot use citrate as sole carbon source
Plesiomonas characteristics
25% to 40% of all infected patients present with: -fever, vomiting, or broth -most common clinical symptom: abdominal pain most cases are self-limiting antimicrobial therapy is indicated in severe and prolonged cases -OXIDASE POSITIVE -Glucose fermenting -Facultatively anaerobic -Gram-negative bacilli --single, pairs, short chains, filamentous forms -Does not have ability to produce gas from glucose -No capsules -Motile
Yersinia enterocolytica
Acquired from contact with swine, cats, dogs -ingestion of contaminated food contacted fecal material -refrigeration is ineffective because it survives in cold temperatures Most common forms of infection -acute gastroenteritis, simulating appendicitis, arthritis, erythema nodosum* *tender red nodules with itching and burning on lower legs (shins)
Salmonella Clinical Infections
Acute gastroenteritis/ food poisoning Typhoid fever Nontyphoid bacteremia Carrier state following Salmonella infection
What are the Virulence and Antigenic Factors of Enterobacteriaceae?
Adherence: prevent being removed from tissue Production of toxins: i.e., food poisoning Invasive enzymes: spreading factors Antibiotic resistance: extended spectrum B-lactamases (ESBL) O antigen (somatic antigen): heat stable antigen located in the cell wall (lipopolysaccharide [LPS]) H antigen: flagella antigen; heat stable K antigen: capsular antigen; heat labile (ex: K1 antigen of E. coli, Vi antigen of Salmonella typhi)
How does Shigella and some Salmonella species appear on Salmonella and Shigella (SS) agar?
Colorless/ Translucent colonies
Salmonella Classification
DNA sequencing: genus has two species: -Salmonella enterica -Salmonella Bongor: rare- found in Chad Africa -usually isolated from cold-blooded animals and the environment
Identification
Determine if Enterobacteriaceae: Gram-negative Oxidase negative -Except for Plesiomonas shigelloides -Always use young colonies from sheep blood agar (SBA) plates Ferment glucose Reduce nitrate to nitrite --Except Photorhabdus and Xenorhabdus
Growth of Enterobacteriaceae on Salmonella and Shigella (SS) agar?
Differentiating agent: lactose Selective agent: brilliant green and bile salts inhibits gram-positive H2S indicator: sodium thiosulfate- colonies will have black center pH indicator: neutral red Lactose positive: red Lactose negative: clear
Microscopic Examination and Culture: Screening Stools
Direct microscopic examination -not very helpful in differentiating species Culture -consider culture media and incubation temperature -Generally, grow quickly and easily Screening stool cultures for pathogens -methods must be appropriate for the recovery and identification of enteric pathogens
Schematic diagram for Enterics: A/A H2S- ; IMViC --++ ; and Motility (37 oC) Positive
Enterobacter spp Serratia spp. DNase -Positive --Serratia sp -Negative --Enterobacter spp.
I am a stool pathogen that causes hemorrhagic diarrhea and in severe cases hemolytic uremic syndrome. My IMViC reaction is ++00 (++--) and my TSI reaction is A/A with gas production. Unlike my cousins, I fail to ferment sorbitol. Who am I?
Enterohemorrhagic E. col (EHEC)
Gastointestinal pathogenic E. coli
Enterotoxigenic E. coli (ETEC) Enteroinvasive E. coli (EIEC) Enteropathogenic E. coli (EPEC) Enterohemorrhagic E. coli (EHEC) Enteradherent: -Enteroaggregative E. coli (EAEC) -Diffusely adherent E. coli (DAEC)
Schematic diagram for Enterics: A/A H2S-
Escherichia coli Klebsiella spp. Enterobacter spp. Serratia spp. IMViC ++-- ---E. coli --++ ---Klebsiella sp ---Enterobacter sp ---Serratia sp
A microbiologist is reading stool culture plates. She sees an organism that has a dry, pink colony with a surrounding "halo" of pink on MAC. What is a good presumptive identification of this organism? a. Enteric pathogen b. Escherichia/Citrobacter-like organism c. Klebsiella/Enterobacter-like organism d. Haemophilus influenza
Escherichia/Citrobacter-like organism
Shigella Epidemiology
Human and large primates are only known reservoirs Transmission: -direct person to person -Fecal-oral route with carriers as the source -Transmitted by flies, fingers, and food or water contaminated by infected persons Personal hygiene plays a major role in transmitting organisms Groups at risk -Children in daycare centers (particularly infants younger than 1 year of age) -individuals living in crowded and inadequate housing -individuals who practice anal-oral sexual activity -known to cause outbreaks on cruise ships
What does IMViC stand for?
I: indole M: Methyl Red V: Voges Proskauer C: Citrate
Klebsiella and Raoultella info
Klebsiella and Raoultella (formelly known as Klebsiella) belong to the tribe Klebsielleae -other members are Enterobacter, Serratia, Pantoea, Cronobacter, and Hafnia Tribe members are found in the intestinal tracts of humans and animals, in soil as free-living organisms, in water, and on plants There organisms are associated with: -various opportunistic and hospital-acquired infections (especially pneumonia, wound infections, and UTIs)
I cause a variety of nosocomial infections. My IMViC reaction is +0++ (+-++). My TSI reaction is A/A, H2S negative. I am nonmotile, PAD negative, and LDC positive. Who am I?
Klebsiella oxytoca
I cause a variety of nosocomial infections. I have the distinct feature of possessing a polysaccharide capsule which protects me from phagocytosis. My IMViC reaction is 00++ (--++). My TSI reaction is A/A H2S negative. I am nonmotile and Phenylalanine deaminase (PAD) negative. Who am I?
Klebsiella pneumoniae
Schematic diagram for Enterics: A/A H2S- and IMViC --++
Klebsiella sp Enterobacter sp Serratia sp Motility (37 degrees Celsius) -Positive: --Enterobacter spp --Serratia spp. -Negative: --Klebsiella spp.
The gram-negative rod is recovered from a catharized urine sample from a nursing home patient. The lactose-negative isolate tested positive for indole, urease, ornithine decarboxylase, and phenylalanine deaminase and negative for H2S. The most probable identification is?
Morganella morganii
I cause wound, urinary, ear, and bacteremic infections. My TSI reaction is A/A, H2S production. I am indole positive, PAD positive, and ornithine decarboxylate negative. Who am I?
Proteus vulgaris
Schematic diagram for Enterics: K/K (no change)
Refer to ID of NLF
K. pneumoniae subsp. rhinoscleromatis
Rhinoscleroma -intense swelling and malformation of the entire face and neck Africa and South America
An organism was inoculated to a TSI tube and gave the following reactions: alkaline slant/acid butt, H2S, gas produced This organism most likely is: a. Klebsiella pneumoniae b. Shigella dysenteriae c. Salmonella typhimurium d. Escherichia coli
Salmonella typhimurium Salmonella is the only distractor that produces H2S. Also, Klebsiella and E coli produce acid/acid reactions in TSI
Characteristics of Xylose lysine deoxycholate XLD agar
Selective (less so than MAC and HE) and Differential Sodium deoxycholate: inhibits gram-positive, some gram-negative Three carbohydrates: sucrose and lactose in excess, and xylose with a phenol red indictor Lysine present to detect lysine decarboxylation Thiosulfate present to detect hydrogen sulfide (H2S)
Enterobacteriaceae Growth on MacConkey (MAC) agar
Selective: bile salts and crystal violet inhibit gram-positive and Differential: lactose fermentation is differential --phenol red is pH indicator
DNase positive indicates
Serratia species
Colorless or Red colonies on Xylose Lysine Deoxycholate (XLD) agar
Shigella
Yesinia pestis Clinical Manifestations
Symptoms of BUBONIC PLAGUE -high fever with painful regional lymph nodes (known as buboes) -Septicemic form occurs when the bacteria spread to the bloodstream Pneumonic plague -Secondary to bubonic plague or the septicemic form when originated proliferate in the bloodstream and respiratory tract
I am the number one cause of UTIs. My IMViC reaction is ++00 (++--). My TSI reaction is A/A, H2S negative with gas production. Who am I?
Uropathogenic E. coli
My infections can manifest in several forms: acute enteritis (most common), an appendicitis-like syndrome and arthritis. My IMViC reaction is ++00 (++--). I ferment sucrose and not lactose. Cold enrichment 25-30 degrees Celsius provides better recovery. Who am I?
Yersinia enterocolytica
I am transmitted to humans by bites of fleas. My gram stain reaction reveals bipolar staining giving a safety pin appearance. My IMViC reaction is ++00 (++--). My TSI reaction is K/A H2S negative. I am nonmotile, PAD negative, urease positive, and ONPG negative. Who am I?
Yersinia pestis
What is only found in uropathogenic E. coli?
aerobactin
What indicates Serratia marcescens?
brick red colonies
What color colonies are lactose negative organisms on Hektoen enteric (HE) agar?
clear/ translucent
Enterotoxigenic E. coli (ETEC) characteristics
diarrhea in infants and adults in tropics and subtropics Traveler's diarrhea (requires large inoculum 10^6 - 10^10 organisms) lasts 1 to 5 days Transmitted via contaminated food and drink Organisms can colonize the small intestine established organisms release toxins heat labile toxin (LT): which leads to hypersecretion of fluids, watery diarrhea heat-stable toxin (ST): leading to hypersecretion Clinical manifestations: -mild, self limiting -watery diarrhea, abdominal cramps, occasional nausea -no vomiting or fever -similar to other diarrheal illnesses and must be differentiated typical diagnosis: -characteristics symptoms and isolation of solely lactose-fermenting organisms on differential media
Serratia odorifera
dirty, must odor resembling that of rotten potatoes two biogroups: -Biogroup 1: respiratory --Positive for sucrose, raffinose, ornithine, and indole (60%) -Biogroup 2: blood and CSF --Negative for sucrose, raffinose, ornithine; positive for indole (50%)
Citrobacter Species- C. koseri
documented pathogen as cause of Nursery outbreaks of neonatal meningitis and brain abscesses
Yellow colonies on Xylose Lysine Deoxycholate (XLD) agar
fermenters or those not producing lysine decarboxylase (Escherichia coli, Citrobacter)
Is Klebsiella typically indole positive or negative?
indole negative
Edwardsiella- E. tarda
E. tarda is the only pathogen Urea: negative Lysine decarboxylase: positive H2S: positive Indole: positive No growth on Simmons citrate medium Clinical manifestations: -bacteremia and wound infections -freshwater fish: both feral and farmed -cold blooded: turtles, snakes, lizards, etc -isolated directly from lakes, rivers, and sewage waters
Characteristics of Shigella Species
Four species: dysenteriae, flexneri, boydii, sonnei Nonmotile Generally do not produce gas from glucose -Except some types of S. flexneri No urease production No H2S in TSI No decarboxylation of lysine Cannot utilize acetate or mucate as carbon source S. sonnei is positive for ONPg and ornithine decarboxylase S. flexneri is negative for these tests
Yesinia enterocolytica Appearance and Culture Media
Gram-negative coccobacilli appearance (bipolar staining) -preferential growth at 25 to 30 degrees Celsisus Motile at 25 degrees Celsius but not at 35 degrees Celsius Cold enrichment can be used to increase recovery in fecal samples Selective media available -Cefsulodin-irgasan-novobiocin (CIN)
Salmonella Species
Gram-negative rods facultatively anaerobic Clear, colorless, non-lactose fermenting on MAC colonies with black centers on HE or XLD Indole negative VP negative Urease negative PAD negative Most produce H2S --exception: Salmonella paratyphi A No growth in potassium cyanide
What color are the colonies of pathogens on Hektoen enteric (HE) agar?
Green to blue-green color with H2S gas creating a black color Salmonella and Shigella
What is the flagella antigen and is it heat stable or labile?
H antigen is the flagella antigen and it is heat stable
What grows in beer wort?
Hafnia alvei Biotype 1
All of the following are symptoms of salmonellosis that may appear 8 to 36 hours after ingestion of contaminated food, except a. nausea b. vomitting c. headache d. watery diarrhea
headache
How does E. coli appear on Salmonella and Shigella (SS) agar?
pink to red colonies
How does Klebsiella appear on Eosin-methylene blue (EMB) agar?
purple black and lactose positive
What color colonies are lactose positive organisms on Hektoen enteric (HE) agar?
salmon colored
K. oxytoca
similar to K. pneumoniae EXCEPT indole positive affects similar sites as K. pnemoniae
Uropathogenic E. coli
Most common cause of UTIs in humans Primary Virulence Factor: production of pili allow attachment to epithelial cells and washed out with urine flow Cytolysins can kill immune cells and inhibit phagocytosis Aerobactin allows bacteria to chelate iron --Aerobactin is a bacterial iron chelating agent found in E. coli. It is a virulence factor enabling E. coli to sequester iron in iron-poor environments such as the urinary tract
E. coli characteristics
Most strains: -Dry pink colonies on MAC plates -B-hemolysis on blood agar plate (BAP) -Motile -Possess adhesive fimbriae and sex pili -possess O, H, K antigens Commonly isolated from colon biota -used as primary marker of fecal contamination in water quality testing
Specimen Collection and Transport
Organisms can be isolated from a wide variety of clinical samples -most often isolated with other organisms Appropriate and immediate transport media important for isolation
Proteus Species- P. mirabilis and P. vulgaris
P. mirabilis is most commonly isolated isolated from urine, wounds, ear, and blood infections, generally produce SWARMING on laboratory media -burnt chocolate odor Lactose negative and PRODUCE UREASE most produce H2S, hydrolyze urea
Differential Tests to identify P. mirabilis and P. vulgaris
P. mirabilis: -indole: negative -ornithine decarboxylase: positive P. vulgaris: -indole: positive -ornithine decarboxylase: negative -other tests of importance: ferments sucrose; Acid/Acid in TSI agar
Key Characteristics of Plesiomonas shigelloides for Presumptive ID
P. shigelloides -Oxidase: positive -Sensitivity to the agent O/129: Sensitive (susceptible) -Inositol fermentation: Positive Similar genera -Oxidase: negative (other enterobacteriaceae) -Sensitivity to the agent O/129: Resistant (aeromonas) -Inositol Fermentation: Negative (Aeromonas and most all Vibrio)
A urine culture had the following culture results: Sheep blood: swarming Columbia CNA: no growth MacConkey: - 1. >100,000 CFU/mL nonlactose-fermenter - 2. >100,000 CFU/mL nonlactose-fermenter with red pigment The isolates from MacConkey agar had the following biochemical reactions: Test Isolate 1 Isolate 2 TSI Alk/acid Alk/acid Urea Positive Negative TDA Positive Negative H2S Positive Negative The organisms are most likely: a. Proteus vulgaris and Enterobacter cloacae b. Proteus mirabilis and Serratia marcescens c. Morganella morganii and Klebsiella pneumoniae d. Providencia stuartii and Serratia liquefaciens
Proteus mirabilis and Serratia marcescens Serratia can produce a red pigment; Proteus mirabilis swarms, is TDA positive and produces H2S
Schematic diagram for Enterics: A/A H2S+
Proteus vulgaris Citrobacter freundii Salmonella Arizonae PAD -Positive --P. vulgaris -Negative --C. freundii --S. Arizonae
Schematic diagram for Enterics: K/A H2S-
Providencia rettgeri Morganella morganii Providencia stuartii Citrobacter spp. Shigella spp. Escherichia coli Providencia alcalifaciens Yersinia pestis Serratia spp. PAD -Positive: --P. rettgeri --M. morganii --P. stuartii --P. alcalifaciens -Negative: --Motility (37 degrees Celsius)
Serologic Grouping (Salmonella and Shigella)
Salmonella -60 types of O antigens --95% are serogroups A through E1 -direct or latex agglutination tests for serogroup Shigella -A through D serogroups
What are the primary human intestinal pathogens that are not normal biota of the intestinal tract?
Salmonella -inhabit the gastrointestinal (GI) tracts of animals -human infection occurs via contaminated or undercooked animal food products Shigella -human carriers coupled with improper sanitary conditions and poor personal hygiene Yersinia -transmitted by wide variety of animals (plague)
Escherichia hermanii
Yellow-pigmented -isolated from cerebrospinal fluid (CSF), wounds, and blood -isolated from foodstuffs such as raw milk and beef Clinical significance still not fully established
Enterobacteriaceae Growth on Hektoen enteric (HE) agar?
Selective: bile salts inhibit gram-positive and some gram-negative and Differential: lactose and sucrose fermentation is differential --most nonpathogens ferment lactose and sucrose and shows an orange color (low pH) --pathogens (salmonella and shigella) green to blue color with H2S gas creating a black precipitant
Enterobacteriaceae Growth on Eosin-methylene blue (EMB) agar
Selective: contains peptones, lactose, sucrose, and two dyes (Eosin Y and Methylene Blue) --both dyes inhibits gram-positive and Differential: Lactose and sucrose fermentation is differential
Extraintestinal infections of E. coli
Septicemia and meningitis -most common in neonates and very young children -gain infection just before or during delivery, and when there is contamination of amniotic fluid -Capsule antigen K1 (predisposition for meningitis)
I am an opportunistic pathogen associated with nosocomial outbreaks. My species ferments lactose slowly, ONPG positive. My IMViC reaction is 00++ (--++), positive for motility and my TSI reaction is A/A H2S negative. I grow a characteristic brick-red type colonies on MacConkey agar. Who am I? only DNase +
Serratia marcescens
Clinical Infection of Shigella Dysentery
Severity of disease varies from asymptomatic to severe Initial symptoms appear 24 to 48 hours after ingestion of the organisms -high fever, chills, abdominal cramps, pain accompanied by tenesmus Organisms multiple in the small intestine move toward the colon -isolation in the colon may occur for 1 to 3 days Organisms invade the colonic tissues -causes an inflammatory reaction -water diarrhea Bloody mucus and numerous leukocytes follow
I am a stool pathogen that forms green/clear colonies on HE agar. I cause bacillary dysentery. I'm Lactose negative on a MacConkey plate. My TSI reaction is K/A H2S negative. I am nonmotile, urease negative, ONPG positive, and PAD negative. Who am I?
Shigella sonnei
Klebsiella and Raoultella Characteristics- Key biochemical results
Simmons Citrate: positive Potassium cyanide broth: negative No H2S production No deanimation of phenylalanine Only a few members hydrolase urea slowly indole: negative (usually) Methyl red: negative VP: positive IMViC: --++
Salmonella enteria
Six subspecies in S. enterica: -S. typhi (least reactive) -S. choleraesuis -S. paratyphi
What is the H2S indicator of Hektoen enteric (HE) agar?
Sodium thiosulfate: colonies will have black center
Salmonella- Typhoid Fever Disease Course
Symptoms develop 9 to 14 days after ingestion of organisms -the larger the inoculum, the shorter the incubation time -first week of disease patients typically develop --fever, malaise, anorexia, lethargy, myalgia, and a dull headache -looks like the flu organisms appear to be resistant to gastric acids -reach the small intestine, invade, and penetrate the intestinal mucosa -leads to constipation organisms gain access to the lymphatic system and are sustained in mesenteric lymph nodes Weeks 2 and 3 of the disease -patient experiences sustained fever with prolonged bacteremia -Organisms invade the gallbladder and Peyer's patches of the bowel; they also reach the intestinal tract via the biliary tract -"Rose spots" appear during week 2 of the fever -infection of the intestinal tract typically transpires resulting in large numbers of organisms --may be isolated from the stool Organisms eventually reach the bloodstream and spread to the: -liver, spleen, bone marrow where they are immediately engulfed by mononuclear phagocytes Organisms multiply intracellulary and are later released into the bloodstream for the second time -febrile episode becomes more evident Seed the bloodstream -spread to spleen, liver, and bone marrow -engulfed by monocytes and grow intracellulary Rereleased into the blood stream -prolonged bacteremia -"Rose spots" in the periumbilical region invasion of the gallbladder and Peyer's patches; release of bacteria into the bowel via the biliary duct -Gallbladder is the foci in long-term infections -Severe infections can cause necrosis of gallbladder and/or Peyer's patches --Hemorrhage and perforation of the bowel Foci of long-term carriage of the organism become the gallbladder -necrosis in the gallbladder may lead to necrotizing cholecystitis and necrosis of the Peyer's patches -Hemorrhage and perforation of the bowel may occur as serious complications Other complications that may occur: -pneumonia, thrombophlebitis, meningitis, osteomyelitis, endocarditis, abscesses
Schematic diagram for Enterics: TSI
TSI Agar -K/A H2S+ --Proteus mirabilis --Salmonella sp. --Citrobacter freundii --Edwardsiella tarda -A/A H2S+ --Proteus vulgaris --Citrobacter freundii --Salmonella Arizonae -K/A H2S- --Providencia rettgeri --Morganella morgani --Providencia stuartii --Citrobacter spp. --Shigella spp. --Escherichia coli --Providencia alcalifaciens --Yersinia pestis --Serratia spp. -A/A H2S- --Escherichia coli --Klebsiella spp. --Enterobacter spp. -Serratia sp. -K/K (no change) --Refer to ID of NLF
A stool culture from a 30-year-old man suffering from bloody mucoid diarrhea gave the following results on differential enteric media: MacConkey agar = clear colonies; XLD agar = clear colonies; Hektoen agar = green colonies; Salmonella-Shigella agar = small, clear colonies Which tests are most appropriate for identification of this enteric pathogen? A. TSI, motility, indole, urease, Shigella typing with polyvalent sera B. TSI, motility, indole, lysine, Salmonella typing with polyvalent sera C. TSI, indole, MR, VP, citrate D. TSI, indole, MR, and urease Who am I, What other biochemical tests would confirm genus and species?
TSI, motility, indole, urease, Shigella typing with polyvalent sera Shigella sp,; serotyping
How does Proteus and Salmonella species appear on Salmonella and Shigella (SS) agar?
Translucent colonies with black centers
Appearance of colonies on Salmonella and Shigella (SS) agar?
Translucent with Black center: Proteus and Salmonella Pink to Red: E. coli Colorless/Translucent: Shigella and some Salmonella species
Differentiating Yersinia Species
Y. pestis -Motility: motile at 18 to 22 degrees Celsius; nonmotile above 22 degrees Celsius -Ornithine decarboxylase: Negative Y. enterolytica -Motility: motile at 25 degrees Celsius; nonmotile (negative) at 35 degrees Celsius -Ornithine decarboxylase: Positive Y. pseudotuberculosis -Motility: Motile at 25 degrees Celsius -Ornithine decarboxylase: Negative
Enteroinvasive E. coli (EIEC) characteristics
affects adults and children -rare in the United States Dysentery with direct penetration, invasion, and destruction of intestinal mucosa very similar to shigellae but require higher amounts of inoculum watery diarrhea with scant stool, pus, mucus, and blood nonmotile and do not ferment lactose (thus very similar to shigellae)
Does Enterobacteriaceae ferment glucose?
all ferment glucose
Enteropathogenic E. coli (EPEC)
associated with: -infantile diarrhea children less than 1 year -nurseries and daycares rare in adults symptoms: -low grade fever -malaise -vomiting -diarrhea (large amounts of mucus without apparent blood)
Citrobacter Species- C. freundii
at least 12 species identified C. freundii -most common --can be isolated in diarrheal stool cultures; role in intestinal disease is not established --known extraintestinal pathogen ---nosocomial UTIs, pneumonias, endocarditis in drug abusers, and intra-abdominal abscesses Biochemical reactions: -H2S production (80%) -fail to ferment lactose (50%) -weak urease activity -ferment lactose? -growth on Simmons citrate agar -MR positive
Pantoea Species- Pantoea agglomerans
formerly Enterobacter agglomerans similar to Klebsiella pneumoniae, so need to differentiate primarily a plant pathogen isolated from contaminated intravenous fluids
Raoultella Species
formerly part of the genera Klebsiella R. ornithinolytica -indole and ornithine decarboxylase positive both species found in urine, respiratory tracts, and blood
Plesiomonas Epidemiology
found in soil and aquatic environments -found in fresh and estuarine waters, subtropical climates widely distributed among warm- and cold-blood animals only one that is OXIDASE POSITIVE
What is the growth pattern of enterobacteriaceae in thioglycolate broth?
growth throughout the tube
Salmonella- Gastroenteritis
food poisoning sources of infection: -primarily poultry, eggs, milk, egg products, handling pets -ingestion of foodstuffs --peanut butter, cantaloupe, puffed rice and wheat cereals, corn and vegetable coated snack, and others -cooking utensils -direct person to person in institutions Symptoms appear 8 to 36 hours after ingestion of contaminated food -nausea, vomiting, fever, chills -accompanied by watery diarrhea and abdominal pain Most infections are self-limiting Antimicrobials of choice in most cases -Chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole
Enterohemorrhagic E. coli (EHEC)
hemorrhagic diarrhea, colitis hemolytic-uremic syndrome (HUS) -low platelets, hemolytic anemia, kidney failure Watery diarrhea progressing to bloody diarrhea without pus -can be fatal (young children and older adults) -undercooked meats, unpasteurized milk, apple cider E. coli O157:H7 -toxins produced by Shiga toxigenic E. coli (STEC) --Verotoxin I (phage mediated) cytotoxin ---Damages Vero cells (African green monkey kidney cells) ---AKA Shiga toxin (Stx) ---Toxin neutralized by Shiga toxin antibodies --Verotoxin II ---not neutralized by Shiga toxin antibodies
Methodologies for Diagnosing Enterohemorrhagic E. coli (EHEC)
identifying serotype and comparing to known Shiga toxin- producing strains Screening stool filtrates for toxin demonstrate a fourfold increase in toxin-neutralizing antibody titer Shiga toxin-producing E. coli (STEC) may be identified in the laboratory by one of three methods: -stool culture on highly differentiated medium, with subsequent serotyping -detecting the Shiga toxin in stool filtrates -demonstration of a fourfold or greater increase in STEC antibody titer Can screen O:157H7 E. coli on sorbitol-containing MAC (SMAC) plate -does not ferment sorbitol in 24 hours and is methylumbelliferyl-B-d-glucuronide (MUG), a commercially available screen biochemical assay, negative Confirmation identification techniques include: -enzyme-linked immunosorbent assay (ELISA), latex agglutination, others Now emergent phenotype is sorbitol fermenting and nonmotile (O157:NM)
What single test best separates Klebsiella oxytoca from Klebsiella pneumoniae?
indole
Red colonies with Black centers on Xylose Lysine Deoxycholate (XLD) agar
initially yellow then revert to red When lysine is decarboxylates, causing alkaline pH (Salmonella)
K. pneumoniae subsp. ozaenae
isolated from nasal secretions and cerebral abscesses -plasmid-mediated ESBLs (extended beta lactamase)
What is the differentiating agent of Salmonella and Shigella (SS) agar?
lactose fermentation +: red -: clear
Are clear colonies on Eosin-methylene blue (EMB) agar lactose positive or negative?
lactose negative
What are the differentiating agents of Hektoen enteric (HE) agar?
lactose, sucrose, and salicin
What is the macroscopic morphology of Enterobacteriaceae?
large, moise, gray colonies some mucoid (Klebsiella and some strains of Enterobacter)
How does E. coli appear on Eosin-methylene blue (EMB) agar?
metallic green and is lactose positive
Yersinia enterocolytica Acute Enteritis
most common form of the infection often affects infants and young children symptoms: -acute gastroenteritis with fever accompanied by headaches, abdominal pain, nausea, diarrhea Stools often contain blood Usually mild and self-limiting
Klebsiella pneumoniae
most common isolate -particularly if gram-negative rods in lower respiratory tract infection of hospitalized patients -also wound, UTIs, liver abscesses, and bacteremia --increase resistance, including carbapenemase moist gray mucoid colonies virulence factor: polysaccharide capsule -prevents phagocytosis and some antimicrobials --capsule and cause bipolar staining lactose positive
What is the pH indicator of Salmonella and Shigella (SS) agar?
neutral red
Escherichia vulneris
newest member -wound infections
General Morphology of Enterobacteriaceae
none have remarkable morphology on routine media (BAP, Choc, and most nonselective media) EXCEPT Klebsiella, Proteus, and some Enterobacter species
What are opportunistic pathogens?
normal flora organisms that are able to cause disease if the host is compromised infections in other "nonnormal" sites --E. coli can cause septicemia, wounds, urinary tract infections (UTIs), meningitis
Providencia Species- P. stuartii
nosocomial burn unit outbreaks also isolated in urine cultures no swarming
What color are the colonies of nonpathogens on Hektoen enteric (HE) agar? What do they ferment?
orange color (low pH) ferment lactose and sucrose
Situation: Abdominal pain, fever, vomiting, and nausea prompted an older male to seek medical attention. A watery stool specimen producing no fecal leukocytes or erythrocytes was cultured, and it grew a predominance of gram-negative fermentative bacilli. The colonies were beta hemolytic on blood agar and cream colored on MacConkey agar. The colonies from the BAP were both oxidase and catalase positive. What reactions rule out Enterobacteriaceae family?
oxidase Enterobacteriaceae are oxidase negative
Is Enterobacteriaceae oxidase positive or negative?
oxidase negative EXCEPT PLESIOMONAS
Quality control of the spot indole test requires the use of ATCC cultures of: a. Pseudomonas aeruginosa / Proteus mirabilis b. Salmonella typhi / Shigella sonnei c. Escherichia coli / Proteus vulgaris d. Escherichia coli / Enterobacter cloacae
Escherichia coli / Enterobacter cloacae Quality control of indole requires both a positive and a negative control. E coli and E cloacae respectively produce a positive and negative reaction with indole.
The stock cultures needed for quality control testing of oxidase production are: a. Escherichia coli / Klebsiella pneumoniae b. Salmonella typhimurium / Escherichia coli c. Escherichia coli / Pseudomonas aeruginosa d. Proteus mirabilis / Escherichia coli
Escherichia coli / Pseudomonas aeruginosa Of the organisms listed only Pseudomonas aeruginosa is oxidase positive
The stock cultures needed for quality control testing of motility are: a. Salmonella typhimurium / Escherichia coli b. Escherichia coli / Pseudomonas aeruginosa c. Serratia marcescens / Escherichia coli d. Klebsiella pneumoniae / Escherichia coli
Klebsiella pneumoniae / Escherichia coli Of the organisms listed only Klebsiella pneumoniae is nonmotile
A TSI tube inoculated with an organism gave the following reactions: alkaline slant, acid butt, no H2S, no gas produced This organism is most likely: a. Yersinia enterocolitica b. Salmonella typhi c. Salmonella enteritidis d. Shigella dysenteriae
Shigella dysenteriae Salmonella produce H2S in TSI and Yersinia produces an acid slant and acid butt. Shigella fits this biochemical profile
An 8-year-old girl was admitted to the hospital with a 3-day history of fever, abdominal pain, diarrhea, and vomiting. A stool culture grew many lactose-negative colonies that yielded the following test results: Oxidase: negative TSI: acid slant/acid butt Indole: negative Urease: positive Ornithine decarboxylase: positive Sucrose: positive H2S: negative Motility at 25 degrees Celsius: positive The most probable identification of this organism is:
Yersinia enterocolitica
A 25 yr. old man who had recently worked as a steward on a transoceanic grain ship presented to the ER with high fever, diarrhea and prostration. Axillary lymph nodes were hemorrhagic and enlarged. A Wayson stain of the aspirate showed bacilli that were bipolar, resembling safety pins. The most likely ID of this organism is: a. Brucella melitensis b. Streptobacillus moniliformis c. Spirillum minus d. Yersinia pestis
Yersinia pestis Yersinia pestis is classically described as having a "safety pin" appearance on Wayson stain. This patient's presentation is classic for bubonic plague.
Plesiomonas shigelloides is a relatively new member of the family Enterobacteriaceae. What characteristic separates it from other members of the Enterbacteriaceae? a. it is oxidase positive b. it ferments glucose c. it produces pyocyanin d. it requires 10% CO2 for growth
it is oxidase positive Enterobacteriaceae ferment glucose and are oxidase negative. Plesiomonas was a member of the Vibrio family in part because it is oxidase positive. However, it was moved to the Enterobacteriaceae family despite its positive oxidase reaction
The Escherichia coli serotype O157:H7 is associated with all the following, except a. hemorrhagic diarrhea b. colitis c. liver damage d. hemolytic-uremic syndrome (HUS)
liver damage
Do all enterobacteriaceace members ferment lactose?
no
Given the presence of blood and white blood cells in stool, which of the following organisms is the most likely pathogen? A) H2S-positive, lysine-positive, citrate-positive, non-lactose fermenter. B) non-motile, urea-negative, non-lactose fermenter. C) H2S-positive, lysine-positive, citrate-negative, non-lactose fermenter. D) PAD-positive, urea-positive non-lactose fermenter.
non-motile, urea-negative, non-lactose fermenter.
Shigella species characteristically are: a. urease positive b. nonmotile c. oxidase positive d. lactose fermenters
nonmotile The biochemical characteristic that best fits Shigella is that it is nonmotile. Shigella are urease negative and oxidase negative. Shigella are lactose nonfermenters
Pus collected from a draining axillary lymph node is sent to the laboratory. A gram-negative rod is observed on Gram stain. Twenty-four hours after incubation at 35°C in CO2, a poorly growing non-lactose fermenter is observed. After 48 hours, the colonies on BAP and MAC were slightly larger. When stained with Methylene blue exhibited a safety pin appearance. The organism proved to beMotility at 35°C: negative Urea: no color changeMotility at 25°C: negative PAD: no color changeVP: no color change Lysine and Ornithine: no color changeIndole: no color change TSI: red clant and yellow buttCitrate: greenThis organism is the causative agent of A) mesenteric lymph adenitis. B) typhoid fever. C) plague. D) gastroenteritis.
plague yersinia pestis safety pin appearance caused by bipolar staining
You have isolated a bacterium from a stool culture that produces clear colonies on sorbitol MacConkey (SMAC) agar. Which of the following would be the most appropriate test to perform next? a. gram stain b. oxidase c. rapid methylumbelliferyl-b-glucuronide (MUG) d. Rapid urease
rapid methylumbelliferyl-b-glucuronide (MUG)