Harr 7.5 - Microbiology - Gram-Positive and Gram-Negative Cocci

A fourfold rise in titer of which antibody is the best indicator of a recent infection with group A β-hemolytic streptococci?

Anti-streptolysin O The antistreptolysin O (ASO) titer is used to indicate a recent infection with group A β-hemolytic streptococci. Streptolysin O may also be produced by some strains of groups C and G streptococci.

A Gram stain of a urethral discharge from a man showing extracellular and intracellular gram-negative diplococci within segmented neutrophils is a presumptive identification for:

Neisseria gonorrhoeae A Gram stain of urethral discharge (in men only) showing typical gonococcal cells in PMNs should be reported "presumptive N. gonorrhoeae, confirmation to follow." With female patients, the normal vaginal flora contain gram-negative cocci and diplococci resembling gonococci and, therefore, no presumptive identification should be reported for N. gonorrhoeae from the vaginal Gram stain smear.

Staphylococcus saprophyticus is best differentiated from Staphylococcus epidermidis by resistance to:

5 μg of novobiocin S. saprophyticus is coagulase negative and resistant to 5 μg of novobiocin. Using the standardized Kirby-Bauer sensitivity procedure, a 6-12 mm zone of growth inhibition is considered resistant. Susceptible strains measure 16-27 mm (inhibition) zones.

Nonpathogenic Moraxella spp. capable of growing on selective media for Neisseria can be differentiated from Neisseria spp. by which test?

10-unit penicillin disk Moraxella spp. are oxidase and catalase positive, as are the gonococci. Neisseria spp. and M. catarrhalis will keep their typical coccal morphology after overnight incubation on blood agar with a 10-unit penicillin disk (CO2 incubation). Other Moraxella species form long filaments or long spindle-shaped cells when grown near a 10-unit penicillin disk.

β-Hemolytic streptococci, not of group A or B, usually exhibit which of the following reactions?

Bacitracin: Susceptible or resistant Trimethoprim-sulfamethoxazole: Susceptible Streptococci that are not group A or B may be either resistant or susceptible to bacitracin but are usually susceptible to SXT.

Which test is used to differentiate the viridans streptococci from the group D streptococci and enterococci?

Bile esculin test The bile esculin test differentiates those bacteria that can hydrolyze esculin and also grow in the presence of 4% bile salts or 40% bile. The bile esculin slant is inoculated on the surface and incubated for 24-48 hours in a nonCO2 incubator. Group D streptococci (enterococci and nonenterococci) are positive, causing blackening of half or more of the slant within 48 hours. Viridans streptococci are negative (do not grow or hydrolyze esculin).

A false-positive CAMP test for the presumptive identification of group B streptococci may occur if the plate is incubated in a(n):

Candle jar or CO2 incubator The CAMP (hemolytic phenomenon first described by Christie, Atkins, and Munch-Petersen in 1944) test refers to a hemolytic interaction that is seen on a blood agar plate between the β-hemolysins produced by most strains of S. aureus and an extracellular protein produced by both hemolytic and nonhemolytic isolates of group B streptococci. When performing a CAMP test, the plate must be placed in an ambient air incubator at 35°C-37°C. Group A streptococci may be CAMP positive if the plate is incubated in a candle jar, high CO2 atmosphere, or anaerobically.

The test used most often to separate the Micrococcaceae family from the Streptococcaceae family is:

Catalase The catalase test (utilizing a 3% hydrogen peroxide [H2O2] solution stored in a brown bottle under refrigeration) is positive for the four genera belonging to the Micrococcaceae family: Planococcus, Micrococcus, Stomatococcus, and Staphylococcus. Members of the Streptococcaceae family are negative. Planococcus spp. are associated with marine life and not human infections. Stomatococcus spp. are implicated in endocarditis following cardiac catheterization; they are weakly catalase positive and produce white or transparent sticky colonies on agar, which help to differentiate them from Staphylococcus.

Which test is used for the determination of inducible clindamycin resistance in staphylococci and streptococci?

D-zone test By using a 15-μg erythromycin disk adjacent to a 2-μg clindamycin disk in a disk diffusion procedure, a flattening of the clindamycin zone occurs in inducible clindamycin resistance, causing the zone to look like the letter D. This is referred to as being D-zone positive. CLSI recommends that inducible clindamycin resistant strains be reported as resistant with a comment that clindamycin may still be effective in some patients.

Many α-hemolytic streptococci recovered from a wound were found to be penicillin resistant. Given the following results, what is the most likely identification? Bile esculin: + PYR: + 6.5% salt: + Hippurate hydrolysis: + Bile solubility: Neg SXT: resistant

Enterococcus faecalis E. faecalis is resistant to penicillin and ampicillin as well as some of the aminoglycoside antibiotics. Pneumococci, group B streptococci, and S. bovis are PYR negative.

The salt tolerance test (6.5% salt broth) is used to presumptively identify:

Enterococcus faecalis Enterococcus faecalis will grow in 6.5% salt and the nonenterococci (S. bovis and S. equinus) will not. This test distinguishes the enterococci group from S. bovis and S. equinus (nonenterococci group). Both groups grow on bile esculin agar.

β-Hemolytic streptococci, more than 50,000 col/mL, were isolated from a urinary tract catheter urine specimen. Given the following reactions, what is the most likely identification? CAMP test = Neg Bile solubility = Neg PYR = + SXT = Resistant Optochin = Resistant Hippurate hydrolysis = ± 6.5% salt = + Bile esculin = + Bacitracin = Resistant

Enterococcus faecalis Group A streptococci are sensitive to bacitracin and negative for bile esculin and 6.5% salt broth. Group B streptococci will grow in 6.5% salt broth but are negative for bile esculin and PYR. The nongroup A, B, or D streptococci will not grow in 6.5% salt broth and are sensitive to SXT. Some group D streptococci will hydrolyze hippurate. Enterococcus faecalis is positive for bile esculin, 6.5% salt broth, and PYR.

Which one of the following organisms is a known producer of β-lactamase-producing strains, and should be tested (screened) by a commercial β-lactamase assay prior to susceptibility testing?

Enterococcus spp. A test for β-lactamase production should be performed on all isolates of Staphylococcus and Enterococcus as well as N. gonorrhoeae and H. influenzae. Some organisms may appear to be penicillin/ampicillin susceptible by the conventional Kirby-Bauer disk diffusion method for β-lactamase producers, but still produce small amounts of β-lactamase. An example is heteroresistant mecA-positive S. aureus (the mecA gene confers penicillin resistance and is present in all MRSA strains).

Streptococcus species exhibit which of the following properties?

Facultative anaerobe, oxidase negative, catalase negative Streptococcus species are facultative anaerobes that grow aerobically as well, and are oxidase and catalase negative. In order to demonstrate streptolysin O on blood agar, it is best to stab the agar to create anaerobiosis because streptolysin O is oxygen labile.

Micrococcus and Staphylococcus species are differentiated by which test(s)?

Fermentation of glucose (OF tube) Both micrococci and staphylococci are catalase-positive and gram-positive cocci. On direct smears, they both appear as pairs, short chains (resembling Streptococcus spp.), or clusters. However, the micrococci fail to produce acid from glucose under anaerobic conditions. The OF tube reactions are:

Strains of Staphylococcus species resistant to the β-lactam antibiotics by standardized disk diffusion and broth microdilution susceptibility methods are called:

Heteroresistant Methicillin-resistant S. aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE) are called heteroresistant. This refers to two subpopulations in a culture, one that is susceptible and the other that is resistant to antibiotic(s). The resistant population grows more slowly than the susceptible one and can be overlooked. Therefore, the more resistant subpopulation should be promoted growthwise by using a neutral pH (7.0-7.4), cooler incubation temperatures (30°C-35°C), the addition of 2%-4% NaCl, and incubation up to 48 hours.

Which of the following enzymes contribute to the virulence of S. aureus?

Hyaluronidase and β-lactamase In addition to coagulase, the virulence of S. aureus is attributed to hyaluronidase, which damages the intercellular matrix (basement membrane) of tissues. β-Lactamase-producing strains are able to inactivate penicillin and ampicillin, making the organism resistant to these antibiotics. Lecithinase is not produced by S. aureus, and urease is not a virulence factor.

The β-galactosidase test aids in the identification of which Neisseria species?

N. lactamica N. lactamica utilizes lactose by producing the enzyme β-galactosidase. All other Neisseria spp. that grow on MTM media are lactose negative.

A pure culture of β-hemolytic streptococci recovered from a leg wound ulcer gave the following reactions: CAMP test = Neg Bile esculin = Neg PYR = Neg Optochin = Resistant Hippurate hydrolysis = Neg 6.5% salt = Neg Bacitracin = Resistant SXT = Sensitive The most likely identification is:

Nongroup A, nongroup B, nongroup D streptococci The β-hemolytic streptococci—not of groups A, B, or D—are sensitive to SXT and may be either sensitive or resistant to bacitracin. Groups A and B are both resistant to SXT. Group A and Enterococcus faecalis are PYR positive. Enterococcus faecalis is also positive for bile esculin and 6.5% salt broth.

Urine cultured from the catheter of an 18-year-old female patient produced more than 100,000 col/mL on a CNA plate. Colonies were catalase positive, coagulase negative by the latex agglutination slide method as well as the tube coagulase test. The best single test for identification is:

Novobiocin susceptibility S. epidermidis and S. saprophyticus are the two possibilities because they are both catalase positive, coagulase negative, urease positive, and ferment lactose. Novobiocin susceptibility is the test of choice for differentiating these two species. S. epidermidis is sensitive but S. saprophyticus is resistant to 5 μg of novobiocin.

S. pneumoniae and the viridans streptococci can be differentiated by which test?

Optochin disk test, 5 μg/mL or less Optochin at a concentration of 5 μg/mL or less inhibits the growth of S. pneumoniae but not viridans streptococci. However, Optochin at a concentration in excess of 5 μg/mL inhibits viridans streptococci as well. A zone of inhibition of 14 mm or more around the 6-mm disk is considered a presumptive identification of S. pneumoniae. A questionable zone size should be confirmed by performing a bile solubility test.

Staphylococcus epidermidis (coagulase negative) is recovered from which of the following sources?

Prosthetic heart valves, intravenous catheters, and urinary tract S. epidermidis represents 50%-80% of all coagulase-negative Staphylococcus spp. recovered from numerous clinical specimens. It is of special concern in nosocomial infections because of its high resistance to antibiotics.

Slime production is associated with which Staphylococcus species?

S. epidermidis S. epidermidis produces an extracellular slime that enhances the adhesion of these organisms to indwelling plastic catheters. The slime production is considered a virulence factor and is associated with infections from prostheses.

Which Staphylococcus species, in addition to S. aureus, also produces coagulase?

S. intermedius S. intermedius infects mammals and certain birds but not usually humans. Cases involving humans result from animal bites and are most often seen in persons who work closely with animals.

Furazolidone (Furoxone) susceptibility is a test used to differentiate:

Staphylococcus spp. from Micrococcus spp. Staphylococci are susceptible to furazolidone, giving zones of inhibition that are 15 mm or greater. Micrococcus spp. are resistant to furazolidone, giving zones of 6-9 mm. The test is performed as a disk susceptibility procedure using a blood agar plate.

Which two tests best differentiate S. bovis (group D, nonenterococcus) from Streptococcus salivarius?

Starch hydrolysis and acid production from mannitol S. bovis and S. salivarius are physiologically and biochemically similar. They are both PYR and 6.5% salt broth negative and bile esculin positive, but only S. bovis is positive for mannitol and starch reactions. See the following chart.

A Staphylococcus spp. recovered from a wound (cellulitis) was negative for the slide coagulase test (clumping factor) and negative for novobiocin resistance. The next test(s) needed for identification is (are):

Tube coagulase test, β-Hemolysis on blood agar, mannitol salt agar plate S. aureus is novobiocin sensitive and cannot be ruled out by a negative clumping factor test. Most S. aureus produce β-hemolysis on sheep blood agar plates and are mannitol salt positive (produce acid and are not inhibited by the high salt concentration). The tube test should be performed because the slide test was negative.

Trimethoprim-sulfamethoxazole (SXT) disks are used along with bacitracin disks to differentiate which streptococci?

β-Hemolytic streptococci β-Hemolytic streptococci are the only streptococci that should be tested. S. pneumoniae, which is α-hemolytic, is susceptible to small concentrations of bacitracin, as are other α-hemolytic streptococci. SXT is inhibitory to most streptococci except Streptococcus pyogenes and Streptococcus agalactiae. For this reason, SXT is used in a commercially available streptococcal selective agar (SSA) as a primary plating agar for the detection of group A streptococci.

An oxacillin-disk screen test is used to detect Streptococcus pneumonia resistance to penicillin. Using Mueller-Hinton agar with 5% sheep blood and a 1 μg oxacillin disk, what is the recommended inhibition zone size for penicillin susceptibility?

≥20 mm The standard Kirby-Bauer method used for disk diffusion susceptibility testing recommended by CLSI is ≥ 20 mm. The test requires 20-24-hour incubation at 35°C in 5%-7% CO2. If the zone size is ≤ 19 mm, an MIC should be performed on isolates from CSF or blood.

Which group of streptococci is associated with erythrogenic toxin production?

Group A Group A β-hemolytic streptococci are the cause of scarlet fever, and some strains produce toxins (pyrogenic exotoxins A, B, and C) that cause a scarlatiniform rash.

Cystine tryptic digest (CTA) media used for identification of Neisseria spp. should be inoculated and cultured in:

A nonCO2 incubator at 35°C for up to 72 hours CTA agar with 1% carbohydrate and phenol red pH indicator added is used for the identification of Neisseria species. CTA carbohydrates must be placed in an ambient air incubator because a high CO2 concentration may reduce the pH, causing a false-positive (acid) result. The utilization of carbohydrates by some fastidious gonococcal strains may take up to 72 hours in order to produce a color change in the pH indicator.

Which of the following tests should be used to differentiate Staphylococcus aureus from Staphylococcus intermedius?

Acetoin The production of acetoin by S. aureus from glucose or pyruvate differentiates it from S. intermedius, which is also coagulase positive. This test is also called the VP test. Acetoin production is detected by addition of 40% KOH and 1% α-naphthol to the VP test broth after 48 hours of incubation. A distinct pink color within 10 minutes denotes a positive test.

Bacitracin A disks (0.04 unit) are used for the presumptive identification of which group of β-hemolytic streptococci?

Group A The bacitracin disk test is used in conjunction with other confirmatory tests for the β-hemolytic streptococci. In addition to group A, groups C, F, and G are also β-hemolytic and give a positive test for bacitracin (a zone of inhibition of any size). Therefore, a positive test does not confirm the presence of group A β-hemolytic streptococci.

The L-pyrrolidonyl-β-napthylamide (PYR) hydrolysis test is a presumptive test for which streptococci?

Group A and D (enterococcus) streptococci The PYR hydrolysis test is highly specific for group A streptococci and group D enterococci. The test detects the pyrrolidonylarylamidase enzyme, which hydrolyzes PYR.

In addition to Enterococcus faecalis, which other streptococci will grow in 6.5% salt broth?

Group B streptococci Approximately 80% of group B streptococci are capable of growing in 6.5% salt broth; however, they do not hydrolyze esculin or grow in media containing 4% bile salts.

Two blood cultures on a newborn grew β-hemolytic streptococci with the following reactions: CAMP test = + Bile solubility = Neg Bacitracin = Resistant PYR = Neg Trimethoprim-sulfamethoxazole = Resistant Hippurate hydrolysis = + 6.5% salt = + Bile esculin = Neg Which is the most likely identification?

Group B streptococci Group B streptococci (S. agalactiae) are resistant to both bacitracin and SXT. Unlike group A and group D streptococci, the group B streptococci are negative for PYR. With some exceptions, group B streptococci will grow in 6.5% salt broth.

Lysostaphin is used to differentiate Staphylococcus from which other genus?

Micrococcus Lysostaphin is an endopeptidase that cleaves the glycine-rich pentapeptide crossbridges in the staphylococcal cell wall peptidoglycan. The susceptibility of the staphylococci to lysostaphin is used to differentiate them from the micrococci. Staphylococci are susceptible and show a 10-16 mm zone of inhibition, while micrococci are not inhibited.

A gram-positive coccus recovered from a wound ulcer from a 31-year-old diabetic patient showed pale yellow, creamy, β-hemolytic colonies on blood agar. Given the following test results, what is the most likely identification? Catalase = + Glucose OF: positive open tube, negative sealed tube Mannitol salt = Neg Slide coagulase = Neg

Micrococcus spp. Micrococcus spp. utilize glucose oxidatively but not under anaerobic conditions (sealed tube). Staphylococcus spp. utilize glucose oxidatively and anaerobically. The catalase differentiates the Micrococcaceae family (positive) from the Streptococcaceae family (negative).

Bacitracin resistance (0.04 unit) is used to differentiate:

Micrococcus spp. from Staphylococcus spp. A bacitracin disk (0.04 unit) is used to identify group A β-hemolytic streptococci, but it will also differentiate catalase-positive organisms. A zone of 10 mm or greater is considered susceptible. The Staphylococcus species are resistant and grow up to the disk, while Micrococcus species are sensitive.

Which of the following tests will rapidly differentiate micrococci from staphylococci?

Modified oxidase The modified oxidase test is used to rapidly identify catalase-positive gram-positive cocci as Micrococcus spp. (positive) or Staphylococcus spp. (negative). Filter paper disks that are saturated with oxidase reagent (tetramethyl-p-phenylenediamine in dimethylsulfoxide) are used. A colony of the isolate is rubbed onto the paper. Oxidase-positive organisms produce a purple color within 30 sec.

Sputum from a patient with pneumonia produced many colonies of gram-negative diplococci on a chocolate plate that were also present in fewer numbers on MTM after 48 hours. Given the following results, what is the most likely identification? Catalase = + DNase = + Glucose = Neg Lactose = Neg Fructose = Neg Oxidase = + Tributyrin hydrolysis = + Sucrose = Neg Maltose = Neg

Moraxella catarrhalis M. catarrhalis is part of the normal upper respiratory flora but is implicated in lower respiratory infections, including pneumonia. It produces stunted growth on MTM and is DNase positive, characteristics that differentiate it from the other saprophytic Neisseria species.

Variation in colony types seen with fresh isolates of Neisseria gonorrhoeae and sometimes with Neisseria meningitidis are the result of:

Multiple nutritional requirements, pili on the cell surface, use of a transparent medium Upon subculture from a primary plate, various sizes and appearances of gonococci are the result of multiple nutritional requirements, such as arginine-hypoxanthine-uracil (AHU)-requiring strains. Colony size and coloration (or light reflection) are the basis of Kellogg's scheme (types T1 through T5). Types T1 and T2 have pili on the surface and T3, T4, and T5 do not. Transparent media are not used routinely, but opaque and transparent colonial differences of the gonococci can be seen when using it.

MTM medium is used primarily for the selective recovery of which organism from genital specimens?

Neisseria gonorrhoeae Both N. gonorrhoeae and N. meningitidis grow selectively on MTM owing to the addition of vancomycin and colistin, which inhibit gram-positive and gram-negative bacteria, respectively. Trimethoprim is added to inhibit swarming of Proteus spp. because a rectal swab may be used for culture. Nystatin and amphotericin B are used to prevent growth of yeasts and molds from vaginal specimens.

The Superoxol test is used as a rapid presumptive test for:

Neisseria gonorrhoeae N. gonorrhoeae colonies recovered from selective MTM media give an immediate positive reaction (bubbling) when 30% H2O2 is added. The catalase test uses 3% H2O2. This is a presumptive test for N. gonorrhoeae; N. meningitidis and N. lactamica give a weak or delayed bubbling reaction. M. catarrhalis is catalase positive, Superoxol negative, and has a variable growth pattern on MTM.

Gram-negative diplococci recovered from an MTM plate and giving a positive oxidase test can be presumptively identified as:

Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica All of the listed Neisseria spp. grow on MTM and are oxidase positive. N. lactamica is a nonpathogenic component of normal throat flora resembling N. meningitidis but it grows well on selective MTM agar. Presumptive identification of N. meningitidis or N. gonorrhoeae is stated only if the source of the specimen (i.e., urogenital or CSF) is given. The identification must be confirmed by further testing such as carbohydrate utilization tests, DNA tests, or rapid latex slide agglutination tests.

Culture on MTM media of a vaginal swab produced several colonies of gram-negative diplococci that were catalase and oxidase positive and Superoxol negative. Given the following carbohydrate reactions, select the most likely identification. Glucose = + Maltose = + Sucrose = Neg Fructose = Neg Lactose = +

Neisseria lactamica N. lactamica is part of the normal vaginal and throat flora and is the only Neisseria species that grows on MTM that utilizes lactose. Other saprophytic Neisseria spp. may utilize lactose but do not grow on MTM media.

Resistance to which drug categorizes a strain of Staphylococcus aureus as methicillin-resistant Staphylococcus aureus (MRSA)?

Oxacillin Oxacillin is the drug used to screen staphylococci for resistance to antibiotics having the β-lactam ring. Included in this group are penicillin, cephalosporin, monobactam, and carbapenem. MRSA defines strains of staph that are resistant to all of these antibiotic groups. MRSA strains are treated with vancomycin or oxazolidinone.

The quellung test is used to identify which Streptococcus species?

S. pneumoniae A precipitin reaction seen microscopically with methylene blue stain (microprecipitin reaction) occurs between the carbohydrate of the capsule of S. pneumoniae and anticapsular antibody. The antibody may be type specific or polyvalent. Binding of antibodies to the bacteria causes the capsule to swell, identifying the organisms as S. pneumoniae.

Which of the following tests is used routinely to identify Staphylococcus aureus?

Slide coagulase test, tube coagulase test, and latex agglutination The slide coagulase test using rabbit plasma with ethylenediaminetetraacetic acid (EDTA) detects bound coagulase or "clumping factor" on the surface of the cell wall, which reacts with the fibrinogen in the plasma. This test is not positive for all strains of S. aureus, and a negative result must be confirmed by the tube method for detecting "free coagulase" or extracellular coagulase. The tube test is usually positive within 4 hours at 35°C; however, a negative result must then be incubated at room temperature for the remainder of 18-24 hours. Some strains produce coagulase slowly or produce fibrinolysin, which dissolves the clot at 35°C. Latex agglutination procedures utilize fibrinogen and IgG-coated latex beads that detect protein A on the staphylococcal cell wall.

Nutritionally variant streptococci (NVS) requiere specific thiol compounds, cysteine, or the active form of vitamin B6. Which of the following tests supplies these requirements?

Staphylococcal cross-streak test The staphylococcal streak, across the NVS inoculum, provides the nutrients needed. Very small colonies of NVS can be seen growing adjacent to the staphylococcal streak on the blood agar plate in a manner similar to the satellite phenomenon of Haemophilus spp. around S. aureus.

The following results were observed by using a tube coagulase test: Coagulase at 4 hours = + DNase = + Hemolysis on blood agar = β Coagulase at 18 hours = Neg Novobiocin = Sensitive (16-mm zone) Mannitol salt plate = + (acid production) What is the most probable identification?

Staphylococcus aureus S. aureus can produce fibrinolysins that dissolve the clot formed by the coagulase enzyme. The tube method calls for an incubation of 4 hours at 35°C-37°C and 18-24 hours at room temperature. Both must be negative to interpret the result as coagulase negative. This organism is coagulase positive and, therefore, identified as S. aureus.

Toxic shock syndrome is attributed to infection with:

Staphylococcus aureus S. aureus is the organism most often recovered from female patients. These strains produce toxic shock syndrome toxin 1 (TSST-1). Toxic shock syndrome is attributed to the use of certain highly absorbent tampons by menstruating females. The toxin is also recovered from sites other than the genital area and produces fever and life-threatening systemic damage as well as shock.

The bile solubility test causes the lysis of:

Streptococcus pneumoniae colonies on a blood agar plate The bile solubility test can be performed directly by dropping 2% sodium deoxycholate onto a few well-isolated colonies of S. pneumoniae. The bile salts speed up the autolysis observed in pneumococcal cultures. The colonies lyse and disappear when incubated at 35°C for 30 min, leaving a partially hemolyzed area on the plate. The same phenomenon can be seen using a broth culture; addition of 10% deoxycholate to broth containing S. pneumoniae results in visible clearing of the suspension after incubation at 35°C for 3 hours.

Staphylococcus aureus recovered from a wound culture gave the following antibiotic sensitivity pattern by the standardized Kirby-Bauer method (S = sensitive; R = resistant): Penicillin = R Cephalothin = R Vancomycin = S Ampicillin = S Cefoxitin = R Methicillin = R Which is the drug of choice for treating this infection?

Vancomycin Vancomycin, along with rifampin, is used for strains of S. aureus that are resistant to the β-lactams. MRSA strains pose problems when reading the zone sizes for these strains. Their heteroresistance results in a film of growth consisting of very small colonies formed within the defined inhibition zone surrounding the antibiotic disk. Initially, this appears as a mixed culture or contaminant.