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We compared the results obtained with six commercial MIC test systems (Etest, MicroScan, Phoenix, Sensititre, Vitek Legacy, and Vitek 2 systems) and three reference methods (agar dilution, disk diffusion, and vancomycin [VA] agar screen [VScr]) with the results obtained by the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference method for the detection of VA-intermediate Staphylococcus aureus (VISA). A total of 129 S. aureus isolates (VA MICs by previous BMD tests, ≤1 μg/ml [ n = 60 strains], 2 μg/ml [ n = 24], 4 μg/ml [ n = 36], or 8 μg/ml [ n = 9]) were selected from the Centers for Disease Control and Prevention strain collection. The results of BMD with Difco Mueller-Hinton broth were used as the standard for data analysis. Essential agreement (percent ±1 dilution) ranged from 98 to 100% for all methods except the method with the Vitek Legacy system, for which it was 90.6%. Of the six commercial MIC systems tested, the Sensititre, Vitek Legacy, and Vitek 2 systems tended to categorize VISA strains as susceptible (i.e., they undercalled resistance); the MicroScan and Phoenix systems and Etest tended to categorize susceptible strains as VISA; and the Vitek Legacy system tended to categorize VISA strains as resistant (i.e., it overcalled resistance). Disk diffusion categorized all VISA strains as susceptible. No susceptible strains (MICs ≤ 2 μg/ml) grew on the VScr, but all strains for which the VA MICs were 8 μg/ml grew on the VScr. Only 12 (33.3%) strains for which the VA MICs were 4 μg/ml grew on VScr. The differentiation of isolates for which the VA MICs were 2 or 4 μg/ml was difficult for most systems and methods, including the reference methods.

This report describes the results of an 11-laboratory study to determine if a cefoxitin broth microdilution MIC test could predict the presence of mecA in staphylococci. Using breakpoints of ≤4 μg/ml for mecA -negative and ≥6 or 8 μg/ml for mecA -positive isolates, sensitivity and specificity based on mecA or presumed mecA for Staphylococcus aureus at 18 h of incubation were 99.7 to 100% in three cation-adjusted Mueller-Hinton broths tested. For coagulase-negative strains at 24 h of incubation, breakpoints of ≤2 μg/ml for mecA -negative and ≥4 μg/ml for mecA -positive isolates gave sensitivity and specificity of 94 to 99% and 69 to 80%, respectively.

A study conducted by 11 laboratories investigated the ability of four combinations of erythromycin (ERY) and clindamycin (CC) (ERY and CC at 4 and 0.5, 6 and 1, 8 and 1.5, and 0.5 and 2 μg/ml) in a single well of a broth microdilution panel to predict the presence of inducible CC resistance. Each laboratory tested approximately 30 Staphylococcus aureus isolates and 20 coagulase-negative staphylococcus (CoNS) isolates in a panel using cation-adjusted Mueller-Hinton broth from three different manufacturers. Only the strains resistant to ERY and those susceptible or intermediate to CC were included in the analysis ( S. aureus , n = 333; CoNS, n = 97). Results of the D-zone test were used as the gold standard. After an 18-h incubation, the combination of 4 μg/ml ERY and 0.5 μg/ml CC performed the best, with 98 to 100% sensitivity and 100% specificity for both organism groups. After a 24-h incubation, the ERY-CC combinations of 4 and 0.5, 6 and 1, and 8 and 1.5 μg/ml correlated well with the D-zone test.

Accurate prediction of mecA- mediated resistance in Staphylococcus spp. has been a challenge for the clinical microbiologist ever since the discovery of methicillin resistance. In 2004, a cefoxitin disk diffusion test was recommended by the Clinical and Laboratory Standards Institute (formerly NCCLS) for prediction of mecA- mediated resistance in both Staphylococcus aureus and coagulase-negative staphylococci because of its high sensitivity and specificity compared to PCR tests for mecA. It is also much easier to read that the oxacillin disk diffusion test, not requiring close scrutiny of the zone of inhibition or 24 h of incubation. The test is used as a surrogate for oxacillin testing. In other words, when cefoxitin is tested, the results are applied to oxacillin and oxacillin is reported. For resistant strains, the results are applied not only to oxacillin, but also to all other β-lactam agents, which must also be reported as resistant if they are reported. There has been some confusion among clinical microbiologists about when and how the test should be used. We answer some of the questions that have been raised about the test.

Acinetobacter sp. isolates having multidrug resistance (MDR) patterns have become common in many medical centers worldwide, limiting therapeutic options. A five-center study tested 103 contemporary clinical Acinetobacter spp., including MDR strains, by reference broth microdilution and disk diffusion (15-μg disk content) methods against tigecycline. Applying U.S. Food and Drug Administration tigecycline breakpoint criteria for Enterobacteriaceae (susceptibility at ≤2 μg/ml [≤1 μg/ml by the European Committee on Antimicrobial Susceptibility Testing]; disk diffusion breakpoints at ≥19 mm and ≤14 mm) to Acinetobacter spp. led to an unacceptable error rate (23.3%). However, an adjustment of tigecycline disk diffusion breakpoints (susceptible/resistant) to ≥16/≤12 mm reduced intermethod errors to an acceptable level (only 9.7%, all minor).

From January 1996 to May 1999, Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) received 448 nonduplicate clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa that were reported to be imipenem intermediate or resistant. However, broth microdilution (BMD) confirmatory testing at the Project ICARE central laboratory confirmed this result in only 11 of 123 (8.9%) Enterobacteriaceae isolates and 241 of 325 (74.2%) P. aeruginosa isolates. To investigate this overdetection of imipenem resistance, we tested 204 selected isolates from the Project ICARE collection plus five imipenem-resistant challenge strains at the Centers for Disease Control and Prevention against imipenem and meropenem by agar dilution, disk diffusion, Etest (AB BIODISK North America, Inc., Piscataway, N.J.), two MicroScan WalkAway conventional panels (Neg MIC Plus 3 and Neg Urine Combo 3) (Dade MicroScan, Inc., West Sacramento, Calif.), and two Vitek cards (GNS-116 containing meropenem and GNS-F7 containing imipenem) (bioMérieux Vitek, Inc., Durham, N.C.). The results of each test method were compared to the results of BMD testing using in-house-prepared panels. Seven imipenem-resistant and five meropenem-resistant isolates of Enterobacteriaceae and 43 imipenem-resistant and 21 meropenem-resistant isolates of P. aeruginosa were identified by BMD. For Enterobacteriaceae , the imipenem and meropenem test methods produced low numbers of very major and major errors. All test systems in the study produced low numbers of very major and major errors when P. aeruginosa was tested against imipenem and meropenem, except for Vitek testing (major error rate for imipenem, 20%). Further testing conducted in 11 of the participating ICARE hospital laboratories failed to pinpoint the factors responsible for the initial overdetection of imipenem resistance. However, this study demonstrated that carbapenem testing difficulties do exist and that laboratories should consider using a second, independent antimicrobial susceptibility testing method to validate carbapenem-intermediate and -resistant results.

Most methods currently available to clinical microbiology laboratories for phenotypic detection of oxacillin resistance in Staphylococcus aureus , e.g., MIC and disk diffusion methods, perform well if the tests are inoculated and incubated properly. However, in rare circumstances, additional testing for confirmation of susceptibility or resistance may be needed. These circumstances include the occurrence of extremely heterogeneous mecA -determined resistance where conventional methods may categorize the strain as susceptible. In addition, confirmation of borderline resistance determined by mechanisms other than the presence of the mecA gene may need to be verified because treatment with oxacillin may be effective. Several new methods that detect either the mecA gene or the gene product (e.g., penicillin-binding protein 2a, or PBP 2a) have been cleared by the Food and Drug Administration and are now available. These methods, including an easy-to-perform latex agglutination test, may be considered for use by many clinical laboratories when more sophisticated molecular techniques (i.e., PCR assay) are not available.

The need for new antimicrobial agents with activity against Gram-positive organisms has become increasingly important because of emerging resistance. We compared the activity of a new β β β β-lactam antimicrobial agent, RWJ-54428 (MC-02 479), with representatives of other classes of antimicrobial agents against 76 Staphylococcus aureus (including four glycopeptide- intermediate strains), 50 coagulase-negative staphylococci, 20 Enterococcus faecalis, 20 Entero- coccus faecium, 10 Enterococcus gallinarum/Enterococcus casseliflavus, 54 Streptococcus pneumoniae and 22 viridans streptococcal isolates. The MIC 90 of RWJ-54428 was ≤ ≤ ≤ ≤2 mg/L for all groups of bacteria tested except E. faecium. The activity against four strains of glycopeptide- intermediate S. aureus was similar to that for other methicillin-resistant S. aureus isolates (range 0.5-2.0 mg/L).

To define more precisely the inoculation methods to be used in the oxacillin screen test for Staphylococcus aureus , we tested agar screen plates prepared in house with 6 μg of oxacillin/ml and 4% NaCl using the four different inoculation methods that would most likely be used by clinical laboratories. The organisms selected for testing were 19 heteroresistant mecA -producing strains and 41 non- mecA -producing strains for which oxacillin MICs were near the susceptible breakpoint. The inoculation method that was preferred by all four readers and that resulted in the best combination of sensitivity and specificity was a 1-μl loopful of a 0.5 McFarland suspension. A second objective of the study was to then use this method to inoculate plates from five different manufacturers of commercially prepared media. Although all commercial media performed with acceptable sensitivity compared to the reference lot, one of the commercial lots demonstrated a lack of specificity. Those lots of oxacillin screen medium that fail to grow heteroresistant strains can be detected by using S. aureus ATCC 43300 as a positive control in the test and by using transmitted light to carefully examine the plates for any growth. However, lack of specificity with commercial lots may be difficult to detect using any of the current quality control organisms.

The activities of two investigational fluoroquinolones and three fluoroquinolones that are currently marketed were determined for 182 clinical isolates of Streptococcus pneumoniae . The collection included 57 pneumococcal isolates resistant to levofloxacin (MIC ≥ 8 μg/ml) recovered from patients in North America and Europe. All isolates were tested with clinafloxacin, gatifloxacin, gemifloxacin, levofloxacin, and trovafloxacin by the National Committee for Clinical Laboratory Standards broth microdilution and disk diffusion susceptibility test methods. Gemifloxacin demonstrated the greatest activity on a per gram basis, followed by clinafloxacin, trovafloxacin, gatifloxacin, and levofloxacin. Scatterplots of the MICs and disk diffusion zone sizes revealed a well-defined separation of levofloxacin-resistant and -susceptible strains when the isolates were tested against clinafloxacin and gatifloxacin. DNA sequence analyses of the quinolone resistance-determining regions of gyrA , gyrB , parC , and parE from 21 of the levofloxacin-resistant strains identified eight different patterns of amino acid changes. Mutations among the four loci had the least effect on the MICs of gemifloxacin and clinafloxacin, while the MICs of gatifloxacin and trovafloxacin increased by up to six doubling dilutions. These data indicate that the newer fluoroquinolones have greater activities than levofloxacin against pneumococci with mutations in the DNA gyrase or topoisomerase IV genes. Depending upon pharmacokinetics and safety, the greater potency of these agents could provide improved clinical efficacy against levofloxacin-resistant pneumococcal strains.

Klebsiella pneumoniae K6 (ATCC 700603), a clinical isolate, is resistant to ceftazidime and other oxyimino-β-lactams. A consistent reduction in the MICs of oxyimino-β-lactams by at least 3 twofold dilutions in the presence of clavulanic acid confirmed the utility of K. pneumoniae K6 as a quality control strain for extended-spectrum β-lactamase (ESBL) detection. Isoelectric-focusing analysis of crude lysates of K6 demonstrated a single β-lactamase with a pI of 7.8 and a substrate profile showing preferential hydrolysis of cefotaxime compared to ceftazidime. PCR analysis of total bacterial DNA from K6 identified the presence of a bla SHV gene. K6 contained two large plasmids with molecular sizes of approximately 160 and 80 kb. Hybridization of plasmid DNA with a bla SHV -specific probe indicated that a bla SHV gene was encoded on the 80-kb plasmid, which was shown to transfer resistance to ceftazidime in conjugal mating experiments with Escherichia coli HB101. DNA sequencing of this bla SHV -related gene revealed that it differs from bla SHV-1 at nine nucleotides, five of which resulted in amino acid substitutions: Ile to Phe at position 8, Arg to Ser at position 43, Gly to Ala at position 238, and Glu to Lys at position 240. In addition to the production of this novel ESBL, designated SHV-18, analysis of the outer membrane proteins of K6 revealed the loss of the OmpK35 and OmpK37 porins.

A multilaboratory study was undertaken to determine the accuracy of the current National Committee for Clinical Laboratory Standards (NCCLS) oxacillin breakpoints for broth microdilution and disk diffusion testing of coagulase-negative staphylococci (CoNS) by using a PCR assay for mecA as the reference method. Fifty well-characterized strains of CoNS were tested for oxacillin susceptibility by the NCCLS broth microdilution and disk diffusion procedures in 11 laboratories. In addition, organisms were inoculated onto a pair of commercially prepared oxacillin agar screen plates containing 6 μg of oxacillin per ml and 4% NaCl. The results of this study and of several other published reports suggest that, in order to reliably detect the presence of resistance mediated by mecA , the oxacillin MIC breakpoint for defining resistance in CoNS should be lowered from ≥4 to ≥0.5 μg/ml and the breakpoint for susceptibility should be lowered from ≤2 to ≤0.25 μg/ml. In addition, a single disk diffusion breakpoint of ≤17 mm for resistance and ≥18 mm for susceptibility is suggested. Due to the poor sensitivity of the oxacillin agar screen plate for predicting resistance in this study, this test can no longer be recommended for use with CoNS. The proposed interpretive criteria for testing CoNS have been adopted by the NCCLS.

We evaluated the abilities of 10 commercially available antimicrobial susceptibility testing methods and four reference methods (agar dilution, broth microdilution, disk diffusion, and the agar screen plate) to classify enterococci correctly as vancomycin susceptible or resistant using 50 well-characterized strains of enterococci. There was a high level of agreement of category classification data obtained with broth-based systems (Sceptor, MicroMedia, Pasco, and Sensititre), agar dilution, and an antibiotic gradient method (E test) with data obtained by reference broth microdilution; no very major or major errors were seen, and minor errors were < or = 6%. Increased minor error rates were observed with disk diffusion (12%), Alamar (16%), Uniscept (16%), and conventional (overnight) MicroScan panels (16%). The errors were primarily with Enterococcus casseliflavus strains and organisms containing the vanB vancomycin resistance gene. Very major error rates of 10.3 and 20.7% were observed with Vitek and MicroScan Rapid (MS/Rapid) systems, respectively; however, only the MS/Rapid system produced major errors (13.3%). On repeat testing of discrepant isolates, the very major error rate with the Vitek system dropped to 3.4%, while the very major error rate with the MS/Rapid system increased to 27.6%; major errors with the MS/Rapid system were not resolved. Many of the commercial systems had only 4 dilutions of vancomycin, which resulted in up to 84% of values being off scale (e.g., Uniscept). Of the methods tested, most conventional broth- and agar-based methods proved to be highly accurate when incubation was done for a full 24 h, although several of the tests had high minor error rates. Automated systems continued to demonstrate problems in detecting low-level resistance.

The incidence of vancomycin resistance among enterococci is increasing in the United States and elsewhere in the world, but automated susceptibility testing methods have difficulty detecting resistance expressed by certain strains. The agar screening method described by Willey et al. (B. M. Willey, B. N. Kreiswirth, A. E. Simor, G. Williams, S. R. Scriver, A. Phillips, and D. E. Low, J. Clin. Microbiol. 30:1621-1624, 1992) has been proposed as a reliable method for confirming vancomycin resistance. In this study, we investigated various parameters associated with the agar screening method and, on the basis of the findings, established optimum testing conditions for the method. First, to evaluate media and vancomycin concentrations, one laboratory used Mueller-Hinton and brain heart infusion agars supplemented with 4, 6, and 8 micrograms of vancomycin per ml to test 100 genetically characterized enterococcal strains. On the basis of the results obtained, brain heart infusion agar supplemented with 6 micrograms of vancomycin per ml was selected for further study. Subsequently, eight laboratories used the medium to test both reference and clinical isolates. There was very good performance with the reference strains and, among 158 clinical isolates tested, the method demonstrated sensitivity and specificity of 100% and from 96 to 99%, respectively.

To test the ability of clinical laboratories to detect antimicrobial resistance among enterococci, we sent four vancomycin-resistant enterococcal strains and one beta-lactamase-producing enterococcus to all 93 nongovernment, hospital-based clinical laboratories in New Jersey; 76 (82%) participated in the study. Each organism was tested by the laboratory's routine antimicrobial susceptibility testing method. The proportion of laboratories that correctly reported that an isolate was resistant to vancomycin varied according to the resistance level of the isolate: high-level resistance (MIC for Enterococcus faecium = 512 micrograms/ml), 96% of laboratories correct; moderate-level resistance (MIC for E. faecium = 64 micrograms/ml), 27% correct; low-level resistance (MIC for Enterococcus faecalis = 32 micrograms/ml), 16% correct; and intrinsic low-level resistance (MIC for Enterococcus gallinarum = 8 micrograms/ml), 74% correct. The beta-lactamase-producing E. faecalis isolate was identified as resistant to penicillin and ampicillin by 66 and 8% of laboratories, respectively, but only three laboratories recognized that it was a beta-lactamase producer. This survey suggests that many laboratories may fail to detect antimicrobial agent-resistant enterococci.

Mupirocin susceptibility testing of Staphylococcus aureus has become more important as mupirocin is used more widely to suppress or eliminate S. aureus colonization and prevent subsequent health care- and community-associated infections. The present multicenter study evaluated two susceptibility testing screening methods to detect mupirocin high-level resistance (HLR), broth microdilution (BMD) MICs of ≥512 μg/ml, and a 6-mm zone diameter for a disk diffusion (DD) test with a 200-μg disk. Initial testing indicated that with Clinical and Laboratory Standards Institute methods for BMD and DD testing, the optimal conditions for the detection of mupirocin HLR were 24 h of incubation and reading of the DD zone diameters with transmitted light. Using the presence or absence of mupA as the “gold standard” for HLR, the sensitivity and specificity of a single-well 256 μg/ml BMD test were 97 and 99%, respectively, and those for the 200-μg disk test were 98 and 99%, respectively. Testing with two disks, 200 μg and 5 μg, was evaluated for its ability to distinguish HLR isolates (MICs ≥ 512 μg/ml), low-level-resistant (LLR) isolates (MICs = 8 to 256 μg/ml), and susceptible isolates (MICs ≤ 4 μg/ml). Using no zone with both disks as an indication of HLR and no zone with the 5-μg disk plus any zone with the 200-μg disk as LLR, only 3 of the 340 isolates were misclassified, with 3 susceptible isolates being classified as LLR. Use of standardized MIC or disk tests could enable the detection of emerging high- and low-level mupirocin resistance in S. aureus .

We collected 705 isolates of enterococci (1 per patient) from cultures of a variety of anatomic sites from patients at eight tertiary-care hospitals in six geographic regions of the United States. A total of 632 (90%) Enterococcus faecalis, 58 (8%) E. faecium, 5 E. gallinarum, 4 E. avium, 3 E. casseliflavus, 1 E. raffinosus, and 1 E. hirae isolate and 1 biochemical variant of E. faecalis were identified; 606 (86%) of these isolates were associated with clinical infections. The most common sites of isolation were the urinary tract (402 [57%]), nonsurgical wounds (94 [13%]), the bloodstream (74 [10%]), and surgical wounds (62 [9%]). High-level resistance to gentamicin or streptomycin or both was detected in 265 (38%) of the isolates. We identified two E. faecalis isolates resistant to vancomycin (MICs, 32 and 128 micrograms/ml) and 11 beta-lactamase-producing E. faecalis isolates. E. faecium isolates were significantly more resistant than E. faecalis isolates to penicillin, ampicillin, piperacillin, imipenem, and ciprofloxacin (P less than 0.001). The MICs for the 15 non-E. faecalis, non-E. faecium enterococci indicated variable resistance to ciprofloxacin and the penicillins. Antimicrobial susceptibility patterns vary among species of enterococci, and these organisms, while commonly resistant to high-level aminoglycosides, can also acquire resistance to vancomycin or the ability to produce beta-lactamase. Because of these diverse antimicrobial resistance mechanisms, successful treatment and control of enterococcal infections with current antimicrobial agents are becoming increasingly difficult.

Objective: To describe the epidemiology of a cluster of vancomycin-resistant Enterococcus faecium (VAREC) in a cardiothoracic surgery intensive care unit. Design: A case series of patients identified through review of surveillance data on nosocomial infections, review of microbiologic records, and culture survey of patients in the unit. Results: Six patients in the cardiothoracic surgery intensive care unit had VAREC with identical antimicrobic susceptibility patterns over a 6-month period. Four patients were identified with VAREC through prospective surveillance and 2 through retrospective review. Prior vancomycin use was seen more commonly in patients with VAREC (6/6,100%) than in those without VAREC (3/12, 25%) (Fisher’s exact test, p= .01). Six of the 7 patients with prior infection developed VAREC (85.7%). A prior nosocomial infection and prior exposure to vancomycin were found to be important variables in a logistic regression analysis. VAREC also was isolated from the environment. Acombination of cohorting of patients and staff, and modifications of standard contact isolation practices eliminated the presence of VAREC from the cardiothoracic surgery intensive care unit. Conclusions: The results suggest that prior administration of vancomycin, especially in the patient who develops nosocomial infection, can influence the acquisition of vancomycin-resistant enterococci and that VAREC may be transmitted from patient to patient. Using a modification of the standard infection control practice of isolation, we were able to control the spread of this resistant strain of E faecium.

Aerococci can be misidentified as streptococci, enterococci, pediococci, lactococci, or leuconostocs. To distinguish the genus and determine if another species is needed in the present taxon, we analyzed 37 aerococci for cellular fatty acids and compared them with 377 strains of gram-positive cocci, including the species type strains from each of the related genera. The cellular fatty acid profile of aerococci was distinguishable from other genera. Two relatively novel fatty acids found in the aerococci were identified as C16:1 omega 9c and C16:1 omega 9t. Eleven strains of aerococci (including a strain originally identified as "Gaffkya" species) were chosen for DNA-DNA reassociation studies with the type strain Aerococcus viridans ATCC 11563; DNAs from eight of these strains were more than 75% related to the type strain and had 1 to 4% divergence in related sequences. The remaining three strains were 60 to 70% related to the type strain, had 7 to 11.5% divergence, and may represent a second species, Aerococcus genospecies 2. beta-Glucuronidase, alpha-galactosidase, and beta-galactosidase were useful in characterizing the aerococci.

A multilaboratory collaborative study was undertaken to determine whether the anaerobic disk diffusion test of Horn et al. could be performed reproducibly and accurately. Tests with nine different antimicrobial disks were evaluated. Reproducibility of the agar diffusion disk method was similar to that of the reference agar dilution test procedure. The anaerobic disk diffusion procedure was found to be a potentially useful method for testing some antimicrobial agents against rapidly growing anaerobes belonging to the Bacteroides fragilis group. These promising results warrant further investigations and validations.

We reevaluated Enterobacteriaceae disk diffusion breakpoints for the tetracyclines published in the Clinical and Laboratory Standards Institute (CLSI) document M100-S16, which were (susceptible/resistant) ≥19 mm/≤14 mm for tetracycline, ≥16 mm/≤12 mm for doxycycline, and ≥19 mm/≤14 mm for minocycline. A collection of 504 recent clinical isolates of Enterobacteriaceae were tested against these tetracycline compounds by disk diffusion and broth microdilution methods according to CLSI guidelines. Regression line and scattergram plot analyses determined intermethod accuracy for current disk diffusion breakpoints and showed excellent r values of 0.91 to 0.95. However, error rates (minor/major [false-resistant]/very major [false-susceptible]) were 14.9/0.8/0.0% for tetracycline, 11.5/0.4/0.0% for doxycycline, and 30.6/0.7/0.0% for minocycline and only 4.4/0.0/0.0% for tetracycline, 5.6/0.0/0.2% for doxycycline, and 8.3/0.0/0.3% for minocycline when proposed breakpoints were modified to (susceptible/resistant) ≥15 mm/≤11 mm for tetracycline, ≥14 mm/≤10 mm for doxycycline, and ≥16 mm/≤12 mm for minocycline. Listed modifications were recently approved by the CLSI (M100-S17).

Phenotypic methods for detecting mecA-mediated resistance in Staphylococcus aureus include both oxacillin and cefoxitin susceptibility tests; many laboratories perform multiple tests. Conflicting oxacillin and cefoxitin susceptibility results are most likely to occur for isolates that either have reduced susceptibility to oxacillin by a non-mecA-mediated mechanism or are mecA positive but are very heteroresistant. To understand the performance of oxacillin and cefoxitin tests for such isolates, we tested 135 S. aureus isolates using either cefoxitin or oxacillin and compared the results with mecA polymerase chain reaction. These strains either expressed borderline oxacillin MICs (1-4 microg/mL) and had undetermined mecA status or were mecA positive but were not detected by oxacillin broth microdilution (BMD) or disk diffusion (DD) in original testing. For 24-h readings, performance of cefoxitin tests (sensitivity/specificity) were DD (99/100), Etest using < or =6 microg/mL as susceptible (99/98), and Phoenix MIC using < or =4 microg/mL as susceptible (98/100). Using 6 microg/mL of cefoxitin as a screen test in both BMD and agar dilution also worked well (98/98-100). Sensitivity/specificity of oxacillin methods were oxacillin agar screen (BBL: 80/86; Remel, Lenexa, KS: 85/50), DD (91/59), BMD (85/88), MicroScan (89/96), VITEK Legacy (82/93), VITEK 2 (91/73), and Phoenix, (67/96). These results suggest that a cefoxitin test can be used alone to predict mecA-mediated resistance in S. aureus.

In 2005, the Clinical and Laboratory Standards Institute published MIC interpretive criteria for 13 antimicrobial agents used for either therapy or prophylaxis of Neisseria meningitidis infections. The MIC method includes the use of lysed horse blood-supplemented Mueller-Hinton broth with incubation in 5% CO 2 for 20 to 24 h. Since some clinical laboratories might prefer the option of disk diffusion testing for infrequently encountered isolates a multicenter collaborative study was conducted to evaluate the reproducibility of a disk diffusion method for testing isolates of N. meningitidis . Interpretive criteria were developed for 12 antimicrobial agents. Four laboratories tested a common collection of 50 meningococcal strains and then tested 25 unique isolates per laboratory. Isolates were tested using Mueller-Hinton sheep blood agar plates incubated for 20 to 24 h in 5% CO 2 ; they were also tested by the reference broth microdilution method in parallel. Pooling of the MIC and disk diffusion data from the common and unique isolates provided a sufficient sample size to develop susceptible, intermediate, and resistant zone diameter interpretive criteria using the error rate-bounded method for the following agents: chloramphenicol, trimethoprim-sulfamethoxazole, ciprofloxacin, and rifampin. Due to the lack of resistant strains at the present time, “susceptible only” interpretive criteria were proposed for cefotaxime, ceftriaxone, meropenem, azithromycin, and minocycline. The numbers of minor interpretive errors with penicillin and ampicillin disk tests were unacceptably high and precluded recommended testing of those agents by the disk method. However, amdinocillin, an agent that preferentially binds to the altered penicillin binding protein responsible for diminished penicillin susceptibility, has potential utility as a surrogate screening reagent for ampicillin resistance. A disk diffusion breakpoint was derived for nalidixic acid to serve as a surrogate marker for gyrase A mutations associated with diminished fluoroquinolone susceptibility. Disk diffusion testing with meningococci can be performed in a reproducible manner with several antimicrobial agents and represents a practical and cost-effective option for testing sporadic clinical isolates or for surveillance purposes by resource-limited laboratories.

The cefoxitin disk diffusion (DD) test for predicting mecA- mediated oxacillin resistance in staphylococci was assessed during a three-phase study. In phase 1, one laboratory tested 62 and 53 strains of Staphylococcus aureus and coagulase-negative staphylococci (CoNS), respectively. These data were used to choose the provisional cefoxitin DD breakpoints (resistant/susceptible) of ≤19 mm/≥20 mm for S. aureus and ≤24 mm/≥25 mm for CoNS for the next phase of testing. In phase 2, 10 laboratories each tested approximately 40 in-house strains of staphylococci (half of which were S. aureus ) using Mueller-Hinton agar from different manufacturers. In this phase, the sensitivity and specificity, respectively, of the cefoxitin disk test were 98 and 100% for S. aureus and 99 and 96% for CoNS. The cefoxitin DD test performed equivalently to oxacillin broth microdilution (BMD) and to oxacillin DD tests among S. aureus and mecA -positive CoNS strains but gave better results than oxacillin BMD or oxacillin DD for mecA -negative strains of CoNS. The cefoxitin DD test also was much easier to read and did not require the use of transmitted light for detection of resistance. Based on data from the first two phases, the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) adopted the use of the cefoxitin DD test for predicting mecA- mediated oxacillin resistance in staphylococci and revised Table 2C in CLSI document M100-S14 to reflect the change. In the third phase, an additional 61 challenge strains of CoNS for which the oxacillin MICs were 0.5 to 2 μg/ml were tested in a single laboratory to determine the effectiveness of the cefoxitin DD test for this group of borderline-resistant isolates. These data were used to refine the description of the test in CLSI document M100-S15. The cefoxitin DD test is preferred over the oxacillin DD test for predicting mecA -mediated oxacillin resistance in S. aureus and CoNS.

An eight-laboratory study addressed the urgent need for quality control (QC) ranges for susceptibility determination when testing colistin (polymyxin E) and polymyxin B, two polycationic peptide antimicrobial agents, against multidrug-resistant gram-negative bacilli. For Escherichia coli ATCC 25922l, the QC ranges were as follows: for colistin, 0.25 to 1 μg/ml (11 to 17 mm), and for polymyxin B, 0.25 to 2 μg/ml (13 to 19 mm). For Pseudomonas aeruginosa ATCC 27853, the QC ranges were as follows: for colistin, 0.25 to 2 μg/ml (11 to 17 mm), and for polymyxin B, 0.25 to 2 μg/ml (14 to 18 mm). More than 97% of all reported QC results were within these proposed ranges.

Although both broth microdilution (BMD) and disk diffusion (DD) are listed by NCCLS as acceptable methods for testing Acinetobacter spp. for antimicrobial susceptibility, few studies have compared the results generated by the two methods. We tested 196 isolates of Acinetobacter spp. from nine U.S. hospitals and from the Centers for Disease Control culture collection by using BMD and DD and clinically appropriate antimicrobial agents. Categorical results for amikacin, ciprofloxacin, gatifloxacin, gentamicin, imipenem, levofloxacin, meropenem, tobramycin, and trimethoprim-sulfamethoxazole were comparable for the two methods: there was only one very major (VM) error, with tobramycin, and only one major (M) error, with meropenem, when DD results were compared with BMD results. However, VM errors were frequent with the β-lactams and β-lactam-β-lactam inhibitor combinations, while M errors were often observed with tetracyclines. For BMD, tests frequently exhibited subtle growth patterns that were difficult to interpret, especially for β-lactams. If subtle growth (i.e., granular, small button, or “starry” growth) was considered positive, error rates between BMD and DD were unacceptably high for ampicillin-sulbactam (VM error, 9.8%; minor [m] error, 16.1%), piperacillin (VM error, 5.7%; m error, 13.5%), piperacillin-tazobactam (VM error, 9.3%; m error, 12.9%), ceftazidime (VM error, 6.2%; m error, 11.4%), cefepime (VM error, 6.2%; m error, 13.0%), cefotaxime (m error, 21.2%), ceftriaxone (m error, 23.3%), tetracycline (M error, 11.4%; m error, 32.1%), and doxycycline (M error, 2.6%). When subtle growth patterns were ignored, the agreement still did not achieve acceptable levels. To determine if the problems with BMD testing occurred in other laboratories, we sent frozen BMD panels containing β-lactam drugs and nine isolates to six labs with experience in performing BMD and DD. Among these laboratories, cefepime MICs ranged from ≤8 to ≥32 μg/ml for four of the nine strains, confirming the problem in interpreting BMD results. Discrepancies between the categorical interpretations of BMD and DD tests were noted primarily with cefepime and piperacillin, for which the BMD results were typically more resistant. Clinical laboratories should be aware of these discrepancies. At present, there are no data to indicate which method provides more clinically relevant information.

Studies were conducted to validate the use of Enterococcus faecalis ATCC 51299 (which is vancomycin resistant and resistant to high levels of gentamicin and streptomycin) and E. faecalis ATCC 29212 (which is susceptible to vancomycin and against which gentamicin or streptomycin and cell wall-active agents have synergistic kill activity) as controls in an agar screening test for vancomycin resistance and high-level streptomycin and gentamicin resistance and a broth microdilution screening test for high-level streptomycin and gentamicin resistance. Both organisms performed as expected in these tests and will serve as appropriate controls. However, E. faecalis ATCC 29212 was occasionally noted to produce light growth on the vancomycin screening plate with certain lots of agar. Quality control ranges for disk diffusion tests with disks with large amounts of streptomycin (300 micrograms) and gentamicin (120 micrograms) were established for E. faecalis ATCC 29212; zone limits are 16 to 22 mm for gentamicin and 14 to 19 mm for streptomycin. No zones for inhibition were seen when E. faecalis ATCC 51299 was tested with these high-content disks.

Using a set of 55 Staphylococcus aureus challenge organisms, we evaluated six routine methods (broth microdilution, disk diffusion, oxacillin agar screen, MicroScan conventional panels, MicroScan rapid panels, and Vitek cards) currently used in many clinical laboratories and two new rapid methods, Velogene and the MRSA-Screen, that require less than a day to determine the susceptibility of S. aureus to oxacillin. The methods were evaluated by using the presence of the mecA gene, as detected by PCR, as the “gold standard.” The strains included 19 mecA -positive heterogeneously resistant strains of expression class 1 or 2 (demonstrating oxacillin MICs of 4 to >16 μg/ml) and 36 mecA -negative strains. The oxacillin MICs of the latter strains were 0.25 to 4 μg/ml when tested by broth microdilution with 2% NaCl-supplemented cation-adjusted Mueller-Hinton broth as specified by the NCCLS. However, when tested by agar dilution with 4% salt (the conditions used in the oxacillin agar screen method), the oxacillin MICs of 16 of the mecA- negative strains increased to 4 to 8 μg/ml. On initial testing, the percentages of correct results (% sensitivity/% specificity) were as follows: broth microdilution, 100/100; Velogene, 100/100; Vitek, 95/97; oxacillin agar screen, 90/92; disk diffusion, 100/89; MicroScan rapid panels, 90/86; MRSA-Screen, 90/100; and MicroScan conventional, 74/97. The MRSA-Screen sensitivity improved to 100% if agglutination reactions were read at 15 min. Repeat testing improved the performance of some but not all of the systems.

Fluoroquinolone resistance appears to be increasing in many species of bacteria, particularly in those causing nosocomial infections. However, the accuracy of some antimicrobial susceptibility testing methods for detecting fluoroquinolone resistance remains uncertain. Therefore, we compared the accuracy of the results of agar dilution, disk diffusion, MicroScan Walk Away Neg Combo 15 conventional panels, and Vitek GNS-F7 cards to the accuracy of the results of the broth microdilution reference method for detection of ciprofloxacin and ofloxacin resistance in 195 clinical isolates of the family Enterobacteriaceae collected from six U.S. hospitals for a national surveillance project (Project ICARE [Intensive Care Antimicrobial Resistance Epidemiology]). For ciprofloxacin, very major error rates were 0% (disk diffusion and MicroScan), 0.9% (agar dilution), and 2.7% (Vitek), while major error rates ranged from 0% (agar dilution) to 3.7% (MicroScan and Vitek). Minor error rates ranged from 12.3% (agar dilution) to 20.5% (MicroScan). For ofloxacin, no very major errors were observed, and major errors were noted only with MicroScan (3.7% major error rate). Minor error rates ranged from 8.2% (agar dilution) to 18.5% (Vitek). Minor errors for all methods were substantially reduced when results with MICs within ±1 dilution of the broth microdilution reference MIC were excluded from analysis. However, the high number of minor errors by all test systems remains a concern.

Resistance to fluoroquinolone (FQ) antibiotics in Streptococcus pneumoniae has been attributed primarily to specific mutations in the genes for DNA gyrase ( gyrA and gyrB ) and topoisomerase IV ( parC and parE ). Resistance to some FQs can result from a single mutation in one or more of the genes encoding these essential enzymes. A group of 160 clinical isolates of pneumococci was examined in this study, including 36 ofloxacin-resistant isolates (MICs, ≥8 μg/ml) recovered from patients in North America, France, and Belgium. The susceptibilities of all isolates to clinafloxacin, grepafloxacin, levofloxacin, sparfloxacin, and trovafloxacin were examined by the National Committee for Clinical Laboratory Standards reference broth microdilution and disk diffusion susceptibility testing methods. Among the ofloxacin-resistant strains, 32 of 36 were also categorized as resistant to levofloxacin, 35 were resistant to sparfloxacin, 29 were resistant to grepafloxacin, and 19 were resistant to trovafloxacin. In vitro susceptibility to clinafloxacin appeared to be least affected by resistance to the other FQs. Eight isolates with high- and low-level resistance to the newer FQs were selected for DNA sequence analysis of the quinolone resistance-determining regions (QRDRs) of gyrA , gyrB , parC , and parE . The DNA and the inferred amino acid sequences of the resistant strains were compared with the analogous sequences of reference strain S. pneumoniae ATCC 49619 and FQ-susceptible laboratory strain R6. Reduced susceptibilities to grepafloxacin and sparfloxacin (MICs, 1 to 2 μg/ml) and trovafloxacin (MICs, 0.5 to 1 μg/ml) were associated with either a mutation in parC that led to a single amino acid substitution (Ser-79 to Phe or Tyr) or double mutations that involved the genes for both GyrA (Ser-81 to Phe) and ParE (Asp-435 to Asn). High-level resistance to all of the compounds except clinafloxacin was associated with two or more amino acid substitutions involving both GyrA (Ser-81 to Phe) and ParC (Ser-79 to Phe or Ser-80 to Pro and Asp-83 to Tyr). No mutations were observed in the gyrB sequences of resistant strains. These data indicate that mutations in pneumococcal gyrA , parC , and parE genes all contribute to decreased susceptibility to the newer FQs, and genetic analysis of the QRDR of a single gene, either gyrA or parC , is not predictive of pneumococcal resistance to these agents.

Genes encoding streptomycin/spectinomycin adenylyltransferases [ANT(3")(9)] have been reported to exist in gram-negative organisms and Staphylococcus aureus . During a study of high-level aminoglycoside resistance in enterococci, we encountered an isolate of Enterococcus faecalis that was streptomycin resistant but did not appear to contain the 6′-adenylyltransferase gene ( aadE ) when examined by PCR with specific primers. Phosphocellulose paper binding assays indicated the presence of an ANT(3")(9) enzyme. Streptomycin and spectinomycin MICs of 4,000 and 8,000 μg/ml, respectively, were observed for the isolate. PCR primers corresponding to a highly conserved region of the aadA gene were used to amplify a specific 284-bp product. The product hybridized with a digoxigenin-labeled PCR product from E. coli C600(pHP45Ω) known to contain the aadA gene. The aadA gene was transferred via filter matings from the E. faecalis donor to E. faecalis JH2-2. PCR primers designed for analysis of integrons were used to amplify a 1-kb product containing the aadA gene, which was cloned into the vector pCRII and transformed into Escherichia coli DH5-α competent cells. d -Rhodamine dye terminator cycle sequencing was used to determine the gene sequence, which was compared to previously reported sequences of aadA genes. We found the aadA gene in E. faecalis to be identical to the aadA genes reported by Sundström et al. for E. coli plasmid R6-5 (L. Sundström, P. Rådström, G. Swedberg, and O. Sköld, Mol. Gen. Genet. 213:191–201, 1988), by Fling et al. for the aadA within transposon Tn 7 (M. E. Fling, J. Kopf, and C. Richards, Nucleic Acids Res. 13:7095–7106, 1985), and by Hollingshead and Vapnek for E. coli R538-1 (S. Hollingshead and D. Vapnek, Plasmid 13:17–30, 1985). Previous reports of the presence of the aadA gene in enterococci appear to be erroneous and probably describe an aadE gene, since the isolates were reported to be susceptible to spectinomycin.

Seven commercial systems for antimicrobial susceptibility testing of Streptococcus pneumoniae were evaluated by using a challenge set of 55 pneumococcal isolates with a variety of resistance phenotypes and genotypes. Overall, the results produced by the Pasco and Etest methods were found to be acceptable for all drugs tested except for trimethoprim-sulfamethoxazole testing by the Etest. The Just One system for penicillin MIC testing was also judged to be acceptable (minor error rate, 5.5%). Although the Sensititre and MicroTech methods both produced 12.7% minor errors with penicillin, the Sensititre method classified penicillin-intermediate strains as resistant or vice versa, while four of MicroTech's errors were among intermediate strains that were classified as susceptible. The MicroMedia (minor error rate, 16.4%) and MicroScan Rapid (minor error rate, 63.6%) methods produced unacceptably high levels of errors when testing penicillin. Minor error rates for cefotaxime and ceftriaxone ranged from a low of 12.7% (Etest and Sensititre) to a high of 28% (MicroMedia). Error rates were low for erythromycin, tetracycline, and chloramphenicol by most methods with the exception of the MicroScan method, which had a high very major error rate for erythromycin (34.6%). For testing of beta-lactam drugs, the Pasco, Etest, and Just One tests for penicillin are the most accurate methods; the Sensititre method also provided acceptable results.

Since the early 1970s, the synergistic activity of an aminoglycoside with a cell wall-active agent has been predicted by determining the ability of an enterococcus to grow in the presence of high levels of the aminoglycoside (usually > or = 2,000 micrograms/ml). However, a variety of media and concentrations of aminoglycosides has been used for this screening procedure. In the present study, we sought to optimize the agar dilution, broth microdilution, and disk diffusion tests used to detect high-level gentamicin and streptomycin resistance in enterococci. For dilution tests, brain heart infusion agar or broth gave the best growth and performance. For agar dilution, 500 micrograms of gentamicin per ml, 2,000 micrograms of streptomycin per ml, and an inoculum of 1 x 10(6) CFU/ml were optimal, while for broth microdilution, 500 micrograms of gentamicin per ml, 1,000 micrograms of streptomycin per ml, and an inoculum of 5 x 10(5) CFU/ml were best. Growth of more than one colony in the agar dilution test was determined to be the best indicator of high-level resistance. For disk diffusion, Mueller-Hinton agar, 120-micrograms gentamicin disks, and 300-micrograms streptomycin disks with breakpoints of no zone for resistance and > or = 10 mm for susceptibility gave the best sensitivity and specificity if results for strains with zones of 7 to 9 mm are considered inconclusive, indicating that a broth or agar test should be performed to determine susceptibility or resistance.

We examined 105 clinical isolates of glycopeptide-resistant enterococci collected from 31 U.S. hospitals in 14 states during May 1988 to July 1992. The isolates included 82 Enterococcus faecium, 8 E. faecalis, 6 Enterococcus spp., 5 E. gallinarum, 3 E. casseliflavus, and 1 E. raffinosus. The isolates were categorized into the following four phenotypes of glycopeptide resistance on the basis of their MIC patterns: (i) 70 VanA (vancomycin [Vm] MIC, > or = 64 micrograms/ml; teicoplanin [Tei] MIC, 16 to > or = 128 micrograms/ml), (ii) 26 VanB (Vm MIC, 16 to 1,024 micrograms/ml; Tei MIC, < or = 2 micrograms/ml), (iii) 5 VanC (Vm MIC, 4 to 16 micrograms/ml; Tei MIC, < or = 2 micrograms/ml) in E. gallinarum, and (iv) 3 E. casseliflavus and 1 E. raffinosus isolates for which Vm MICs were 4 to 16 micrograms/ml and Tei MICs were < or = 1 micrograms/ml were called unclassified. Of the 101 isolates with the VanA, VanB, and VanC phenotypes, 99 were confirmed by production of a specific 1,030-, 433-, or 796-bp polymerase chain reaction product, respectively, and hybridization with the respective gene probe. The vanA gene was also detected in the E. raffinosus isolate for which the Vm MIC was 16 micrograms/ml and the Tei MIC was 1 microgram/ml. The vanA gene was located on either a 34- or a 60-kb plasmid in all of the U.S. isolates examined. Pulsed-field gel electrophoresis demonstrated both intrahospital and interhospital diversity among Vmr enterococci in the United States and was more useful than plasmid analysis for epidemiologic studies.

Kirby-Bauer disk susceptibility tests with five standard cephalosporin disks were performed on 23 penicillin-resistant Streptococcus pneumoniae isolates for which ceftriaxone MICs were 0.125 to 4 micrograms/ml. Cefuroxime disk inhibition zone diameters distinguished clearly isolates for which ceftriaxone MICs wereor = 2 micrograms/ml from more susceptible strains, whereas cephalothin, ceftizoxime, cefotaxime, and ceftriaxone disks distinguished these isolates less clearly than the cefuroxime disk did.

Since 1988, when the first vancomycin-resistant enterococcus was described, several descriptions of failures of disk diffusion breakpoints to detect low-level vancomycin resistance (MICs, 8 to 32 micrograms/ml) have been published. A four-laboratory collaborative study was undertaken to establish more accurate breakpoints for the disk test. Mueller-Hinton agar was used to perform dilution testing (in three laboratories) and disk diffusion testing (in all laboratories). Results were determined at 18, 24, and 48 h, and zones of inhibition were read using both transmitted and reflected light. One hundred organisms (35 Enterococcus faecalis, 55 E. faecium, and 10 E. gallinarum or E. casseliflavus isolates) were selected to represent vancomycin-susceptible and -resistant phenotypes. Interlaboratory agreement of agar dilution MICs was better at 24 h (91 to 94% within +/- 1 dilution) than at 18 h (76% within +/- 1 dilution). Therefore, 24-h agar dilution MIC results were used as the reference. For disk diffusion, it was critical to note the presence of a haze or colonies inside the zone when interpreting the test, since this correlated better with the results of the agar dilution test. The presence of a haze or inner colonies was best detected by reading the zones with transmitted light and incubating the plates for a full 24 h. When plotted against 24-h agar dilution MICs, breakpoints of /= 17 mm (susceptible) resulted in 58 minor errors (14.5% of total values) and 5 very major errors (2.2% of resistant values or 1.3% of total values). No major errors were seen. Results of repeat testing using a common lot of Mueller-Hinton agar showed 52 minor errors (13.3%) and 4 major errors (4.2% of susceptible values of 1.0% pf total values) but no very major errors. It is recommended that any haze or colonies within the zone be taken into account when determining zones of inhibition and that an MIC test be performed for strains with intermediate zones if vancomycin is being considered for treatment.

Streptococcus pneumoniae isolates resistant to penicillin, chloramphenicol, tetracycline and sulfamethoxazole-trimethroprim are being recovered with increasing frequency in the United States. We analyzed the penicillin-binding proteins (PBPs), multilocus enzyme electrophoresis (MLEE) genotypes, and ribotypes of 22 multiresistant serotype 23F isolates of S. pneumoniae from the United States and 1 isolate each from Spain and South Africa. Also included were seven multiresistant isolates of other serotypes, three penicillin-resistant but chloramphenicol-susceptible serotype 23F isolates, and two penicillin-susceptible isolates (one penicillin-susceptible isolate was serotype 23F). Fifteen of the 22 multiresistant isolates from the United States and the isolates from Spain and South Africa had identical PBP patterns, MLEE profiles, and ribotypes. Six of the remaining seven multiresistant isolates were related by PBP pattern, but demonstrated slightly different MLEE and/or ribotype profiles, possibly because of acquisition of additional resistance markers (four of the six isolates were also resistant to erythromycin). The remaining multiresistant serotype 23F isolate had a unique PBP pattern and ribotype and was only distantly related to the other pneumococcal isolates by MLEE analysis. The PBP patterns, MLEE profiles, and ribotypes of the multiresistant serotype 23F isolates were easily distinguished from those of six multiresistant isolates of other serotypes; three other penicillin-resistant, chloramphenicol-susceptible, serotype 23F isolates; and two penicillin-susceptible isolates. One exception was a multiresistant serotype 19A isolate that was highly related to the clonal group by PBP pattern and MLEE analysis and that had a ribotype similar to those of the other erythromycin-resistant serotype 23F isolates. MLEE analysis and ribotyping were more discriminating than were the PBP patterns in discerning strain differences. These data strongly suggest that a multiresistant clone of S. pneumoniae serotype 23F that is related to multiresistant isolates from Spain and South Africa has become disseminated in the United States. Clinicians should be alerted to the spread of these multiresistant strains in the United States.

Eighty-five strains of vancomycin-resistant gram-positive bacteria from three genera, Leuconostoc, Pediococcus, and Lactobacillus, were tested to determine susceptibility to 24 antimicrobial agents by broth microdilution and to 10 agents by disk diffusion. The MICs of vancomycin and teicoplanin ranged from 64 to greater than 512 micrograms/ml; however, the MICs of daptomycin, a new lipopeptide, were all less than or equal to 0.25 micrograms/ml. None of the organisms were resistant to imipenem, minocycline, chloramphenicol, gentamicin, or daptomycin. The MICs of penicillin were in the moderately susceptible range for all but three strains. Susceptibility to the other agents varied by genus and, in some cases, by species. When disk diffusion results were compared with MICs for drugs recommended for streptococci by the National Committee for Clinical Laboratory Standards, Villanova, Pa., few very major or major errors were obtained, but the number of minor errors was 19.3%. Therefore, we recommended that MIC testing be used instead of disk diffusion testing for these organisms.

Between July 1987 and January 1988, five patients dialyzed at a hemodialysis outpatient clinic developed systemic Mycobacterium chelonae abscessus (MCA) infections. Four of the five patients had arteriovenous graft infections, and two died during antimicrobial therapy. Case-patients were more likely than control-patients to have received high-flux dialysis during the 6 mo before their infection (100% vs. 30%, P = .009). MCA was cultured from the hose connected to a water spray device used for manual reprocessing of high-flux but not regular dialyzers. Renalin was the germicide used to manually disinfect dialyzers for reuse and was prepared at a concentration of 2.5%. Laboratory studies documented survival of MCA from two high-flux dialyzers that were reprocessed in a manner similar to that used in the clinic. Early diagnosis with complete rather than partial graft removal in combination with antimicrobial therapy is recommended for MCA graft infections. In addition, 2.5% Renalin did not appear to ensure complete killing of MCA in high-flux dialyzers that were manually reprocessed at this hemodialysis clinic.

As pointed out by Dr. Walsh and colleagues in the July 2001 issue of the Journal of Clinical Microbiology (4), the detection of staphylococci with reduced susceptibility to vancomycin is an important issue for clinical laboratories. In their study, Dr. Walsh et al. evaluated several methods for detection of these strains using a population-analysis profile-area under the curve ratio method as their “gold standard.” Two NCCLS reference methods, broth microdilution and agar dilution, were included in their analysis. They state that both were performed “precisely” as described in the most recent NCCLS document, M7-A5 (2, 3). However, the incubation time for both tests in their study was only 18 h, whereas current NCCLS recommendations specify that tests for both oxacillin and vancomycin should be incubated for a full 24 h. We suspect that the sensitivity of both reference methods would have improved had the readings been made at 24 h rather than at 18 h as was done in their study. Incubation for a full 24 h appears to be important not just for NCCLS reference microdilution susceptibility tests but also for primary isolation plates (1), as pointed out in a study by other investigators in the same issue of the journal.

One hundred twenty-three patients with nonpulmonary infections due to Mycobacterium fortuitum or Mycobacterium chelonei were treated by wound debridement and with chemotherapy on the basis of in vitro susceptibilities of the organism. Of 76 patients with infections caused by M. fortuitum, 13 required no therapy or were adequately treated with surgery alone. Patients with active localized disease received single drug therapy (usually with a sulfonamide) for a mean period of 10.6 weeks for cellulitis and seven months for osteomyelitis. Patients with extensive disease received amikacin or amikacin plus cefoxitin (mean, four weeks) followed by a sulfonamide (mean, six months). The 47 patients with infections caused by M. chelonei received no therapy or were treated with surgery alone (6); with amikacin (10), erythromycin (6), doxycycline (3), or cefoxitin (1); or with amikacin plus cefoxitin followed by cefoxitin alone for a total of 10-12 weeks (20); or other multiple-drug regimens (1). Surgery was performed on 74 (60%) patients. Schlichter tests or serum drug levels were determined for 81 (66%) patients. Response to therapy was excellent; 68 (90%) infections with M. fortuitum and 34 (72%) with M. chelonei were successfully treated. Cultures became negative within six weeks of chemotherapy, except for sternal osteomyelitis, for which cultures were not negative until up to 14 weeks. Follow-up for a mean period of 12 months following therapy was possible in 80% of cases. Relapses were rare except in patients with disseminated disease, and drug resistance developed in only one patient. These studies demonstrate the value of routine susceptibility testing of these mycobacterial species and the benefit of chemotherapy on the basis of in vitro susceptibilities.

Seventeen cases of otitis media caused by Mycobacterium chelonae were detected among patients seen at a single ear-nose-and-throat (ENT) office (Office A) in Louisiana between May 5 and September 15, 1987. All the patients had a tympanotomy tube or tubes in place or had one or more tympanic-membrane perforations, with chronic otorrhea that was unresponsive to standard therapy with antimicrobial agents. Middle-ear exploration in six patients revealed abundant granulation tissue; multiple granulomas and acid-fast bacilli were demonstrated on a section of tissue from one patient with a nonhealing mastoidectomy incision. Thirteen of the 14 ear isolates obtained from patients seen in Office A had the same unusual pattern of high-level resistance to aminoglycosides. M. chelonae and other nontuberculous mycobacteria were recovered from several sources of water in Office A, as well as in another ENT office (Office B) in a neighboring city that was visited by the index patient. Only one additional case was detected in Office B during the same period. Otologic instruments in Office A were cleaned in an ultrasonic bath with tap water and a liquid detergent; the contents of the bath were changed only once weekly. Instruments in Office B were placed in boiling water between patient examinations. This outbreak establishes M. chelonae as an agent of otitis media and underscores the need for high-level disinfection or sterilization of ENT instruments between examinations to prevent the transmission of this organism to patients in the office setting.

Most media used for susceptibility testing contain sulfonamide inhibitors that make them unacceptable for testing sulfonamides. The major substance that inhibits sulfonamides has been identified as thymidine, and recent efforts to remove it from Mueller-Hinton medium have made it possible to perform susceptibility tests with sulfamethoxazole-trimethoprim by broth microdilution. Some lots of Mueller-Hinton broth are thymidine free, but some contain small amounts of thymidine and require the addition of thymidine phosphorylase or lysed horse blood which contains thymidine phosphorylase. Some lots may contain too much thymidine so that its activity cannot be reversed by adding the recommended amont of thymidine phosphorylase. Therefore the suitability of a medium must be determined by testing with control strains. Some organism, for example enterococci, cannot be tested in thymidine phosphorylase-treated media because thymine works in the same way thymidine does to inhibit the action of sulfamethoxazole-trimethoprim.

The name Providencia heimbachae sp. nov. is proposed for a group of organisms that were isolated from the feces of penguins in the Federal Republic of Germany and from a cow in the United States. P. heimbachae strains are gram-negative, oxidase-negative, fermentative, rod-shaped organisms that grow on MacConkey agar, as well as other media that are selective for members of the Enterobacteriaceae. The 13 P. heimbachae isolates which we studied gave negative results for the following tests: indole production, Voges-Proskauer, Simmons citrate, H2S production on triple sugar iron and Kligler agars, urease, lysine decarboxylase, arginine dihydrolase, gelatinase, growth in the presence of KCN, malonate, acid production from L-arabinose, cellobiose, dulcitol, erythritol, lactose, melezitose, melibiose, a-methylglucoside, raffinose, salicin, D-sorbitol, L-sorbose, starch, D-tagatose, trehalose, D-turanose, and D-xylose (15% positive after 7 days), esculin hydrolysis, acetate, lipase, deoxyribonuclease, and o-nitrophenyl-β-D-galactopyranoside. The strains were positive for the following tests: catalase, nitrate reductase, methy1 red, phenylalanine deaminase, acid production from D-glucose, adonitol, D-arabitol, D-fructose, D-galactose, D-mannose, and L-rhamnose, and tyrosine clearing. Acid production from glycerol, myo-inositol, maltose, and D-mannitol was delayed; gas was produced from D-glucose, D-galactose, and D-mannose in small amounts or not at all (54% positive after 7 days). Motility at 36°C was variable (46% positive after 2 days; 85% positive after 7 days). Deoxyribonucleic acids from 12 other strains of P. heimbachae were highly related (91 to 100% related in reactions assayed on hydroxyapatite at 60 and 75°C) to 32P-labeled deoxyribonucleic acid from the proposed type strain (strain ATCC 35613). Labeled deoxyribonucleic acid from this type strain was 22 to 45% related at 60°C and 4 to 31% related at 75°C to deoxyribonucleic acids from four other Providencia species. The levels of relatedness of P. heimbachae to Proteus species ranged from 10 to 13% in 60°C reactions. P. heimbachae could be differentiated from the other Providencia species by the following characteristics: negative tests for Simmons citrate, urease, indole production, and acid production from trehalose and positive tests for acid production from adonitol, D-arabitol, D-galactose, and L-rhamnose. All of the P. heimbachae strains which we studied were resistant to tetracycline, and most strains were resistant to cephalothin.

Nucleotide sequences homologous to the transposon-specific region and the tetM determinant mediating tetracycline resistance of the multiresistance transposon Tn1545, originating in Streptococcus pneumoniae, were detected by hybridization in the first multiresistant pneumococcus reported in the United States. The sequences were localized to a common 25-kb EcoRI restriction fragment of the chromosome.