Disk Diffusion Antimicrobial Susceptibility Testing Research Articles

EUCAST evaluation of 21 brands of Mueller–Hinton dehydrated media for disc diffusion testing

ObjectivesMueller–Hinton (MH) agar is recommended by EUCAST and CLSI for disc diffusion antimicrobial susceptibility testing. Using EUCAST methodology, we evaluated the performance of 21 internationally available brands of dehydrated MH agar from 17 manufacturers. MethodsMH plates were prepared in-house and evaluated against four quality control (QC) strains tested in triplicate, using EUCAST disc diffusion methodology. This resulted in 30 disc–QC strain combinations and 90 readings per MH brand. All brands were tested blindly and in parallel. Results were evaluated against targets and ranges in the EUCAST QC tables. The agar depth, pH and concentration of five cations were measured for all brands. ResultsSix brands of MH agar (Bio-Rad, Biolife, Oxoid, Sigma MH 2, BD BBL MH II and CRITERION) demonstrated excellent performance, with ≥99% of zone diameter readings within QC ranges and ≥70% on target ±1 mm. The poorest performance was seen for Biolab and Merck MH, with 10% (9/90) and 23% (21/90) of readings outside the QC ranges, respectively. Of all readings, 4.9% (93/1890) were out of range, mainly related to trimethoprim sulfamethoxazole (n = 25), aminoglycosides (n = 25) and fluoroquinolones (n = 15). The cation content differed considerably between the agars, and for four brands pH values were outside the acceptable range 7.2–7.4. DiscussionsThis study evaluated the performance and content of 21 brands of MH dehydrated media. Six brands showed excellent performance with all investigated antimicrobial classes. Others exhibited problems with one or more classes of agents. This could partly be explained by differences in concentration of specific chemical components and pH.

Breakpoint beware: reliance on historical breakpoints for Enterobacteriaceae leads to discrepancies in interpretation of susceptibility testing for carbapenems and cephalosporins and gaps in detection of carbapenem-resistant organisms.

Carbapenem-resistant Enterobacteriaceae (CRE) are an important public health and infection prevention threat. CRE are typically detected via phenotypic antimicrobial susceptibility testing (AST), for which interpretive standards were modified in recent years. Our objective was to measure the impact of breakpoint changes on AST interpretation for CRE. Zone sizes from disk diffusion AST for Enterobacteriaceae isolates recovered from clinical cultures over a 1-year period (n = 10,183) and CRE from clinical and environmental sources from the USA and Pakistan (n = 342) were evaluated. Results were interpreted according to historical (CLSI M100-S19) and current (CLSI M100-S29) breakpoints. Interpretive errors were calculated according to the FDA definitions. Using current breakpoints as the reference standard, 56 (17%) very major (false susceptibility) errors occurred for cefepime and 13 (45%) very major errors for meropenem interpretation using historical breakpoints in clinical isolates of Enterobacteriaceae, corresponding to 12 carbapenemase-producing CRE that would have been missed during the 1-year period. For confirmed blaKPC CP-CRE clinical and environmental isolates (n = 149), the very major error rate for historic breakpoints was 8%, 30%, 63%, and 0% for cefepime, meropenem, imipenem, and ertapenem, respectively. For blaKPC isolates, the use of historical breakpoints would have led to 42 (28%) reports of false susceptibility to meropenem. Failure to adopt updated AST breakpoints may lead to reports of false susceptibility for antimicrobials commonly used to treat Gram-negative infections and preclude recognition of CRE. Such errors could negatively impact patient care and hamper infection control and public health efforts.

The potential of fosfomycin for multi-drug resistant sepsis: an analysis of in vitro activity against invasive paediatric Gram-negative bacteria.

Antimicrobial resistance (AMR) is of increasing global concern, threatening to undermine recent progress in reducing child and neonatal mortality. Repurposing older antimicrobials is a prominent strategy to combat multidrug-resistant sepsis. A potential agent is fosfomycin, however, there is scarce data regarding its in vitro activity and pharmacokinetics in the paediatric population. We analysed a contemporary, systematically collected archive of community-acquired (CA) and hospital-acquired (HA) paediatric Gram-negative bacteraemia isolates for their susceptibility to fosfomcyin. MICs were determined using agar serial dilution methods and validated by disk diffusion testing where breakpoints are available. Disk diffusion antimicrobial susceptibility testing was also conducted for current empirical therapies (ampicillin, gentamicin, ceftriaxone) and amikacin (proposed in the literature as a new combination empirical therapeutic option). Fosfomycin was highly active against invasive Gram-negative isolates, including 90 % (202/224) of Enterobacteriaceae and 96 % (22/23) of Pseudomonas spp. Fosfomycin showed high sensitivity against both CA isolates (94 %, 142/151) and HA isolates (81 %, 78/96; P =0.0015). CA isolates were significantly more likely to be susceptible to fosfomycin than the current first-line empirical therapy (96 % vs 59 %, P <0.0001). Extended spectrum β-lactamases (ESBL) production was detected in 34 % (85/247) of isolates with no significant difference in fosfomycin susceptibility between ESBL-positive or -negative isolates [73/85 (86 %) vs 147/162 (91 %) respectively, P =0.245]. All isolates were susceptible to a fosfomycin-amikacin combination. Gram-negative paediatric bacteraemia isolates are highly susceptible to fosfomycin, which could be combined with aminoglycosides as a new, carbapenem-sparing regimen to achieve excellent coverage to treat antimicrobial-resistant neonatal and paediatric sepsis.

Identification of Subsets of Enteroaggregative Escherichia coli Associated with Diarrheal Disease among Under 5 Years of Age Children from Rural Gambia.

.Enteroaggregative Escherichia coli (EAEC) cause acute and persistent diarrhea, mostly in children worldwide. Outbreaks of diarrhea caused by EAEC have been described, including a large outbreak caused by a Shiga toxin expressing strain. This study investigated the association of EAEC virulence factors with diarrhea in children less than 5 years. We characterized 428 EAEC strains isolated from stool samples obtained from moderate-to-severe diarrhea cases (157) and healthy controls (217) children aged 0–59 months recruited over 3 years as part of the Global Enteric Multicenter Study (GEMS) in The Gambia. Four sets of multiplex polymerase chain reaction were applied to detect 21 EAEC-virulence genes from confirmed EAEC strains that target pCVD432 (aatA) and AAIC (aaiC). In addition, Kirby-Bauer disc diffusion antimicrobial susceptibility testing was performed on 88 EAEC strains following Clinical Laboratory Standard Institute guidelines. We observed that the plasmid-encoded enterotoxin [odds ratio (OR): 6.9, 95% confidence interval (CI): 2.06–29.20, P < 0.001], aggregative adherence fimbriae/I fimbriae (aggA) [OR: 2.2, 95% CI: 1.16–4.29, P = 0.008], and hexosyltransferase (capU) [OR: 1.9, 95% CI 1.02–3.51, P = 0.028] were associated with moderate-to-severe diarrhea among children < 12 months old but not in the older age strata (> 12 months). Our data suggest that some EAEC-virulent factors have age-specific associations with moderate-to-severe diarrhea in infants. Furthermore, our study showed that 85% and 72% of EAEC strains tested were resistant to sulphamethoxazole-trimethoprim and ampicillin, respectively. Sulphamethoxazole-trimethoprim and ampicillin are among the first-line antibiotics used for the treatment of diarrhea in The Gambia.

Can Ceftazidime-Avibactam and Aztreonam Overcome β-Lactam Resistance Conferred by Metallo-β-Lactamases in Enterobacteriaceae?

Based upon knowledge of the hydrolytic profile of major β-lactamases found in Gram-negative bacteria, we tested the efficacy of the combination of ceftazidime-avibactam (CAZ-AVI) with aztreonam (ATM) against carbapenem-resistant enteric bacteria possessing metallo-β-lactamases (MBLs). Disk diffusion and agar-based antimicrobial susceptibility testing were initially performed to determine the in vitro efficacy of a unique combination of CAZ-AVI and ATM against 21 representative Enterobacteriaceae isolates with a complex molecular background that included blaIMP, blaNDM, blaOXA-48, blaCTX-M, blaAmpC, and combinations thereof. Time-kill assays were conducted, and the in vivo efficacy of this combination was assessed in a murine neutropenic thigh infection model. By disk diffusion assay, all 21 isolates were resistant to CAZ-AVI alone, and 19/21 were resistant to ATM. The in vitro activity of CAZ-AVI in combination with ATM against diverse Enterobacteriaceae possessing MBLs was demonstrated in 17/21 isolates, where the zone of inhibition was ≥21 mm. All isolates demonstrated a reduction in CAZ-AVI agar dilution MICs with the addition of ATM. At 2 h, time-kill assays demonstrated a ≥4-log10-CFU decrease for all groups that had CAZ-AVI with ATM (8 μg/ml) added, compared to the group treated with CAZ-AVI alone. In the murine neutropenic thigh infection model, an almost 4-log10-CFU reduction was noted at 24 h for CAZ-AVI (32 mg/kg every 8 h [q8h]) plus ATM (32 mg/kg q8h) versus CAZ-AVI (32 mg/kg q8h) alone. The data presented herein require us to carefully consider this new therapeutic combination to treat infections caused by MBL-producing Enterobacteriaceae.

Renaissance of Conventional First-Line Antibiotics in Salmonella enterica Clinical Isolates: Assessment of MICs for Therapeutic Antimicrobials in Enteric Fever Cases from Nepal.

Enteric fever caused by Salmonella enterica is a life-threatening systemic illness of gastrointestinal tract especially in tropical countries. Antimicrobial therapy is generally indicated but resistance towards commonly used antibiotics has limited their therapeutic usefulness. Therefore, we aimed to determine the antimicrobial susceptibility pattern by minimum inhibitory concentration method of common therapeutic regimens against Salmonella enterica from enteric fever clinical cases. Salmonella enterica clinical isolates recovered from the patients with suspected enteric fever whose blood samples were submitted to microbiology laboratory of Manmohan Memorial Community Hospital, Kathmandu, from March 2016 to August 2016, were studied. These isolates were subjected to antimicrobial susceptibility testing against common therapeutic antimicrobials by Kirby-Bauer disk diffusion method. The minimum inhibitory concentration of ciprofloxacin, azithromycin, chloramphenicol, and cefixime was determined by Agar dilution method based on the latest CLSI protocol. A total of 88 isolates of Salmonella enterica were recovered from blood samples of enteric fever cases. Out of them, 74 (84.09%) were Salmonella Typhi and 14 (15.91%) were Salmonella Paratyphi A. On Kirby-Bauer disk diffusion antimicrobial susceptibility testing, entire isolates were susceptible to cotrimoxazole, cefixime, ceftriaxone, azithromycin, and chloramphenicol. Sixty-four (72.7%) Salmonella enterica isolates were nalidixic acid resistant and nonsusceptible to ciprofloxacin and levofloxacin. On MIC determination, four Salmonella isolates were ciprofloxacin resistant with MIC 1 µg/ml and two isolates were ciprofloxacin intermediate with MIC 0.5 µg/ml. The MIC range of azithromycin was from 0.125 µg/ml to 2.0 µg/ml, whereas that for chloramphenicol was 2.0 µg/ml–8.0 µg/ml and for cefixime was 0.0075–0.5 µg/ml, respectively. Despite global surge of antimicrobial resistance among Salmonella enterica clinical isolates, the level of drug resistance in our study was not so high. However, higher level of NARST strains limits therapeutic use of fluoroquinolones and necessitates the routine monitoring of such resistance determinants in order to effectively and rationally manage enteric fever cases.

Enteric Fever Caused by Salmonella enterica Serovars with Reduced Susceptibility of Fluoroquinolones at a Community Based Teaching Hospital of Nepal.

Enteric fever continues to be an important public health problem especially in developing countries of the tropical region including Nepal. In this study, we aimed to investigate the incidence of enteric fever associated with Salmonella enterica and determine its antimicrobial susceptibilities to therapeutic antimicrobials in a community based teaching hospital of Nepal. A total of 2,304 blood samples from suspected enteric fever patients attending Manmohan Memorial Teaching Hospital were processed with standard microbiological methods for the isolation and identification of bacterial pathogens. The Salmonella enterica clinical strains were subjected to antimicrobial susceptibility testing by Kirby–Bauer disk diffusion method, and the results were interpreted according to the criteria suggested by the Clinical and Laboratory Standards Institute (CLSI). A total of 245 (10.6%) cases of enteric fever associated with Salmonella enterica were confirmed by blood culture. Out of them, 162 (66.1%) were caused by Salmonella Typhi and 83 (33.9%) by Salmonella Paratyphi. On Kirby–Bauer disk diffusion antimicrobial susceptibility testing, Salmonella isolates were highly susceptible to cefixime (100%), ceftriaxone (100%), ampicillin (97.9%), cotrimoxazole (94.6%), azithromycin (96.7%), tetracycline (95.5%), and chloramphenicol (97.5%), respectively. Two hundred twenty-six (92.2%) of Salmonella isolates were nalidixic acid resistant with reduced susceptibility to ciprofloxacin (36.7%) and ofloxacin (54.8%), respectively. Although the rate of MDR Salmonella strains was very low (<5%), their reduced susceptibility to fluoroquinolones has restricted their routine empirical use. Third generation cephalosporins are the safest choice for empirical use but ampicillin, cotrimoxazole, azithromycin, and chloramphenicol can be effective alternatives.

Evaluation of an Automated System for Reading and Interpreting Disk Diffusion Antimicrobial Susceptibility Testing of Fastidious Bacteria.

Results of disk diffusion antimicrobial susceptibility testing depend on individual visual reading of inhibition zone diameters. Therefore, automated reading using camera systems might represent a useful tool for standardization. In this study, the ADAGIO automated system (Bio-Rad) was evaluated for reading disk diffusion tests of fastidious bacteria. 144 clinical isolates (68 β-haemolytic streptococci, 28 Streptococcus pneumoniae, 18 viridans group streptococci, 13 Haemophilus influenzae, 7 Moraxella catarrhalis, and 10 Campylobacter jejuni) were tested on Mueller-Hinton agar supplemented with 5% defibrinated horse blood and 20 mg/L β-NAD (MH-F, Oxoid) according to EUCAST. Plates were read manually with a ruler and automatically using the ADAGIO system. Inhibition zone diameters, indicated by the automated system, were visually controlled and adjusted, if necessary. Among 1548 isolate-antibiotic combinations, comparison of automated vs. manual reading yielded categorical agreement (CA) without visual adjustment of the automatically determined zone diameters in 81.4%. In 20% (309 of 1548) of tests it was deemed necessary to adjust the automatically determined zone diameter after visual control. After adjustment, CA was 94.8%; very major errors (false susceptible interpretation), major errors (false resistant interpretation) and minor errors (false categorization involving intermediate result), calculated according to the ISO 20776–2 guideline, accounted to 13.7% (13 of 95 resistant results), 3.3% (47 of 1424 susceptible results) and 1.4% (21 of 1548 total results), respectively, compared to manual reading. The ADAGIO system allowed for automated reading of disk diffusion testing in fastidious bacteria and, after visual validation of the automated results, yielded good categorical agreement with manual reading.

An Overview of Mastitis Management and Therapy in Dairy Goats

An Overview of Mastitis Management and Therapy in Dairy Goats This study surveys dairy goat practices, and determines the bacterial pathogens responsible for goat mastitis from samples submitted to the Hill Farm Mastitis Laboratory. Goat milk samples (4,490) for analysis were submitted frozen to the laboratory from 67 farms across the southern United States. All organisms were identified using standard microbiological procedures. Staphylococcus species were identified using the API STAPH-TRAC System. Disk diffusion antimicrobial susceptibility testing, and susceptible or resistant determinations were performed following the guidelines established by the Clinical Laboratory Standards Institute. A total of 4,490 milk samples resulted in 1,033 bacterial isolates for evaluation.

Extraintestinal Pathogenic and Antimicrobial-Resistant Escherichia coli Contamination of 56 Public Restrooms in the Greater Minneapolis-St. Paul Metropolitan Area

How extraintestinal pathogenic Escherichia coli (ExPEC) and antimicrobial-resistant E. coli disseminate through the population is undefined. We studied public restrooms for contamination with E. coli and ExPEC in relation to source and extensively characterized the E. coli isolates. For this, we cultured 1,120 environmental samples from 56 public restrooms in 33 establishments (obtained from 10 cities in the greater Minneapolis-St. Paul, MN, metropolitan area in 2003) for E. coli and compared ecological data with culture results. Isolates underwent virulence genotyping, phylotyping, clonal typing, pulsed-field gel electrophoresis (PFGE), and disk diffusion antimicrobial susceptibility testing. Overall, 168 samples (15% from 89% of restrooms) fluoresced, indicating presumptive E. coli: 25 samples (2.2% from 32% of restrooms) yielded E. coli isolates, and 10 samples (0.9% from 16% of restrooms) contained ExPEC. Restroom category and cleanliness level significantly predicted only fluorescence, gender predicted fluorescence and E. coli, and feces-like material and toilet-associated sites predicted all three endpoints. Of the 25 E. coli isolates, 7 (28%) were from phylogenetic group B2(virulence-associated), and 8 (32%) were ExPEC. ExPEC isolates more commonly represented group B2 (50% versus 18%) and had significantly higher virulence gene scores than non-ExPEC isolates. Six isolates (24%) exhibited ≥3-class antibiotic resistance, 10 (40%) represented classic human-associated sequence types, and one closely resembled reference human clinical isolates by pulsed-field gel electrophoresis. Thus, E. coli, ExPEC, and antimicrobial-resistant E. coli sporadically contaminate public restrooms, in ways corresponding with restroom characteristics and within-restroom sites. Such restroom-source E. coli strains likely reflect human fecal contamination, may pose a health threat, and may contribute to population-wide dissemination of such strains.

Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories

With the support of ESCMID and European countries, EUCAST has developed a disk diffusion test with zone diameter breakpoints correlated with the EUCAST clinical MIC breakpoints. The development of the EUCAST disk diffusion method and quality control criteria are described, together with guidance on quality control and implementation of the method in clinical microbiology laboratories. The method includes the use of Mueller–Hinton agar without supplements for non-fastidious organisms and with 5% mechanically defibrinated horse blood and 20 mg/L β-NAD for fastidious organisms, a standardized inoculum resulting in confluent growth, an incubation time of 16–20 h, a reading guide on how to read zone diameters on individual species-agent combinations and zone diameter breakpoints calibrated to the EUCAST clinical MIC breakpoints. EUCAST recommendations are described in detail and updated regularly on the EUCAST website (http://www.eucast.org).

A comparison of Neo-Sensitabs™ tablets and paper discs in disc diffusion antimicrobial susceptibility testing

A comparative evaluation of Neo-SensiitabsTM (Rosco, Taastrup, Denmark) and BBL paper discs (Becton Dickinson, Cockeysville, MD) according to the CLSI Performance Standards for Antimicrobial Susceptibility Testing was done. A total of 303 clinical isolates were included: 60 Enterobacteriaceae (including ESBLs isolates), 63 Pseudomonas aeruginosa, 63 Staphylococcus aureus, 33 Streptococcus pneumoniae, 51 Enterococcus faecalis and 33 Beta haemolytic streptococci. Strains were tested using the disc diffusion method, including both discs and tablets. minimal inhibitory concentrations (MICs) were determined by E test (AB Biodisk, Solna, Sweden). The results were analyzed by linear regression and Pearson's correlation coefficient. The Pearson correlation coefficient of the inhibition zone diameter, related to the discs and tablets, had a high value (0.824 to 0.998). Based on the CLSI categorization of antimicrobial susceptibility (S-I-R), agreement was found within the range of 78.79 to 100%. Overall percentage of 3.31% of minor error and 0.38% of major error was observed. All of the 12 major errors occurred when there was no intermediate category. The major errors were found within isolates with SR discrepancies, and minor errors (1,64/1,67% for Neosensitabs and BBL, respectively) within isolates with IS-IR discrepancies. Antibiotic tablet sensitivity for ESBL detection was 97.14% compared to paper discs. Neo-Sensitabs™ tablets showed a high inter-correlation with BBL paper discs which indicated the possibility of using tablets as an alternative to paper discs. Key words: Disc diffusion, paper discs, Neo-Sensitabs™ tablets.

Comparison of Rosco Neo-Sensitabs with Oxoid paper disks in EUCAST disk diffusion antimicrobial susceptibility testing on Mueller–Hinton agar

This study compared Neo-Sensitabs with Oxoid paper disks using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) disk diffusion antimicrobial susceptibility test on Mueller-Hinton agar. The EUCAST-recommended quality control strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212) (Part I) and clinical isolates (Part II) were investigated. In Part I of the study, 27 combinations of antimicrobial agents were tested on four quality control strains repeatedly up to 60 times and zone diameters of tablets and disks were compared. In Part II of the study, 351 clinical isolates were included to cover a broad range of species, as well as resistance mechanisms. In Part I, four major deviations (>1 mm outside quality control ranges) were observed with Neo-Sensitabs. In one case with P. aeruginosa ATCC 27853 (meropenem), there was a corresponding major deviation (2 mm) with the Oxoid disk. The three remaining major deviations with Neo-Sensitabs were observed with meropenem (2 mm) in E. coli ATCC 25922 and with ciprofloxacin (2 mm) and gentamicin (3 mm) in P. aeruginosa ATCC 27853. For Oxoid disks, there were only minor deviations (=1 mm outside quality control ranges) in these three cases. In Part II, there were six discrepancies, susceptible versus resistant, in 3,533 comparisons between the two methods with the clinical isolates. The Rosco Neo-Sensitabs appear to be a possible alternative to Oxoid paper disks for EUCAST disk diffusion antimicrobial susceptibility testing on Mueller-Hinton agar.

Antimicrobial susceptibility and synergy studies of cystic fibrosis sputum by direct sputum sensitivity testing

Standard disc diffusion antimicrobial susceptibility testing (C+S) on individual Pseudomonas aeruginosa colonial morphotypes cultured from cystic fibrosis (CF) sputum has questionable clinical relevance. Direct sputum sensitivity testing (DSST) is a whole-sputum susceptibility test that removes bias associated with selecting individual colonial morphotypes. We sought to determine whether, in principle, the results from DSST support the possibility of improved clinical relevance compared with C+S. Individual (DSSTi) and combination (DSST) susceptibility to gentamicin, tobramycin, ceftazidime and meropenem were determined on 130 sputum samples referred from CF subjects with antibiotic-resistant chronic Gram-negative endobronchial infection. DSSTi and concurrent C+S were compared for categorical susceptibility, synergistic combinations were evaluated and the combination DSST efficacy index (DEI) calculated. Meropenem and tobramycin were the most active individual antibiotics by DSSTi on 89 P. aeruginosa-predominant samples, with 62% of samples sensitive to each. C+S and DSSTi showed poor agreement (κ ranging from 0.02 to 0.6), discordance ranging from 20% (meropenem) to 49% (tobramycin), with DSSTi demonstrating both increased susceptibility and increased resistance. The combination that most frequently had the highest DEI was tobramycin + meropenem, occurring in 76% of samples. DSSTi appears to be reproducible, yields different antimicrobial susceptibility results from C+S without simply identifying the most resistant isolates and DSST identifies the most effective in vitro antibiotic combinations, providing preliminary proof of concept of the potentially improved clinical relevance of whole-sputum testing. Future studies will determine whether these potential theoretical advantages translate into clinical benefits.

Gastroenteritis in US Marines during Operation Iraqi Freedom

Approximately 83,000 US Marines participated in the opening phase of Operation Iraqi Freedom in Spring 2003. A Navy Preventive Medicine laboratory was set up in Ad Diwaniyah, Iraq, to provide clinical diagnostic support for Marine medical units during a period of repositioning in south-central Iraq. Specimen collection boxes were sent to >30 primary care medical stations handling 500-900 personnel each. The laboratory had capability to detect many different disease agents, especially those causing febrile illness. Diarrheal stool diagnostic evaluation included plating and biochemical identification, antigen serologic testing, fluorescent antibody antigen detection, disk diffusion antimicrobial susceptibility testing, enzyme immunoassay, and reverse-transcriptase polymerase chain reaction for norovirus (NV). Confirmation and sequencing work for NV was done at Cincinnati Children's Hospital Medical Center (Ohio). By far the most common reason for infectious disease sick call visits was gastrointestinal illness; no other symptoms had equivalent impact. An enteropathogen was detected in 57 (44%) of 129 stool samples, with NV detected in 30 stool samples (23%) obtained from 14 different battalion or similar-sized units; next in frequency were Shigella flexneri and Shigella sonnei, which were isolated from 26 stool samples (20%) obtained from 15 units. Sequencing the NV RNA polymerase gene demonstrated that NV strains represented 7 genetic clusters, including 2 strains from genogroup I and 5 from genogroup II. Ciprofloxacin was effective in vitro against most bacterial agents, but neither doxycyline (which was taken daily as the antimalarial prophylaxis dose) nor trimethoprim-sulfamethoxazole were effective. Multiple strains of Shigella species and NV predominated, probably because they do not require a large inoculum to cause infection. Otherwise, personnel remained free of infectious illness during this phase of the conflict, because other infectious agents were rare or absent.

Development of interpretive criteria and quality control limits for broth microdilution and disk diffusion antimicrobial susceptibility testing of Streptococcus pneumoniae.

A five-center collaborative study was undertaken to develop quality control and specific interpretive criteria for susceptibility testing of Streptococcus pneumoniae against 12 antimicrobial agents. MICs were determined for 248 pneumococcal clinical isolates (with an emphasis on resistant strains) by use of the National Committee for Clinical Laboratory Standards (NCCLS)-recommended broth microdilution procedure incorporating lysed horse blood-supplemented Mueller-Hinton broth. NCCLS disk diffusion testing was also performed for each isolate by using Mueller-Hinton sheep blood agar incubated in 5% CO2. Repetitive testing of S. pneumoniae ATCC 49619 with different sources and lots of media and disks allowed development of quality control ranges which encompassed approximately 95% of MIC and zone size values observed in the study. Good intra- and interlaboratory reproducibilities were seen with these testing methods and all of the drugs examined. On the basis of the results of this study, MIC interpretive criteria are proposed for 11 agents. Comparisons of MICs and disk diffusion zone sizes allowed disk diffusion zone size interpretive criteria to be proposed for five drugs and confirmed the use of the oxacillin disk test for prediction of penicillin susceptibility among pneumococci. Excessive numbers of minor-category interpretive errors precludes recommendation at this time of the disk diffusion method for testing of pneumococci against five of the drugs. Use of these proposed quality control and interpretive criteria should provide for reproducible test results and allow recognition of recently emerging resistance among pneumococcal clinical isolates.

Antimicrobial susceptibility testing of Neisseria gonorrhoeae and implications for epidemiology and therapy.

Antimicrobial susceptibility testing (AST) of Neisseria gonorrhoeae has been under development since the early days of antimicrobial agents. However, it is rarely applied to clinical isolates today. The history of the various in vitro tests to determine the susceptibility of N. gonorrhoeae to antibiotics is rich with evidence that these results predict response to therapy for almost all agents tested. Further, AST is a useful and important aspect of strain characterization and disease epidemiology in conjunction with the more specific but laborious techniques of auxotyping, serotyping, and plasmid analysis. Current technology has overcome many of the objections to AST for N. gonorrhoeae with standardization of test media and the development of an accurate disk diffusion AST method that is suited to most clinical laboratories regardless of volume or level of technical expertise. Ironically, the very low level of resistance to the current primary treatment strategy in the United States, ceftriaxone or another potent cephalosporin, makes the use of AST somewhat superfluous.

Antimicrobial susceptibility of clinical isolates of Haemophilus influenzae to ampicillin-sulbactam

A total of 1092 clinical isolates of Haemophilus influenzae (306 type b; 786 non-type-b), from five medical centers were obtained during 1987 and 1988. Disk diffusion antimicrobial susceptibilities were obtained for all isolates, and broth microdilution susceptibilities were obtained for 502 isolates. Beta-lactamase was produced by 34.3% of type-b and 22.1% of non-type-b isolates, with some geographic variations. Using disk diffusion antimicrobial susceptibility testing, all isolates were susceptible to ampicillin-sulbactam, ceftriaxone, cefuroxime, and rifampin; two isolates were resistant to chloramphenicol. Whether tested using a fixed ratio of ampicillin to sulbactam of 2:1 or a fixed concentration of sulbactam, the ampicillin-sulbactam combination demonstrated good activity against clinical isolates of H. influenzae. Only 8 of the 1092 isolates did not produce beta-lactamase but demonstrated MICs of greater than or equal to 2 micrograms/ml for ampicillin.

Antimicrobial susceptibility testing of less commonly isolated Haemophilus species using Haemophilus test medium

Haemophilus test medium (HTM) was developed recently for dilution and disk diffusion antimicrobial agent susceptibility testing of Haemophilus influenzae. The application of HTM to the testing of other, less frequently encountered Haemophilus species recovered from humans was evaluated in this study by using commercially prepared HTM (BBL Microbiology Systems, Cockeysville, Md.) in broth microdilution and agar disk diffusion susceptibility tests with 18 antimicrobial agents. A total of 93.3% of 90 isolates belonging to six Haemophilus species provided acceptable growth in HTM agar disk diffusion tests, while only 63.3% (57 of 90) provided acceptable growth in the broth microdilution tests. However, HTM agar dilution testing provided an alternative method for those strains (primarily H. haemolyticus) which failed to grow adequately in broth. Based on the latest National Committee for Clinical Laboratory Standards guidelines (standard M2-T4) for interpretation of HTM disk tests of H. influenzae, the overall very major, major, and minor errors for all 18 drugs and six species tested were 0.2, 0.7, and 3.4%, respectively. Thus, the use of HTM in agar or broth susceptibility tests can be recommended for testing the less commonly encountered Haemophilus species by using the same test conditions and interpretive guidelines developed for H. influenzae.

Antimicrobial susceptibility testing of Haemophilus influenzae Improvement of accuracy of the disc diffusion test

A national quality control study was performed in 1986 to investigate the standard of performance of the disc diffusion antimicrobial susceptibility testing of Haemophilus influenzae in Sweden. The accuracy of susceptibility interpretations was unacceptably low. A new standardized method for susceptibility testing of H. influenzae was then worked out, and a new method of setting interpretive zone breakpoints was introduced. The susceptibility category of the main population of clinical isolates was determined according to the MIC50 of the strains. The zone histograms of clinical isolates from five reference laboratories were used for the calculation of interpretive breakpoints. For instance, for susceptible strains the mean of the combined zone values from these laboratories +/- 2 S.D. covered the zone range of the susceptible group, and one more S.D. below covered the intermediate/indeterminate group. The new zone breakpoints would place the main population of clinical isolates in the correct susceptibility group, and even make it possible to detect strains with different degrees of reduced susceptibility in the routine test. In a follow-up quality control study in 1988 the interpretive errors for clinical isolates were eliminated for all antibiotics except doxycycline in some laboratories. Laboratory-related zone breakpoints for doxycycline calculated by the single strain regression analysis method led to correct susceptibility interpretations also in these cases.