Susceptibility Testing Of Haemophilus Influenzae Research Articles

Ampicillin susceptibility testing of Haemophilus influenzae in the routine clinical laboratory by the EUCAST methodology compared to broth microdilution and the presence of ftsI gene mutations

ObjectivesTo investigate the prevalence of ampicillin resistance in Haemophilus influenzae and the diagnostic accuracy of the EUCAST recommended disc diffusion method to detect the increasingly prevalent ampicillin resistance due to the presence of PBP3 alterations based on mutations in the ftsI gene. MethodsDuring a 6-month period all consecutive non-duplicate H. influenzae isolates were prospectively collected and stored. MICs of ampicillin were determined by broth microdilution (BMD). PCR was performed to detect mutations in the ftsI gene. Results of routine disc diffusion susceptibility testing, including the penicillin screening test in accordance with the current EUCAST methodology, as well as additional Etest results, were compared to the BMD as the reference method. ResultsIn 102 isolates, the prevalence of ampicillin resistance was 28% (29/102) by BMD. There was a good correlation between MICs of ampicillin and the presence of a β-lactamase and/or an ftsI gene mutation. The prevalence of ampicillin resistance was overestimated using the EUCAST method (33% (34/102)) and underestimated when an additional Etest was used (24% (24/102)) (not significant). The sensitivity and specificity of the EUCAST methodology for the detection of ampicillin resistance were 97% ((28/29); 95% CI, 82–100%) and 92% ((67/73); 95% CI, 83–97%), respectively. ConclusionsThe prevalence of ampicillin resistance was 28%, as determined by BMD. Although the overall diagnostic accuracy of the EUCAST ampicillin disc diffusion was high, misclassification of ampicillin susceptibility may still occur.

Isolation and Antimicrobial Susceptibility Testing of Haemophilus influenzae in a Tertiary Care Hospital, a 6-Year Study (2016 to 2021)

Objective: To determine the prevalence and antimicrobial susceptibility of Haemophilus influenzae at Taksin Hospital. Materials and Methods: The susceptibilities of clinical isolates to six antimicrobial agents, ampicillin (AP), cefuroxime (CXM), cefotaxime (CTX), ciprofloxacin (CIP), trimethoprim-sulfamethoxazole (SXT), and meropenem (MEM), between 2016 and 2021 were evaluated by the standard disk diffusion method. Results: One hundred thirty-eight H. influenzae clinical isolates were found in different patients. There were 63.04% male patients. The ages of patients ranged from two months to 93 years old (mean±SD: 65.67±20.58). H. influenzae was mostly found in patients older than 50 (60.14%) years old. In children, H. influenzae was mostly found in patients younger than 1 to 10 (16.67%) years old. Unduplicated H. influenzae from different patients were isolated from the sputum (71.01%), blood (11.59%), pus from eyes (10.14%), and urethra (2.90%). H. influenzae demonstrated 100% susceptibility to CTX, CIP, and MEM, but less susceptibility to CXM (93.48%), AP (49.28%), and SXT (53.62%). Conclusion: The present study results suggested the importance of monitoring the prevalence of H. influenzae at a hospital in Bangkok. The antibiogram of susceptibility helps provide guidelines for clinician to consider empirical treatment. Keywords: Haemophilus influenzae; Drug resistance, Thailand

Detection of N526K-substituted penicillin-binding protein 3 conferring low-level mutational resistance to -lactam antibiotics in Haemophilus influenzae by disc diffusion testing on Mueller-Hinton agar according to EUCAST guidelines

EUCAST has recently authorized a new disc diffusion test for routine antimicrobial susceptibility testing of Haemophilus influenzae, calibrated to EUCAST MIC breakpoints. We investigated whether disc diffusion testing as recommended by EUCAST could discriminate strains of H. influenzae carrying the N526K substitution in penicillin-binding protein 3 from the wild-type population. A total of 170 recent clinical isolates, genetically characterized for the presence of acquired and mutational resistance mechanisms, were tested by disc diffusion of β-lactam antibiotics on supplemented Mueller-Hinton agar. Tentative epidemiological breakpoint values for the presence of the N526K substitution were suggested for various β-lactams, and the performances were calculated. Epidemiological cut-off values of 19/20 mm for ampicillin (2 μg) and 11/12 mm for benzylpenicillin (1 U) accurately categorized 96% of the study strains, and outperformed cephalosporin-containing discs in the discrimination of mutational resistance in β-lactamase-non-producing isolates. Current EUCAST interpretative criteria for the categorization of clinical resistance showed concordance between resistance rates based on MIC and zone diameter breakpoints for both ampicillin and cefuroxime, but categorization of individual isolates was not consistent. Disc diffusion testing of H. influenzae accurately identified β-lactamase-non-producing isolates with the N526K substitution by use of discs containing low amounts of penicillins. Cephalosporin-containing discs could detect mutational resistance in β-lactamase-producing isolates, but performed with reduced specificity.

A report from the committee on microbroth dilution antimicrobial susceptibility testing

In 1968, the Japanese Society of Chemotherapy (JSC) established an agar dilution method for MIC determinations of aerobic bacteria [1]. In 1979, the JSC established an agar dilution method for MIC determinations of anaerobic bacteria. Furthermore, a revision of the agar dilution standard was made in 1980, and this served as the standard method for MIC determinations for an extensive period of time in Japan. In 1990, the committee established a standard microbroth dilution method for MIC determinations. This method was modified in 1992 for fastidious aerobic and anaerobic bacteria [2]. In the development of standards for antimicrobial susceptibility testing, there has been a movement to incorporate pharmacologic as well as clinical considerations with respect to in vitro antimicrobial susceptibility interpretations. This trend reflects the need to respond to the emergence and spread of resistant microorganisms as well as a greater understanding of pharmacokinetics and pharmacodynamics and how it relates to improved patient outcome. The Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility (EUCAST) are among the professional organizations that proactively revise antimicrobial susceptibility testing on a regular basis. JSC has also been revising its standards. In 2007, the agar dilution method was revised. After the exclusion of selected organisms from the CLSI agar dilution standard, this method was adopted by the JSC [3]. With respect to revising the standard method for microbroth dilution antimicrobial susceptibility testing, a committee chaired by Keizo Yamaguchi was formed in January 2008. The subcommittee’s first task was to critically examine the methods for the antimicrobial susceptibility testing of Haemophilus influenzae. Although JSC and CLSI microbroth dilution methods both use a Mueller– Hinton base, the supplements differ. This difference had raised concerns among some Japanese clinical microbiology professionals that there were many issues with the CLSI method with respect to reported difficulties in measuring endpoints and the poor growth of some strains. Our first action was to send questionnaires to the members of the JSC and the Japanese Society for Clinical Microbiology to better understand the technical problems experienced in clinical microbiology laboratories regarding microbroth dilution testing of H. influenzae. From the responses to the questionnaire, it was clear that a major problem in routine testing was the poor growth of Members of the Microdilution Susceptibility Standards Committee: committee chairperson: Keizo Yamaguchi, committee members: Yoichi Hirakata, Intetsu Kobayashi, Masanari Ikedo, Akira Ohno.

Disc diffusion susceptibility testing of Haemophilus influenzae by NCCLS methodology using low-strength ampicillin and co-amoxiclav discs

The objective of this multicentre study was to define the accuracy and reproducibility of the NCCLS disc diffusion method for Haemophilus influenzae against ampicillin and co-amoxiclav in Finnish clinical microbiology laboratories. Special attention was paid to the ability of the laboratories to detect beta-lactamase-negative ampicillin-resistant (BLNAR) strains. Three BLNAR and two beta-lactamase-negative ampicillin-susceptible isolates (BLNAS)-originating from the American Type Culture Collection (ATCC) and UK National External Quality Assessment (UKNEQAS) schemes-were included in this study. Susceptibility tests for these isolates were performed in 26 clinical microbiology laboratories, in accordance with NCCLS guidelines. Additionally, low-strength discs for ampicillin (2 microg) and co-amoxiclav (3 microg) were tested. The low-strength discs for ampicillin and co-amoxiclav categorized more accurately BLNAR and BLNAS H. influenzae isolates than did the high-strength discs recommended by the NCCLS. In addition, the high-strength discs produced more major errors than the low-strength discs (22 versus six for ampicillin and 40 versus seven for co-amoxiclav). Great variation occurred in the method regardless of the antibiotic concentration of the discs. The use of low-content ampicillin and co-amoxiclav discs is recommended for the susceptibility testing of H. influenzae. Interpretative criteria of S > or = 17 mm and R < or = 13 mm for both discs are suggested.

Evaluation of the WIDER I system for antimicrobial susceptibility testing of clinical isolates of Haemophilus influenzae and Streptococcus pneumoniae

The aim of this study was to evaluate the WIDER I system for susceptibility testing of Haemophilus influenzae and Streptococcus pneumoniae. MICs of 12 antimicrobials against 42 H. influenzae and 58 S. pneumoniae strains were determined using 1W MIC panels and compared with those obtained by microdilution. Overall essential agreements were >99%. Very major errors were not detected. Major errors occurred with ampicillin (1.7% H. influenzae). Minor errors were 2.3% (amoxicillin-clavulanate, cefuroxime, chloramphenicol), 7.1% (ampicillin) and 16.7% (clarithromycin) for H. influenzae, and 1.7% (chloramphenicol, erythromycin, meropenem), 3.4% (amoxicillin-clavulanate, cefuroxime, tetracycline) and 8.6% (levofloxacin) for S. pneumoniae. The WIDER I system is a reliable method for susceptibility testing of H. influenzae and S. pneumoniae.

Susceptibility testing of Haemophilus influenzae to clarithromycin.

Susceptibility testing of Haemophilus influenzae to the macrolide compounds has often been problematic. This is as a result of the inability of many isolates of H. influenzae to grow without the addition of 5% CO2 to the incubation atmosphere and the subsequent detrimental effect that CO2 has on the activity of the macrolide group of compounds. This report describes refinements and recommendations for susceptibility testing of H. influenzae to the macrolide clarithromycin.

Variability in susceptibilities of Haemophilus influenzae to clarithromycin and azithromycin due to medium pH.

The National Committee for Clinical Laboratory Standards (NCCLS) methods for susceptibility testing of Haemophilus influenzae in Haemophilus test medium allow a pH range of 7.2 to 7.4. However, it is known that bacteria may appear to be less susceptible to macrolides at lower pHs. Forty-four strains of H. influenzae were tested for their susceptibilities to clarithromycin and azithromycin by the disk diffusion and broth microdilution methods. The isolates appeared to be less susceptible at pH 7.2 than at pH 7.4 by both methods. Clarithromycin was less active at pH 7.2 against 43% of the isolates by the disk diffusion method and against 52% of the isolates by the broth microdilution method. Similarly, azithromycin was less active at pH 7.2 against 41 and 45% of the isolates by the disk diffusion and broth microdilution methods, respectively. Forty-two isolates were classified as clarithromycin susceptible and all isolates were classified as azithromycin susceptible by the disk diffusion method, regardless of the medium pH. However, only 21 isolates were clarithromycin susceptible at pH 7.2 and 34 isolates were susceptible at pH 7.4 by the broth microdilution method, even though quality control results indicated valid testing at both pHs. This study indicated that the results of tests of the susceptibility of H. influenzae with clarithromycin and azithromycin are highly dependent on the pH of the medium. Test results and their interpretations varied even when the medium pH was within the NCCLS-approved range and, coupled with the current NCCLS breakpoint of 8 microg/ml in the case of clarithromycin, may explain some of the observed discordances between the disk diffusion and broth microdilution methods.

Susceptibility testing of Haemophilus influenzae--an international collaborative study in quality assessment.

In order to compare the prevalence of antibiotic resistance in different geographical areas, it is necessary to ensure that agreement is achieved between laboratories on the assignment of strains to 'susceptible' and 'resistant' categories. An international quality assessment study, involving 15 laboratories in eight countries, was performed to investigate the standard of performance of the susceptibility testing of Haemophilus influenzae. One hundred and fifty strains of H. influenzae were distributed from the London Hospital Medical College (LHMC) to all laboratories who were asked to test the susceptibility of the strains to ampicillin, chloramphenicol, tetracycline, trimethoprim, cephalosporins and ciprofloxacin. Laboratories were also asked to provide the details of methodology to test the susceptibility. Significant discrepancy between the LHMC and the participating laboratories appeared in the detection of resistance to ampicillin (especially beta-lactamase-negative strains resistant to ampicillin) as well as the assignment of susceptibility and resistance to chloramphenicol, tetracycline and trimethoprim. Often these reflected the use of inappropriate breakpoints which led to erroneous assignment of susceptibility. Other variations including disc content, medium and supplement, inoculum as well as failure to measure zone sizes properly also led to some repeating anomalies.

Surveillance of susceptibility testing methodologies for Haemophilus influenzae in Canada, including evaluation of disk diffusion test.

We surveyed 75 clinical laboratories to determine if National Committee for Clinical Laboratory Standards (NCCLS) were being used for the susceptibility testing of Haemophilus influenzae. Of the 66 laboratories that performed susceptibility testing, all claimed to follow current NCCLS guidelines. However, upon further questioning, only 23, all of which used disk diffusion testing, accurately interpreted and followed the guidelines. Proficiency testing of 22 of these laboratories found that an unacceptable number of interpretive errors (> 10%) occurred. These results query the merit of routine disk diffusion susceptibility testing of H. influenzae to beta-lactam agents.

In vitro susceptibility test practices with Haemophilus influenzae among college of American Pathologists survey participants in the United States

Questionnaire results from 5233 clinical microbiology laboratories participating in the College of American Pathologists (CAP) survey program in the United States were used to establish current standards of practice with respect to in vitro susceptibility testing of Haemophilus influenzae. The results of this CAP survey indicated that the recently developed National Committee for Clinical Laboratory Standards (NCCLS) guidelines for H. influenzae susceptibility tests have been widely adopted, particularly with regard to the medium used to perform susceptibility tests. Haemophilus test medium (HTM) is now the most commonly used medium and there exists a general level of satisfaction (≈80%) with medium performance. Specific methodologic recommendations of the NCCLS, however, are often not being followed, for example, length and atmosphere of incubation and means of preparing inocula. β-Lactamase assays constitute a very commonly employed means of assessing ampicillin activity. Among susceptibility test methods, disk diffusion (82.2%) is much more commonly used compared with broth microdilution (17.8%) procedures. Data are provided regarding the most commonly tested antimicrobials as well as some of the problems encounted when using current NCCLS methods for susceptibility tests with H. influenzae. Finally, the CAP survey questionnaire revealed that many laboratories have applied HTM to susceptibility tests with other fastidious bacteria such as pathogenic Neisseria sp., streptococci, and Moraxella catarrhalis.

In vitro susceptibility testing of Haemophilus influenzae: review of new National Committee for Clinical Laboratory Standards recommendations

In vitro susceptibility testing of Haemophilus influenzae: review of new National Committee for Clinical Laboratory Standards recommendations

Problems with current recommendations for susceptibility testing of Haemophilus influenzae

We compared results of MIC and disk susceptibility tests on Haemophilus test medium (HTM) and those on comparative media. Ampicillin MICs were determined with seven ampicillin-resistant, non-beta-lactamase-producing (AmprNBLP) isolates by using HTM and supplemented brain heart infusion (sBHI) agar. Ampicillin and amoxicillin-clavulanate disk tests with 16 AmprNBLP strains, 18 ampicillin-susceptible (Amps) isolates, and 17 ampicillin-resistant, beta-lactamase-producing (AmprBLP) strains were performed by using five media: laboratory-prepared HTM (PHTM), commercial HTM (CHTM), sBHI, enriched chocolate agar, and Mueller-Hinton chocolate agar. We observed that five of seven and three of seven AmprNBLP strains were misclassified as susceptible with PHTM (MIC, less than 2 micrograms/ml) with inocula of 10(3) and 10(5) CFU, respectively, but were resistant with sBHI (MIC, greater than or equal to 2 micrograms/ml). Whereas Mueller-Hinton chocolate agar and enriched chocolate agar plates supported the growth of all 51 strains by the disk tests, 37% (19 of 51) and 8% (4 of 51) of strains did not grow on PHTM and CHTM, respectively. Lack of growth on PHTM was observed for all three phenotypes; 7 of 18 Amps, 4 of 17 AmprBLP, and 8 of 16 AmprNBLP strains did not grow. The four strains that did not grow on CHTM were all AmprNBLP isolates. Zone sizes were significantly larger on PHTM than on the other media. Of the strains that were evaluable by the new National Committee for Clinical Laboratory Standards guidelines with either PHTM or CHTM, all Amps strains were classified as susceptible. Among the AmprBLP strains, CHTM correctly identified all as resistant, whereas PHTM detected two isolates to be intermediate. Among the AmprNBLP strains, CHTM and PHTM misclassified four (33%) and five (62%) isolates, respectively, as susceptible; an additional isolate was identified as intermediate on both media. We conclude that there is strain-dependent growth on HTM, that adoption of this medium for routine Haemophilus susceptibility testing is problematic due to this growth variability, and that detection of AmprNBLP isolates would be unreliable.

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.

Use of Haemophilus test medium for broth microdilution antimicrobial susceptibility testing of Streptococcus pneumoniae

A recently described medium (Haemophilus test medium [HTM]) for antimicrobial susceptibility testing of Haemophilus influenzae was evaluated in this study for broth microdilution testing of Streptococcus pneumoniae. A total of 137 clinical isolates was tested against 11 antimicrobial agents, using Mueller-Hinton broth supplemented with 3% lysed horse blood in parallel with HTM. Inocula of 5 X 10(5) CFU/ml and incubation for 20 to 24 h were used with both media. All isolates of S. pneumoniae produced acceptable growth in both media, and MICs determined in HTM agreed closely with those determined in lysed horse blood. Drugs which provided a MIC within 1 log2 concentration difference in both media included penicillin (100%), ampicillin (98.0%), amoxicillin-clavulanate (100%), ampicillin-sulbactam (100%), cephalexin (98.9%), cefaclor (96.8%), cefuroxime (99.0%), chloramphenicol (96.2%), tetracycline (96.2%), and erythromycin (100%). HTM MICs with trimethoprim-sulfamethoxazole were 1 to 2 log2 concentration increments higher in 92.0% of isolates than MICs determined in lysed horse blood. Based on the results of this study, HTM appears to represent a promising alternative medium for broth microdilution susceptibility testing of S. pneumoniae.

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.

Improved medium for antimicrobial susceptibility testing of Haemophilus influenzae.

The need for complex growth media has complicated routine susceptibility testing of Haemophilus influenzae because of antagonism of certain antimicrobial agents by the medium or because of difficulties in interpretation of growth endpoints. Haemophilus test medium (HTM) is a simple, transparent medium for broth- or agar-based tests with H. influenzae. HTM incorporates Mueller-Hinton medium with additions of 15 micrograms of hematin per ml, 15 micrograms of NAD per ml, and 5 mg of yeast extract per ml as growth-promoting additives. Agar or broth microdilution MICs of 10 antimicrobial agents for a collection of 179 H. influenzae isolates determined by using HTM compared favorably with MICs determined by the conventional agar or broth dilution methods recommended by the National Committee for Clinical Laboratory Standards. Disk diffusion tests performed with HTM allowed accurate categorization of susceptible and resistant strains and were easier to interpret than tests performed with Mueller-Hinton chocolate agar. A particular advantage of HTM was the reliability of broth- or agar-based test results with trimethoprim-sulfamethoxazole. The results of the study suggest modification of current National Committee for Clinical Laboratory Standards MIC-interpretive criteria for H. influenzae with amoxicillin-clavulanate, chloramphenicol, and trimethoprim-sulfamethoxazole. Error rate-bounded analysis of MICs and disk diffusion zone sizes also suggest modified zone-interpretive criteria for ampicillin, amoxicillin-clavulanate, chloramphenicol, and tetracycline with HTM or conventional media. Interpretive zone sizes are newly proposed for cefaclor and rifampin disk diffusion tests.

Antimicrobial susceptibility testing of Haemophilus influenzae: trial organised as part of United Kingdom national external quality assessment scheme for microbiology.

Six strains of Haemophilus influenzae were distributed to 417 United Kingdom laboratories who were asked to test susceptibility of the strains to ampicillin, augmentin, tetracycline, chloramphenicol, and trimethoprim and to test for beta lactamase production. Laboratories were also asked to provide details of their methods by completing a questionnaire. The incidence of reports recording sensitive strains as resistant was 8% (ampicillin), 7% (augmentin), 3% (tetracycline), 1% (chloramphenicol), and 12% (trimethoprim). The incidence of reports recording resistant strains as sensitive was 9% (ampicillin), (2% with beta lactamase producing strains, 24% with non-beta lactamase producing strains), 51% (augmentin), 10% (tetracycline), 20% (chloramphenicol), and 3% (trimethoprim). High error rates were associated with several methods or practices. These included use of general purpose growth media rather than susceptibility testing media and failure to add lysed blood to the media when testing trimethoprim susceptibility; standardise the inoculum; use suitable control strains; and the use of high content discs for testing chloramphenicol, tetracycline, and ampicillin.

Susceptibility testing of Haemophilus influenzae: Some good news—some not so good

Susceptibility testing of Haemophilus influenzae: Some good news—some not so good