Comparative Analysis of the Vitek 2 Antifungal Susceptibility System and E-test with the CLSI M27-A3 Broth Microdilution Method for Susceptibility Testing of Indian Clinical Isolates of Cryptococcus neoformans

The emergence of antifungal resistance among Cryptococcus neoformans isolates is a matter of great concern. The Clinical and Laboratory Standards Institute (CLSI) broth microdilution reference method (BMD) for antifungal susceptibility testing of C. neoformans is tedious and time-consuming. Consequently, there is a greater need for a reproducible in vitro susceptibility testing method for use in clinical microbiology laboratories. By random amplified polymorphic DNA analysis, the 62 Indian clinical isolates were characterized as Cryptococcus neoformans var. grubii. We evaluated the susceptibilities of these isolates for amphotericin B (AMB) and fluconazole (FLC) by two commercial techniques, i.e., Vitek 2 and E-test against the CLSI M27-A3 BMD. The essential agreement (EA) between the Vitek 2 and E-test with the reference procedure for FLC was similar (82.2%). For AMB, EA of 92 and 76% was obtained with E-test and Vitek 2. Excellent categorical agreement (CA) (98.3% and 100% by Vitek 2 and E-test, respectively) was obtained for AMB. The CA for FLC was 81 and 77.4% by Vitek 2 and E-test. We conclude that both E-test and Vitek 2 system have acceptable levels of accuracy for susceptibility testing of both the drugs. Both of them could identify fluconazole-resistant strains. Vitek 2 could be used for testing susceptibility of voriconazole and 5-flucytosine also at the same time.     

Antimicrobial resistance and genomic screening of clinical isolates of thermophilic Campylobacter spp. from south-east Queensland,Australia

AIM: To screen 90 clinical isolates of thermophilic Campylobacter species for putative resistance to ampicillin, erythromycin and tetracycline and perform numerical analysis to determine isolate relatedness. METHODS AND RESULTS: Disc diffusion, E-test MIC and agar dilution methods were performed. Disc diffusion testing showed 87 (97%) isolates appeared resistant to ampicillin at 10 microg; 14 (16%) resistant to tetracycline at 30 microg; and three (3.4%) resistant to erythromycin at 15 microg. E-test MICs showed a range of 0.5 to >256 mg l(-1) for ampicillin; 16 to >256 mg l(-1) for tetracycline; and >256 mg l(-1) for erythromycin. E-test showed 68% correlation (+/-1 log2 dilution) with agar dilution for ampicillin, 100% for erythromycin and 64% for tetracycline. Disc diffusion testing showed 100% correlation with agar dilution for erythromycin and tetracycline, and 77% for ampicillin. Numerical analyses of restriction endonuclease (RE) fragment profiles suggested a high level of isolate variation. CONCLUSION: The incidence of resistance of thermophilic Campylobacter spp. to erythromycin and tetracycline is low in south-east Queensland. SIGNIFICANCE AND IMPACT OF THE STUDY: Disc diffusion susceptibility testing may be used to screen thermophilic Campylobacter spp. for putative resistance to erythromycin and tetracycline. Agar dilution should be used to determine ampicillin susceptibility.     

Quantifying antimicrobial resistance at veal calf farms

This study was performed to determine a sampling strategy to quantify the prevalence of antimicrobial resistance on veal calf farms, based on the variation in antimicrobial resistance within and between calves on five farms. Faecal samples from 50 healthy calves (10 calves/farm) were collected. From each individual sample and one pooled faecal sample per farm, 90 selected Escherichia coli isolates were tested for their resistance against 25 mg/L amoxicillin, 25 mg/L tetracycline, 0.5 mg/L cefotaxime, 0.125 mg/L ciprofloxacin and 8/152 mg/L trimethoprim/sulfamethoxazole (tmp/s) by replica plating. From each faecal sample another 10 selected E. coli isolates were tested for their resistance by broth microdilution as a reference. Logistic regression analysis was performed to compare the odds of testing an isolate resistant between both test methods (replica plating vs. broth microdilution) and to evaluate the effect of pooling faecal samples. Bootstrap analysis was used to investigate the precision of the estimated prevalence of resistance to each antimicrobial obtained by several simulated sampling strategies. Replica plating showed similar odds of E. coli isolates tested resistant compared to broth microdilution, except for ciprofloxacin (OR 0.29, p≤0.05). Pooled samples showed in general lower odds of an isolate being resistant compared to individual samples, although these differences were not significant. Bootstrap analysis showed that within each antimicrobial the various compositions of a pooled sample provided consistent estimates for the mean proportion of resistant isolates. Sampling strategies should be based on the variation in resistance among isolates within faecal samples and between faecal samples, which may vary by antimicrobial. In our study, the optimal sampling strategy from the perspective of precision of the estimated levels of resistance and practicality consists of a pooled faecal sample from 20 individual animals, of which 90 isolates are tested for their susceptibility by replica plating.     

Susceptibility of Brazilian Staphylococcal Strains to Glycopeptides Evaluated by Different Testing Methods

Reports of staphylococci with reduced susceptibility to glycopeptides are cause for concern. This study evaluated the susceptibility of 84 staphylococci clinical isolates to glycopeptides by the disk diffusion, agar dilution, E-test, and BHIA screening methods. Vancomycin agar dilution showed all strains presented minimum inhibitory concentration (MIC) ranging from 0.5 to 2 μg/ml, and the E-test showed similar results. Teicoplanin agar dilution test showed MICs ranging from ≤ 0.5 to 2 μg/ml for Staphylococcus aureus and MICs ranging from <0.25 to 32 μg/ml for coagulase-negative Staphylococcus (CNS). Ten CNS isolates presented MICs ranging from 8 to 32 μg/ml for agar dilution and/or E-test. All the staphylococci were susceptible to vancomycin by the disk diffusion test (DDT), but two CNS isolates presented intermediate resistance to teicoplanin by the DDT and MICs of susceptibility, with two other CNS strains, teicoplanin-susceptible by the DDT, presented MICs of intermediate resistance. On the vancomycin-containing agar, 20 CNS isolates were able to grow, but no S. aureus strain. All these isolates showed MICs to teicoplanin (4–32 μg/ml) higher than those isolates that did not grow on the agar screen plate. PFGE of chromosomal SmaI digests showed a wide diversity of these CNS strains, without any predominance of a single PFGE pattern. Received: 25 May 2001 / Accepted: 9 July 2001     

体外诱导耐万古霉素金黄色葡萄球菌及分子机制

【目的】通过前期体外诱导获得耐万古霉素金黄色葡萄球菌,从基因突变方面对万古霉素耐药性菌株进行研究。【方法】通过低浓度万古霉素逐步诱导13株敏感性金黄色葡萄球菌,用琼脂稀释法和E-test法检测所有菌株对万古霉素的耐药性(最低抑菌浓度,MIC),PCR扩增与万古霉素耐药性密切相关的4个重要基因:rpo B、vra S、gra R和gra S,并测序分析,比较诱导前后不同菌株的基因序列。【结果】通过60 d的体外诱导实验,13株对万古霉素敏感性金黄色葡萄球菌中有6株被诱导为中介耐药金黄色葡萄球菌(Vancomycin intermediate Staphylococcus aureus,VISA),7株菌被诱导之后对万古霉素仍处于敏感状态,MIC4 mg/L。检测诱导前后所有菌株的rpo B、vra S、gra R和gra S基因发现:有3株VISA的rpo B基因同时有L466S和H481N的突变,gra S基因同时有R232K的突变。【结论】对万古霉素敏感的金黄色葡萄球菌经过较长时间的体外诱导可发展为VISA。在已检测的重要基因中,rpo B和gra S的突变对耐药性的发展很可能起关键作用,而vra S和gra R对这一过程没有显著影响。     

Ampicillin resistance and penicillin-binding proteins of Haemophilus influenzae

Penicillin-binding protein (PBP) alterations have been associated with non-beta-lactamase-mediated ampicillin resistance in Haemophilus influenzae. We evaluated the PBP profiles of several ampicillin-susceptible and -resistant clinical isolates of H. influenzae to determine how consistently the described alterations occurred, and to document the reproducibility of the PBP profiles for this species. The MIC of ampicillin ranged from 0.06 to 0.13 microgram ml-1 for the susceptible isolates at an inoculum of 100,000 c.f.u. when tested by broth dilution, and was 0.5 microgram ml-1 for all four isolates when tested by agar dilution. The MIC for the resistant isolates ranged from 4 to 8 micrograms ml-1 when tested by broth dilution, and from 1.5 to 16 micrograms ml-1 when tested by agar dilution. At least eight distinct PBPs with molecular masses ranging from 27 to 90 kDa were detected both in cell membrane preparations and whole cell (in vivo) binding assays done on cells in the exponential growth phase. PBP variability was evident both in the ampicillin-susceptible and -resistant isolates; however, much greater variability existed within the four resistant strains. The differences in PBP patterns included (1) electrophoretic mobility, (2) binding capacity for the antibiotic and (3) the presence of additional PBPs in two of the resistant isolates. However, decreased binding capacity was consistently demonstrated in PBP 5 (56 kDa) of all of the resistant isolates. Saturation curves with both penicillin and ampicillin indicated that PBP 5 had decreased affinity for the antibiotics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of Real-Time PCR with Disk Diffusion, Agar Screen and E-test Methods for Detection of Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen. Our main objective was to compare oxacillin disk test, oxacillin E-test, and oxacillin agar screen for detection of methicillin resistance in S. aureus, using real-time PCR for mecA as the “gold standard” comparison assay. 196 S. aureus isolates were identified out of 284 Staphylococcus isolates. These isolates were screened for MRSA with several methods: disk diffusion, agar screen (6.0 μg/ml), oxacillin E-test, and real-time PCR for detection of mecA gene. Of the 196 S. aureus isolates tested, 96 isolates (49%) were mecA-positive and 100 isolates (51%) mecA-negative. All methods tested had a statistically significant agreement with real-time PCR. E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen method were 98 and 99%, respectively and sensitivity and specificity of oxacillin disk diffusion method were 95 and 93%, respectively. In the present study, oxacillin E-test is proposed as the best phenotypic method. For economic reasons, the oxacillin agar screen method (6.0 μg/ml), which is suitable for the detection of MRSA, is recommended due to its accuracy and low cost.     

Characterization of glycopeptides, aminoglycosides and macrolide resistance among Enterococcus faecalis and Enterococcus faecium isolates from hospitals in Tehran

The prevalence of glycopeptides, aminoglycosides and erythromycin resistance among Enterococcus faecalis and Enterococcus faecium was investigated. The susceptibility of 326 enterococcal hospital isolates to amikacin, kanamycin, netilmicin and tobramycin were determined using disk diffusion method. The minimum inhibitory concentration (MIC) of vancomycin, teicoplanin, gentamicin, streptomycin, and erythromycin were determined by microbroth dilution method. The genes encoding aminoglycoside modifying enzymes described as AMEs genes, erythromycin-resistant methylase (erm) and vancomycin-resistant were targeted by multiplex-PCR reaction. High level resistance (HLR) to gentamicin and streptomycin among enterococci isolates were 52% and 72% respectively. The most prevalent of AMEs genes were aac (6')-Ie aph (2") (63%) followed by aph (3')-IIIa (37%). The erythromycin resistance was 45% and 41% of isolates were positive for ermB gene. The ermA gene was found in 5% of isolates whereas the ermC gene was not detected in any isolates. The prevalence of vancomycin resistant enterococci (VRE) was 12% consisting of E. faecalis (6%) and E. faecium (22%) and all of them were VanA Phenotype. The results demonstrated that AMEs, erm and van genes are common in enterococci isolated in Tehran. Furthermore our results show an increase in the rate of vancomycin resistance among enterococci isolates in Iran.     

Comparison of disc diffusion and epsilometer (E-test) testing techniques to determine antimicrobial susceptibiliy of Campylobacter isolates of food and human clinical origin

The antibiotic resistance profiles of 75 Campylobacter isolates of food and human clinical origin was determined by two agar diffusion susceptibility methods; disc diffusion and epsilometer-test (E-test). The most common therapeutic antimicrobials, erythromycin, ciprofloxacin and tetracycline were studied, along with chloramphenicol, ampicillin and naladixic acid. The resistance observed for each antimicrobial, as determined by both of methods, were statistically compared using Fisher two-tailed analysis.Of the six antimicrobials studied only two were shown to have statistically different patterns when resistance was compared by disc diffusion and E-test. The percentage of isolates resistant to clinically relevant antimicrobials using both techniques ranged from 6.6 to 21.3% for erythromycin, 25.3–26.6% for tetracycline and 33.3–36.0% for ciprofloxacin. The prevalence of multi-drug resistant (MDR) campylobacters (isolates resistant to 2 or more antimicrobials) for both disc diffusion and E-test was 44%. It can be concluded that, for four of the six antimicrobials assessed, antimicrobial resistance prevalences could be equally determined by either of the methods studied.     

Evaluation of methods for detecting oxacillin resistance in coagulase-negative staphylococci including cefoxitin disc diffusion

The aim of this study was to evaluate the accuracy of cefoxitin disc diffusion as a prediction of oxacillin resistance in coagulase-negative staphylococci (CoNS), and also to compare genotypic and phenotypic methods for detecting this resistance property. A total of 151 clinical CoNS isolates were tested by PCR for the presence of the mecA gene (gold standard method). The isolate susceptibilities were determined by the disc diffusion method with oxacillin (1 microg) and cefoxitin (30 microg) and by the agar dilution method for cefoxitin and oxacillin. Although none of the techniques showed 100% sensitivity and 100% specificity, the cefoxitin disc diffusion and oxacillin agar dilution were the best methods for detecting resistance to oxacillin among CoNS as these methods produced the best negative and positive predictive values. A combination of methods can be used routinely to identify resistance to oxacillin in CoNS.     

Broth dilution and disc diffusion methods in the susceptibility testing of pathogenic Candida albicans against four antimycotics

Susceptibility of Candida albicans isolated from mouthrinse specimens during episodes of acute pseudomembranous candidiasis in patients with haematological malignancies was tested by the broth dilution and disc diffusion methods using 24 and 48 h of incubation. The time factor did not significantly affect the results with 5-fluorocytosine. With amphotericin B, prolonged incubation doubled the geometric mean MIC of C. albicans as well as the number of isolates with intermediate sensitivity. With the shorter incubation in the disc diffusion assay, few isolates showed lowered sensitivity to clotrimazole; at 48 h, however, this figure was as high as 54%. The yeasts were highly sensitive to ketoconazole at 24 h, whereas at 48 h the results were bizarre. At 24 h, correlations between disc diffusion and broth dilution methods were satisfactory, with clotrimazole and ketoconazole accounting for most of the discordancies. Accordingly, the disc diffusion results should be recorded at 24 h.     

Determination of antibiotic resistance and resistance plasmids of clinical Enterococcus species

To determine the antibiotic resistance pattern and resistance plasmids, we studied 23 antibiotic-resistant clinical isolates of Enterococcus spp. which caused infection in Bayindir-Ankara Hospital, Turkey. Biochemical and physiological identification tests were applied by the Vitek system and compared with the results of protein profiles by SDS-PAGE. From 23 isolates, 20 were identified as E. faecalis, 2 as E. faecium and 1 as E. gallinarum. Twenty four antibiotics belong to 10 different groups were used in susceptibility tests. Multiple antibiotic resistance was determined in 10 of 23 Enterococcus spp. Overall resistance to the used antibiotics was 47.3% and low level resistance was 16.6%. Among the isolates tested, 8.7% demonstrated high level gentamicin resistance, 17.4% demonstrated high level streptomycin resistance, and 43.5% demonstrated penicillin resistance. High level vancomycin resistant Enterococcus spp. rate was 34.8%, and 60.9% exhibited low level resistance to vancomycin and teicoplanin. They contain plasmids which varied in numbers between 1 and 11 and the plasmid sizes ranged from 2.08 to 56.15 kb. In curing experiments with acriflavine, two different plasmids were shown in different molecular sizes of 33.49 and 13.6 kb while the first determined glycopeptide and penicillin resistance, the second one determined either glycopeptide or penicillin resistance in two different E. faecalis strains. On the other hand, a 22.58 kb plasmid, determining kanamycin resistance, was detected in an E. faecium strain. After the curing experiments, an elimination of 37.17 and 44.47 kDa protein bands was shown in E. faecium EFA1 and E. faecalis EFA13 in SDS-PAGE, respectively. This survey indicates the increase of antibiotic-resistant enterococci, especially to vancomycin in our hospital isolates.     

纹带棒状杆菌耐药特征研究

分析我国山东地区临床分离的纹带棒状杆菌对常用药物的敏感性,并对其耐药机制进行探讨。收集该省某三甲医院临床样本,用哥伦比亚血平板培养基对纹带棒状杆菌进行分离培养,用微量肉汤稀释法检测12种常用抗生素对纹带棒状杆菌的最低抑菌浓度(minimal inhibitory concentration, MIC)。采用聚合酶链式反应(polymerase chain reaction,PCR)对耐药菌株的耐药基因[aph(3”)-Ⅰb、aph(6)-Ⅰd、aac(6’)-Ⅰb, tetW, ermX, gyrA]进行扩增、测序比对,以探讨相关的耐药机制。通过实时荧光定量PCR(quantitative real-time PCR, qPCR)对耐药基因ermX、tetW进行定量分析,采用公式2^－ΔΔCt计算基因相对表达量差异倍数。结果显示,共分离出纹带棒状杆菌83株,且均为多重耐药菌。对万古霉素、利奈唑胺表现为完全敏感;对红霉素、环丙沙星、克林霉素表现出完全耐药,对四环素的耐药率为86.7%(72/83),对庆大霉素的耐药率为38.6%(32/83);ermX基因的检出率为100%(83/83),tetW基因的检出率为88.0%(73/83),aph(3”)-Ⅰb基因的检出率为36.1%(30/83),aph(6)-Ⅰd基因的检出率为37.3%(31/83),aac(6’)-Ⅰb基因的检出率为15.7%(13/83)。ermX和tetW基因的相对表达量均有所变化,但变化不明显。内在型耐药(cMLS)是菌株对红霉素耐药的潜在机制。结果提示,本研究调查医院的纹带棒状杆菌耐药严重,耐药谱较为广泛,须引起医院和实验室的重视。     

Conjugal transferability of multiple resistance in Shigella strains

Shigella strains isolated in Japan between 1971 and 1979 were surveyed for drug resistance and distribution of R plasmids. Of 2,510 strains, 89.3% were resistant to either one or various combinations of four drugs, tetracycline, chloramphenicol, streptomycin, and sulfanilamide. About 66% of the Shigella isolates were quadruply resistant. The frequency of isolation of R plasmids from quadruply resistant Shigella strains was the highest when compared with other strains resistant to various combinations of the four drugs. The conjugal transferability of 204 quadruply resistant strains isolated between 1977 and 1979 was tested by various mixed-culture methods. Among the total strains examined, 70.6% carried transferable resistance when tested by the conventional broth culture method, 90.2% transferred their resistance when, in addition the replica-plating method was used and 97.5% could transfer their resistance when the membrane filter method was also used. Although the remaining five strains could not transfer their resistance by any of the mixed culture methods, the drug resistance of four of the five strains was mobilized by the concomitant presence of F-tet or T-kan plasmid. These results indicate that almost all of the quadruple resistance in Shigella isolates was mediated by plasmid.     

Comparative study of in vitro methods to analyse the antifungal activity of propolis against yeasts isolated from patients with superficial mycoses

AIM: To test a total of 15 strains belonging to four species of yeasts by different in vitro methods against propolis and itraconazole (ITC). METHODS AND RESULTS: Three methods were compared for susceptibility testing of yeast isolates to propolis: disc diffusion method, agar dilution method and National Committee for Clinical Laboratory Standards (NCCLS, M27A) broth microdilution method. ITC was selected as the antifungal agent for comparison study. Using the broth microdilution method, the geometric mean for MIC (microg ml(-1)) with regard to all isolates was < or =0.06 for propolis and < or =0.35 for ITC. The broth microdilution and the agar dilution methods were in good agreement (75%) for propolis against yeasts isolated from patients with superficial mycoses. Using the diffusion method, all strains showed a broad zone of inhibition at the first available reading time (24 or 48 h). An increase of MIC values was accompanied by a decrease of growth inhibition zone diameter. A favourable correlation was found between MIC and inhibition zone around the disc for propolis sample and the correlation coefficient was: r = -0.626 (P < 0.01). CONCLUSIONS: This study suggests the potential value of the agar dilution and disc diffusion method as a convenient alternative method for testing of yeasts to propolis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that propolis and ITC were very active against yeasts from patients with superficial mycoses. The other prominent finding in this study is that RPMI 1640 with L-glutamine was the available broth for the in vitro susceptibility testing of yeasts.     

Comparison of microdilution method and E-test procedure in susceptibility testing of caspofungin against Candida non-albicans species

It is widely known that systemic and mucosal candidiasis caused by Candida non-albicans strains endangers the lives of hospitalised patients since these pathogens are extremely difficult to defeat by commonly used antifungal agents. The present study determined the in vitro activities of a novel antimicotic drug - caspofungin - against 76 Candida non-albicans isolates by means of the CLSI reference method for broth dilution antifungal susceptibility testing of yeasts and the E-test procedure for comparison. Caspofungin was efficacious against the majority of strains tested, with the average MICs90 evaluated by the microdilution method and E-tests amounting to 1 mg/l and 0.5 mg/l, respectively. Since the agreement between MICs within +/-2 dilutions obtained by these two techniques was 92% (Kappa coefficient of 0.92), the E-test procedure seems to be a reliable alternative to the broth microdilution method and may provide another choice for clinical laboratories.     

Genotypic diversity, antimicrobial resistance, and virulence factors of human isolates and probiotic cultures constituting two intraspecific groups of Enterococcus faecium isolates

The intraspecific relationships among a collection of Enterococcus faecium isolates comprising probiotic cultures and human clinical isolates were investigated through the combined use of two high-resolution DNA-fingerprinting techniques. In addition, the incidences of antimicrobial resistance and virulence traits were investigated. A total of 128 E. faecium isolates from human clinical or nonclinical sources or used as probiotic cultures were subjected to fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting and pulsed-field gel electrophoresis (PFGE) analysis of SmaI macrorestriction patterns. Susceptibilities to 16 antimicrobial agents were tested using broth microdilution, and the presence of the corresponding resistance genes was investigated using PCR. Multiplex PCR was used to detect the presence of the enterococcal virulence genes asa1, gelE, cylA, esp, and hyl. The results of the study showed that two intraspecific genomic groups (I and II) were obtained in FAFLP analysis. PFGE analysis demonstrated high variability within these two groups but also indicated that some probiotic cultures were indistinguishable and that a number of clinical isolates may be reisolations of commercial probiotic cultures. Compared to group II, which contained the majority of the probiotic isolates and fewer human clinical isolates, higher phenotypic and genotypic resistance frequencies were observed in group I. Two probiotic isolates were phenotypically resistant to erythromycin, one of which contained an erm(B) gene that was not transferable to enterococcal recipients. None of the probiotic E. faecium isolates demonstrated the presence of the tested virulence genes. The previously reported observation that E. faecium consists of two intraspecific genomic groups was further substantiated by FAFLP fingerprinting of 128 isolates. In combination with antimicrobial resistance and virulence testing, this grouping might represent an additional criterion in assessing the safety of new potential probiotic E. faecium isolates.     

Antimicrobial resistance profiles and genetic characterisation of macrolide resistant isolates of Streptococcus agalactiae

In this study, 100 clinical isolates of Streptococcus agalactiae recovered from genitourinary tract specimens of non-pregnant individuals living in Rio de Janeiro were submitted for antimicrobial susceptibility testing, detection of macrolide resistance genes and evaluation of the genetic diversity of erythromycin-resistant isolates. By agar diffusion method, all isolates were susceptible to ceftazidime, penicillin and vancomycin. Isolates were resistant to levofloxacin (1%), clindamycin (5%), erythromycin (11%) and tetracycline (83%) and were intermediated to erythromycin (4%) and tetracycline (6%). Erythromycin-resistant and intermediated isolates presented the following phenotypes: M (n = 3), constitutive macrolide-lincosamide-streptogramin B (MLS B, n = 5) and inductive MLS B (n = 7). Determinants of macrolide resistance genes, erm and mef, were detected in isolates presenting MLS B and M phenotypes, respectively. Randomly amplified polymorphic DNA profiles of erythromycin-resistant isolates were clustered into two major groups of similarity.     

Association of MexAB-OprM with intrinsic resistance ofPseudomonas aeruginosa to aminoglycosides

MexAB-OprM is known to pump out mostly lipophilic and amphiphilic drugs. But in low-ionic-strength medium, nutrient broth (NB), this pump has been shown to contribute to hydrophilic antibiotic (aminoglycosides) resistance, via active efflux. The association of the MexAB-OprM efflux system to aminoglycosides resistance inPseudomonas aeruginosa were assessed using a drug susceptibility test carried out in NB, in presence and absence of protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) by 23 multidrug resistant strains were selected from 104 clinical isolates ofP. aeruginosa. Active efflux was assessed using EtBr accumulation assays. PCR was used to identify themexAB-oprM and MexAB-OprM-dependent efflux of aminoglycosides and the results were confirmed by continuous fluorescence assay. A multidrug resistant mutant ofmexAB-oprM, derivative of PAO1, was selected by ciprofloxacin and subjected to the same analysis as described above for the clinical isolates. In this study, CCCP reduced the level of MICs in at least 1 dilution. Ethidium bromide accumulation assays confirmed the presence of efflux mechanism in all clinical isolates and PCR demonstrated that 17% of our isolates had themexAB-oprM operon. Results of aminoglycosides accumulation showed, in addition to amphiphilic antibiotics in NB medium, MexAB-OprM extrudes aminoglycosides (hydrophilic) drugs.     

Comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of Helicobacter cinaedi

The aim of this study was to establish a broth microdilution method for antimicrobial susceptibility testing of Helicobacter cinaedi and to assess the prevalence and mechanisms of fluoroquinolone resistance in Japanese clinical isolates. A broth microdilution method using modified Levinthal broth was developed and compared with the agar dilution method for testing susceptibility to ampicillin, gentamicin, tetracycline and ciprofloxacin. The minimum inhibitory concentrations obtained by these two methods were almost the same for all the antibiotics tested, demonstrating the broth microdilution method to be a suitable and reliable technique for antimicrobial susceptibility testing. A broth microdilution method for antimicrobial susceptibility test for H. cinaedi was established. This method is expected to help improve treatment.