Imipenem-EDTA disk method for differentiation of metallo-beta-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp

Rapid detection of metallo-beta-lactamase (MBL)-producing gram-negative bacilli is necessary to prevent their dissemination. The method using a disk with imipenem plus 750 micro g of EDTA differentiated all MBL-producing pseudomonads, and the sensitivity and specificity for acinetobacters were 95.7 and 91.0%, respectively. The imipenem-EDTA disks were stable for 12 and 16 weeks at 4 and -20 degrees C, respectively.     

Evaluation of 5 rapid colorimetric and immunochromatographic tests for the detection of carbapenemases in Pseudomonas aeruginosa and Acinetobacter spp. isolates

European Journal of Clinical Microbiology & Infectious Diseases -     

Evaluation of the Hodge test and the imipenem-EDTA double-disk synergy test for differentiating metallo-beta-lactamase-producing isolates of Pseudomonas spp. and Acinetobacter spp

Gram-negative bacilli with acquired metallo-beta-lactamase (MBL) production have been increasingly reported in some countries, necessitating their detection. The aim of this study was to evaluate the performance of the Hodge test and those of the imipenem (IPM)-EDTA, ceftazidime (CAZ)-mercaptopropionic acid (MPA), and CAZ-sodium mercaptoacetic acid (SMA) double-disk synergy tests (DDSTs). The efficiencies of testing CAZ-resistant and IPM-nonsusceptible isolates were also compared. Strains used for the evaluation were known IMP-1 and VIM-2 MBL-producing isolates and consecutive and CAZ-nonsusceptible isolates of pseudomonads and acinetobacters. The performance of the Hodge test was improved by addition of zinc sulfate (140 microg/disk) to an IPM disk. In DDSTs, EDTA (ca. 1,900 microg) disks were better at detecting MBL-producing strains among pseudomonads, while MPA (3 microl) and SMA (3 mg) disks performed better for acinetobacters. EDTA (ca. 750 microg)-plus-SMA (ca. 2 mg) disks performed better than EDTA, MPA, or SMA disks with both organisms. CAZ-SMA DDSTs failed to detect 22 of 80 (28%) MBL-producing acinetobacters. In conclusion, use of an IPM disk and an EDTA (750 microg)-plus-SMA (2 mg) disk improves performance, and testing IPM-nonsusceptible isolates rather than CAZ-resistant isolates could reduce screening work. Further evaluation of the test is required for the detection of other types of MBL-producing gram-negative bacilli.     

Molecular surveillance of European quinolone-resistant clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp. using automated ribotyping

Nosocomial isolates of Pseudomonas aeruginosa and Acinetobacter spp. exhibit high rates of resistance to antibiotics and are often multidrug resistant. In a previous study (D. Milatovic, A. Fluit, S. Brisse, J. Verhoef, and F. J. Schmitz, Antimicrob. Agents Chemother. 44:1102-1107, 2000), isolates of these species that were resistant to sitafloxacin, a new advanced-generation fluoroquinolone with a high potency and a broad spectrum of antimicrobial activity, were found in high proportion in 23 European hospitals. Here, we investigate the clonal diversity of the 155 P. aeruginosa and 145 Acinetobacter spp. sitafloxacin-resistant isolates from that study by automated ribotyping. Numerous ribogroups (sets of isolates with indistinguishable ribotypes) were found among isolates of P. aeruginosa (n = 34) and Acinetobacter spp. (n = 16), but the majority of the isolates belonged to a limited number of major ribogroups. Sitafloxacin-resistant isolates (MICs > 2 mg/liter, used as a provisional breakpoint) showed increased concomitant resistance to piperacillin, piperacillin-tazobactam, ceftriaxone, ceftazidime, amikacin, gentamicin, and imipenem. The major ribogroups were repeatedly found in isolates from several European hospitals; these isolates showed higher levels of resistance to gentamicin and imipenem, and some of them appeared to correspond to previously described multidrug-resistant international clones of P. aeruginosa (serotype O:12) and Acinetobacter baumannii (clones I and II). Automated ribotyping, when used in combination with more discriminatory typing methods, may be a convenient library typing system for monitoring future epidemiological dynamics of geographically widespread multidrug-resistant bacterial clones.     

Evaluation of Etest and disk diffusion methods compared with broth microdilution antifungal susceptibility testing of clinical isolates of Candida spp. against posaconazole

We performed Etest, disk diffusion, and broth microdilution susceptibility testing of 2,171 clinical isolates of Candida spp. against posaconazole. By using provisional breakpoints for comparison purposes only, the categorical agreement between the agar-based methods and broth microdilution results ranged from 93 to 98%, with <1% very major errors. The essential agreement (within 2 well dilutions) between the Etest and broth microdilution methods was 94%. These agar-based methods hold promise as simple and reliable methods for determination of the posaconzole susceptibilities of Candida spp.     

Evaluation of Mast-ID 15 system for identification of fresh clinical isolates of Enterobacteriaceae and Acinetobacter.

AIMS: To assess the accuracy of the Mast-ID 15 system compared with API 20 E for the identification of stock and fresh clinical strains of Enterobacteriaceae and Acinetobacter spp; to compare the accuracy of 19 pin and 36 pin multipoint inoculator heads. METHODS: One hundred frozen stock cultures of Enterobacteriaceae and Acinetobacter spp which had previously been identified by the API 20E were classified by the Mast-ID using 19 and 36 pin multipoint inoculator heads. Reproducibility was determined by testing 36 randomly selected organisms in duplicate. Four hundred and sixty nine consecutive fresh clinical isolates of Enterobacteriaceae and Acinetobacter spp were identified by the Mast-ID using a 36 pin multipoint inoculator and by the API 20E. Reproducibility for the fresh isolates was determined by testing 96 randomly selected strains in duplicate. RESULTS: The Mast-ID 15 identified 82% and 85% of frozen strains to species level and reproducibility was 80% and 86% using 19 and 36 pin inoculator heads, respectively. Of the 469 fresh clinical isolates, the Mast-ID identified 70% of strains to species level; 19% were not identified and 11% were identified incorrectly by comparison with the API 20E. The Mast-ID achieved a reproducibility level of 80% with the fresh clinical isolates. CONCLUSIONS: The use of a 36 pin multipoint inoculator head in preference to the standard 19 pin head for the Mast-ID was advantageous as it allowed greater numbers of strains to be identified at a reduced cost. Unfortunately, in our hands, the Mast-ID system was insufficiently accurate for routine use in the clinical laboratory. Modifications to some of the problematic tests may result in a sufficient increase in accuracy and reproducibility to make the system beneficial in the routine clinical laboratory.     

Detection of integrons in worldwide nosocomial isolates of Acinetobacter spp.

Objective: To examine the distribution of integrons in genotypically unrelated worldwide multiresistant clinical isolates of Acinetobacter spp.
Methods: The presence and genetic location of class 1, 2 and 3 integrons were examined in a genotypically heterogeneous collection of 25 nosocomial isolates of Acinetobacter spp., from 15 locations in 11 different countries worldwide, by hybridization and PCR-based methods. Class 1 integron structures were characterized genetically by a PCR mapping technique.
Results: Class 1 integrons were detected in 17 of the 25 Acinetobacter isolates examined. Only one isolate contained a class 2 integron. No class 3 integrons were detected. The integrons varied in size and in the number of inserted cassettes, but similar integrons were found in genotypically distinct isolates from different locations worldwide. These structures were integrated into the chromosome in all isolates where they were detected, although some integrons were capable of subsequent transfer or mobilization. Genes coding for aminoglycoside-modifying enzymes formed the predominant cassettes identified within the integrons.
Conclusions: Clinical isolates of Acinetobacter spp. from diverse locations seem to share resistance mechanisms acquired from other genera by a variety of mechanisms, including dissemination of integrons. Once integrons are incorporated into the bacterial genome, Acinetobacter spp. are potentially able to act as a reservoir of resistance genes for other species and genera.     

Evaluation of Etest method for determining voriconazole and amphotericin B MICs for 162 clinical isolates of Cryptococcus neoformans

The performance of the Etest for voriconazole and amphotericin B susceptibility testing of 162 isolates of Cryptococcus neoformans was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 72 h at 35 degrees C. MICs were determined by Etest for all 162 isolates with RPMI 1640 agar containing 2% glucose (RPG agar) and were read after incubation for 72 h at 35 degrees C. The Etest results for both voriconazole and amphotericin B correlated well with reference MICs. Agreement was 94% for voriconazole and 99% for amphotericin B. When discrepancy was noted between the results obtained by Etest and broth microdilution for voriconazole, the Etest generally provided a higher MIC. The Etest method using RPG agar appears to be a useful method for determining the susceptibility of C. neoformans to voriconazole and amphotericin B.     

Evaluation of Etest for susceptibility testing of multidrug-resistant isolates of Mycobacterium tuberculosis

To prescribe effective treatment schemes for patients with tuberculosis, more-efficient susceptibility testing techniques for Mycobacterium tuberculosis are needed, especially in regions with multidrug resistance. Etest (AB BIODISK, Solna, Sweden) is a simple technique that provides quantitative drug susceptibility results for M. tuberculosis in 5 to 10 days from a culture grown at low cost. The performance of Etest was compared to that of the reference proportion method, using 95 M. tuberculosis clinical isolates of which 42.1% (40 of 95) were resistant to at least one antibiotic by the reference method. Overall agreement between Etest and the reference method was 98.9% (94 of 95) for detection of multidrug resistance; for resistance to individual drugs, agreement was 97.9% (93 of 95) for rifampin, 96.0% (92 of 95) for ethambutol, 94.7% (90 of 95) for isoniazid, and 85.3% (81 of 95) for streptomycin. This study supports the utility of Etest for timely detection of drug resistance in M. tuberculosis and for use in tuberculosis control programs.     

blaVIM-2 and blaVIM-7 carbapenemase-producing Pseudomonas aeruginosa isolates detected in a tertiary care medical center in the United States: report from the MYSTIC program

Two Pseudomonas aeruginosa strains resistant to beta-lactams, fluoroquinolones, aminoglycosides, tetracyclines, and carbapenems and susceptible only to polymyxin B (MIC 相似文献   

Dissemination and genetic context analysis of blaVIM-6 among Pseudomonas aeruginosa isolates in Asian-Pacific Nations

VIM-6, previously reported in two strains from Singapore recovered in 2000, was detected in 16 isolates collected in 2006 in India (12 isolates), Indonesia (two), Korea and the Philippines (one each). High genetic variability was observed among VIM-6-producing isolates (12 ribotypes and 11 pulsed-field gel electrophoresis types), but clones were observed in India and Indonesia; bla_VIM-6-carrying integrons of 3.9 kb and 5 kb were detected, and two of five Indian hospitals yielded isolates with both integrons. These two integrons, bla_VIM-6 was located in the first position, followed by bla_OXA-10 and aacA4. The 5-kb integrons also harboured aadAl and a 331-bp open reading frame encoding a putative efflux pump.     

Survey of amphotericin B susceptibility of Candida clinical isolates determined by Etest.

BACKGROUND AND PURPOSE: The minimal inhibitory concentrations (MICs) of amphotericin B (AmB) determined by the National Committee for Clinical Laboratory Standards (NCCLS; NCCLS document M27-A) broth dilution method are in a relatively narrow ranges and this may lead to underestimation of the AmB-resistant rate in clinical isolates. We evaluated in vitro susceptibility of clinical isolates of Candida spp. to AmB using Etest and determined the distribution of AmB MICs in different species. METHODS: We used the Etest (AB Biodisk, Solna, Sweden) to evaluate the MICs of Candida isolates randomly collected during 2001-2003 in a teaching hospital. RESULTS: Of the 572 isolates evaluated, Candida albicans (50.7%) was the most common species, followed by Candida tropicalis (23.9%), Candida parapsilosis (13.1%), Candida glabrata (9.4%), Candida krusei (1.9%), and Candida guilliermondii (0.9%). The majority of isolates were from blood (85%). The minimal concentrations of AmB required to inhibit 50%/90% of the isolates (MIC(50)/MIC(90)) were 0.19/0.38 microg/mL for C. krusei, 0.125/0.38 microg/mL for C. glabrata, 0.094/0.25 microg/mL for C. tropicalis, 0.032/0.19 microg/mL for C. albicans, 0.016/0.125 microg/mL for C. parapsilosis, and 0.023/0.032 microg/mL for C. guilliermondii. Only 1 blood isolate of C. glabrata was resistant to AmB (MIC > or =1 microg/mL) [0.17%]. 18.2% of isolates were less susceptible to AmB (MIC > or =0.19 microg/mL) with the highest rates for C. krusei (63.6%), followed by C. glabrata (37.0%), C. tropicalis (29.9%), C. albicans (11.0%), C. parapsilosis (5.3%), and C. guilliermondii (0%). More isolates collected from patients with hematologic malignancy were less susceptible to AmB than those collected from those with other diseases (30.5% vs 15.4%, p<0.05). CONCLUSION: This study demonstrated that AmB resistance remains rare at this hospital in Candida clinical isolates despite increasing use of this agent during the past 4 decades.     

Clinical importance of extended-spectrum beta-lactamase (PER-1-type)-producing Acinetobacter spp. and Pseudomonas aeruginosa strains.

Recently, an extended-spectrum beta-lactamase (PER-1) was found to be disseminated among Acinetobacter spp. and Pseudomonasaeruginosa isolates in Turkey. A population-based cohort study was conducted to elucidate predictive mortality factors in patients with nosocomial infections caused by Acinetobacter spp. and P. aeruginosa, with particular reference to PER-1-type extended-spectrum beta-lactamase (ESBL) production. The study group comprised 16 and 21 non-survivors and 82 and 126 survivors in cohorts infected with Acinetobacter and P. aeruginosa, respectively. In the Acinetobacter-infected cohort, nosocomial pneumonia, hypotension and infection with a PER-positive isolate were independent predictors of mortality. In the P. aeruginosa-infected cohort, impaired consciousness, a PER-positive isolate, male sex and (with a negative relative risk) urinary tract infection were independent predictors of death. This study demonstrated the relationship of PER-1-type ESBL-producing Acinetobacter spp. and P. aeruginosa with poor clinical outcome.     

Determination of antimicrobial susceptibility patterns of Nocardia spp. from clinical specimens by Etest

Susceptibilities to 11 antimicrobial agents were determined by Etest for 93 Nocardia isolates from clinical specimens and 15 type strains belonging to different Nocardia spp. All isolates were susceptible to trimethoprim-sulphamethoxazole, amikacin and linezolid, but susceptibilities of the various Nocardia spp. to beta-lactams, aminoglycosides, ciprofloxacin and clarithromycin varied markedly. Overall, there was a good correlation between the drug resistance patterns and the species identification established by conventional phenotypic tests and 16S rDNA sequencing. Among the different species encountered, Nocardia farcinica and Nocardia brasiliensis displayed the most multiresistant profiles, with resistance to imipenem occurring mainly among isolates of N. brasiliensis and Nocardia abscessus. The species variability in susceptibility profiles and the numerous recent taxonomic changes means that in-vitro susceptibility tests may be a complementary tool for the identification of Nocardia isolates from human clinical specimens. Further studies on a larger number of species from more diverse geographical sources, including species that are found less commonly among clinical isolates, are required to validate and extend the results.     

Enterotoxicity of clinical and environmental isolates of Aeromonas spp.

Of 147 isolates of three species of Aeromonas, 54 were from clinical and 93 from environmental sources. When tested for enterotoxin production, most of the isolates (56%) caused accumulation of fluid in rabbit ileal loops (RILs). Although large proportions of clinical and environmental isolates of A. caviae (55% and 65%, respectively) elicited such a response in RILs, isolates of A. hydrophila and A. sobria produced significantly more fluid (p less than 0.05). Furthermore, the environmental strains of A. hydrophila and A. sobria produced more fluid than the clinical isolates (p less than 0.05). The strains of Aeromonas spp. that caused little or no fluid accumulation in the initial experiments became enterotoxin producers after 1-3 passages through RILs, regardless of their source, and showed gradual enhancement of fluid outpouring after each passage. The present study suggests that all strains of these species of Aeromonas are potentially enterotoxigenic, whether from clinical or environmental sources.     

Evaluation of Etest for testing antimicrobial susceptibilities of Neisseria gonorrhoeae isolates with different growth media.

The MICs for 101 isolates of Neisseria gonorrhoeae obtained by Etest (AB-Biodisk, Solna, Sweden) and the agar dilution method on GC medium base supplemented with 1% Kellog's defined supplement (GCMB) were compared. The overall percent agreement (within 1 log2 dilution) between methods was greater than 97.9. The Pearson's correlation coefficients for penicillin, tetracycline, erythromycin, and ceftriaxone for the two methods were 0.98, 0.97, 0.93, and 0.93 (P = 0.001), respectively, for comparisons on GCMB. The overall percent agreement was lower when hemoglobin-supplemented GCMB was used. Etest is an attractive alternative to the agar dilution method for gonococcal antimicrobial susceptibility testing and should be further analyzed in multicenter studies.     

Evaluation of Etest method for determining fluconazole and voriconazole MICs for 279 clinical isolates of Candida species infrequently isolated from blood

The performance of Etest in fluconazole and voriconazole testing of 279 isolates of uncommon Candida spp. was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS)-approved standard broth microdilution (BMD) method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48 h at 35 degrees C. The isolates include Candida krusei, C. lusitaniae, C. guilliermondii, C. kefyr, C. rugosa, C. lipolytica, C. pelliculosa, C. dubliniensis, C. famata, C. zeylanoides, C. inconspicua, and C. norvegensis. Overall agreement between Etest and BMD MICs was 96% for fluconazole and 95% for voriconazole. Where a discrepancy was observed between Etest and the reference method, the Etest tended to give lower values with both fluconazole and voriconazole. The Etest method using RPMI agar appears to be a useful method for determining fluconazole and voriconazole susceptibilities of uncommon species of Candida.     

Evaluation of the Etest ESBL and the BD Phoenix,VITEK 1, and VITEK 2 automated instruments for detection of extended-spectrum beta-lactamases in multiresistant Escherichia coli and Klebsiella spp

Seventy-four isolates of multiresistant Escherichia coli and Klebsiella spp. recovered during a 3-year period and 17 control strains with genotypically identified beta-lactamases were tested for the production of extended-spectrum beta-lactamases (ESBLs) by using the Etest and the VITEK 1, VITEK 2, and Phoenix automated instruments. The use of the Etest was evaluated by investigating its accuracy in detecting the ESBLs of the control strains and by comparing interpretation results of laboratory technicians and experts. The accuracy of the Etest was 94%. With the Etest as the reference for the clinical strains and the genotype as the reference for the control strains, the automated instruments detected the ESBLs with accuracies of 78% (VITEK 2), 83% (VITEK 1), and 89% (Phoenix). No significant difference between the systems with regard to the control strains was detected. The VITEK 2 did, however, perform less well than the Phoenix (P = 0.03) on the collection of clinical isolates, mainly because of its high percentage of indeterminate test results (11%). No significant difference between the performances of the VITEK 1 and either the VITEK 2 or the Phoenix was found. However, because of its associated BDXpert system the Phoenix showed the best performance. The Etest was found to be an accurate test but was limited by its indeterminate results (4%), its inability to differentiate between K1 hyperproduction and ESBLs, questionable guidelines concerning mutants inside the inhibition zones, and the inability of the technicians to recognize subtle zone deformations.     

Reliability of the E-test method for detection of colistin resistance in clinical isolates of Acinetobacter baumannii

We compared the E-test to the broth microdilution method for testing the susceptibility of 115 clinical isolates of Acinetobacter baumannii to colistin. Twenty-two (19.1%) strains were resistant to colistin and 93 (80.8%) strains were susceptible according to the reference broth microdilution method. A categorical agreement of 98.2% was found, with only two (1.7%) very major errors. Agreement within 1 twofold dilution between the E-test and the broth microdilution was 16.5%. Complete agreement was found for the strains for which MICs fell within the range of 0.25 to 1 microg of colistin/ml. However, there was poor concordance, particularly in extreme dilutions with higher MICs by the E-test method.