Characterization of mutations in the rpoB gene associated with rifampin resistance in Rhodococcus equi isolated from foals

Treatment with a combination of erythromycin and rifampin has considerably improved survival rates of foals and immunocompromised patients suffering from severe pneumonia caused by Rhodococcus equi. Frequently, because of monotherapy, emergence of rifampin-resistant strains has been responsible for treatment failure. Using consensus oligonucleotides, we have amplified and sequenced the rifampin resistance (Rif(r))-determining regions of 12 rifampin-resistant R. equi strains isolated from three foals and of mutants selected in vitro from R. equi ATCC 3701, a rifampin-susceptible strain. The deduced amino acid sequences compared to those of four rifampin-susceptible R. equi strains showed several types of mutations. In 3 of the 10 strains isolated from one foal, His526Asn (Escherichia coli numbering) and Asp516Val mutations were associated with low-level resistance (rifampin MIC, 2 to 8 microg/ml), whereas His526Asp conferred high-level resistance (rifampin MIC, 128 microg/ml) in the 7 remaining strains. In strains from the two other foals, His526Asp and Ser531Leu mutations were found to be associated with high-level and low-level resistance, respectively. The in vitro mutants, highly resistant to rifampin, harbored His526Tyr and His526Arg substitutions. As described in other bacterial genera, His526, Ser531, and Asp516 are critical residues for rifampin resistance in R. equi, and the resistance levels are dependent on both the location and the nature of the substitution.     

Laboratory detection of Haemophilus influenzae with decreased susceptibility to nalidixic acid, ciprofloxacin, levofloxacin, and moxifloxacin due to GyrA and ParC mutations

The detection of clinical isolates with decreased fluoroquinolone susceptibilities and a resistance mechanism is of epidemiological and clinical interest. We studied the susceptibilities of 62 clinical isolates and 2 American Type Culture Collection reference strains of Haemophilus influenzae to ciprofloxacin, levofloxacin, moxifloxacin, and nalidixic acid by the microdilution and disk diffusion methods. The ciprofloxacin MICs for 34 of the isolates were >/=0.12 micro g/ml (range, 0.12 to 32 micro g/ml), and the ciprofloxacin MICs for 28 matched control isolates were /=0.5 micro g/ml and the vast majority of those for which nalidixic acid MICs were >/=32 micro g/ml exhibited amino acid changes in GyrA and ParC. Nalidixic acid and the other three fluoroquinolones studied could be used to screen H. influenzae isolates for the detection of decreased susceptibilities to quinolones due to the acquisition of two amino acid changes in the QRDRs of GyrA and ParC (sensitivity, >95%; specificity, >80%).     

Molecular epidemiology of extended-spectrum beta-lactamase-producing,fluoroquinolone-resistant isolates of Klebsiella pneumoniae in Taiwan

Strains of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) have emerged worldwide. Concomitant ciprofloxacin resistance with ESBL production in K. pneumoniae isolates would severely restrict treatment options. Among 39 (18.5%) of 211 ESBL-KP isolates resistant to ciprofloxacin (MIC, >/=4 micro g/ml), 37 (95%) were high level resistant (MIC, >/=16 micro g/ml). These isolates were also cross resistant to the newer fluoroquinolones, including levofloxacin, gatifloxacin, gemifloxacin, and garenoxacin (BMS 284756). Sitafloxacin was most active against these ciprofloxacin-resistant ESBL-KP isolates with MICs for 67% of the isolates being 相似文献   

Fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae from Asian countries: ANSORP study

Seventeen clinical isolates of Streptococcus pneumoniae showing reduced susceptibility to ciprofloxacin (MIC >/= 4 micro g/ml) collected from eight different Asian countries were analyzed by antimicrobial susceptibility, serotyping, pulsed-field gel electrophoresis (PFGE), and DNA sequencing of the quinolone resistance-determining regions (QRDRs) in gyrA, gyrB, parC, and parE. All isolates but one showed more than one amino acid alteration in QRDRs of four responsible genes. Ile460 --> Val in parE was the most common mutation. Data suggest that Lys137 --> Asn in parC may be a primary step in the development of high-level and multiple FQ resistance. An additional mutation of Ser81 --> Phe in gyrA resulted in high-level resistance to ciprofloxacin, levofloxacin, and gatifloxacin, whereas Ser79 --> Phe in parC may exert an important role in the development of moxifloxacin resistance. Two novel amino acid changes in gyrB, Ala390 --> Val and Asn423 --> Thr, were found. Data from PFGE suggest an introduction and local spread of multiple resistant Spain(23F)-1 clone in Hong Kong, but isolates from other Asian countries were not related to this clone.     

Emergence of rifampin-resistant Rhodococcus equi in an infected foal.

To investigate the emergence of rifampin resistance in Rhodococcus equi strains isolated from foals and their environment in Japan, we compared the in vitro antimicrobial susceptibilities to rifampin of 640 isolates from 64 infected foals and 98 soil isolates from their horse-breeding farms. As a control, 39 human isolates from patients with and without AIDS were also tested for susceptibility to rifampin. All of the isolates showed rifampin sensitivity, except isolates from one infected foal and two patients with AIDS that showed rifampin resistance. To investigate the emergence of rifampin-resistant R. equi in the infected foal, which had received rifampin monotherapy for a month before euthanasia, 99 isolates of R. equi from the lesions and 20 isolates from the intestinal contents of the one foal with rifampin-resistant organisms were analyzed for rifampin susceptibilities, pathogenicities, and ribotypes. Of the 99 isolates from the lesions, all of which were virulent R. equi strains containing a virulence plasmid with a size of 85 or 90 kb, 90 (91%) isolates were rifampin resistant (MIC, > or = 12.5 microg/ml). On the other hand, of the 20 isolates from the intestinal contents, 11 (55%) isolates showed rifampin resistance (MIC, > or = 25 microg/ml), and 5 of them were avirulent R. equi strains. Among these 101 rifampin-resistant R. equi isolates with and without virulence plasmids characterized by ribotyping, 58 were type I, 20 were type II, 11 were type III, and 12 were type IV. These results demonstrated that at least eight different rifampin-resistant R. equi strains emerged concurrently and respectively from the different lesions and intestinal contents of the infected foal.     

Caspofungin activity against clinical isolates of fluconazole-resistant Candida

A total of 7,837 clinical isolates of Candida were tested against fluconazole, and 351 resistant (fluconazole MIC >/=64 micro g/ml) isolates were identified (4% of the total tested). All fluconazole-resistant isolates were inhibited by caspofungin at concentrations that can be exceeded by standard doses (MIC at which 90% of the isolates were inhibited, 1 micro g/ml; 99% of the MICs were 相似文献   

Molecular characterization of gentamicin-resistant Enterococci in the United States: evidence of spread from animals to humans through food

We evaluated the molecular mechanism for resistance of 360 enterococci for which the gentamicin MICs were >/=128 micro g/ml. The aac(6')-Ie-aph(2")-Ia, aph(2")-Ic, and aph(2")-Id genes were identified by PCR in isolates from animals, food, and humans. The aph(2")-Ib gene was not identified in any of the isolates. Two Enterococcus faecalis isolates (MICs > 1,024 micro g/ml) from animals failed to generate a PCR product for any of the genes tested and likely contain a new unidentified aminoglycoside resistance gene. Pulsed-field gel electrophoresis (PFGE) analysis showed a diversity of strains. However, 1 human and 18 pork E. faecalis isolates from Michigan with the aac(6')-Ie-aph(2")-Ia gene had related PFGE patterns and 2 E. faecalis isolates from Oregon (1 human and 1 grocery store chicken isolate) had indistinguishable PFGE patterns. We found that when a gentamicin-resistant gene was present in resistant enterococci from animals, that gene was also present in enterococci isolated from food products of the same animal species. Although these data indicate much diversity among gentamicin-resistant enterococci, the data also suggest similarities in gentamicin resistance among enterococci isolated from humans, retail food, and farm animals from geographically diverse areas and provide evidence of the spread of gentamicin-resistant enterococci from animals to humans through the food supply.     

Molecular analysis of isoniazid and rifampin resistance in Mycobacterium tuberculosis isolates recovered from Barcelona

We studied the presence of mutations in the whole katG gene and specific regions of the oxyR-ahpC and mabA-inhA regulatory region in 61 Mycobacterium tuberculosis isoniazid-resistant isolates. An 81-bp region of the rpoB gene was also sequenced in 17 rifampin-resistant strains. Alterations in the katG gene were detected in 55% of the isolates. Mutation in codon 315 was the most prevalent (32%). Strains showed a high level of resistance, and most maintained a substantial catalase-peroxidase activity. Three strains with an isoniazid MIC of >or=32 microg/ml lacked catalase-peroxidase activity. Two of them had deletions in the catalytic domain of the KatG protein. One strain with deletion and three strains with mutations in the C-terminal domain showed low-level resistance and conserved the catalase-peroxidase activity. Mutations in the mabA-inhA regulatory region were identified in 32% of the isolates. All had low-level resistance, and the vast majority conserved catalase-peroxidase activity. Seventeen percent of the isoniazid-resistant isolates had no detectable alterations at the studied loci. Resistance to rifampin was associated with mutations in the 81-bp of the rpoB gene in all cases. IS6110 analysis indicated that recent transmission contributed substantially to the emergence of isoniazid- resistant tuberculosis in Barcelona through short transmission chains. A rapid genotypic assay, including the 315-katG codon and the -15 nucleotide of the mabA-inhA regulatory region, may cover 62% of isoniazid- resistant strains in Barcelona. In contrast, the targeting of the 81-bp region of rpoB would detect all our rifampin-resistant isolates.     

Emergence of Klebsiella pneumoniae isolates producing inducible DHA-1 beta-lactamase in a university hospital in Taiwan

Ten nonrepetitive clinical isolates of Klebsiella pneumoniae exhibiting an unusual inducible beta-lactam resistance phenotype were identified between January 1999 and September 2001 in a university hospital in Taiwan. In the presence of 2 micro g of clavulanic acid, the isolates showed a one to four twofold concentration increase in the MICs of ceftazidime, cefotaxime, and aztreonam but remained susceptible to cefepime (MICs, 相似文献   

In vitro antimicrobial susceptibility of viridans streptococci isolated from blood cultures

The susceptibility of 211 viridans streptococci isolated from blood cultures to eight antimicrobial agents was determined. All the isolates were susceptible to cefotaxime, ceftriaxone, imipenem and vancomycin. Thirty eight percent of the isolates were resistant to penicillin (MICs 0.25 µg/ml). Tetracycline resistance was found in 41 % of the isolates and in 7 % of these strains tetracycline resistance was combined with erythromycin resistance. FiveStreptococcus mitis isolates exhibited increased (MIC 64 µg/ml and 128 µg/ml) or high-level (MIC 500 µg/ml) resistance to gentamicin, kanamycin and tobramycin. Four of these isolates were also resistant to penicillin (MICs 16–32 µg/m). In vitro synergy was not demonstrated for combinations of penicillin and gentamicin against threeStreptococcus mitis isolates with gentamicin MICs of 1000, 128 and 64 µg/ml. Results of this study indicate the importance of monitoring antibiotic resistance trends in viridans streptococci particularly with respect to penicillin and aminoglycoside resistance.     

Divergent rifamycin susceptibilities of Clostridium difficile strains in Canada and Italy and predictive accuracy of rifampin Etest for rifamycin resistance

We tested the activities of rifampin (RIF) and rifaximin (RFX) against 180 Clostridium difficile clinical isolates selected from Canadian and Italian culture collections. MICs were determined by CLSI agar dilution for both drugs and by Etest for RIF. Sixteen of 85 Italian isolates (18.8%) showed high-level resistance to both rifamycins (MICs, >16 μg/ml), compared to 2 of 95 (2.1%) Canadian isolates. Two new rpoB mutations were identified in rifamycin-resistant isolates. RIF susceptibility by Etest correlated completely with susceptibility to both rifamycins determined by agar dilution.     

Replacement of Candida albicans with C. dubliniensis in human immunodeficiency virus-infected patients with oropharyngeal candidiasis treated with fluconazole

Candida dubliniensis is an opportunistic yeast that has been increasingly implicated in oropharyngeal candidiasis (OPC) in human immunodeficiency virus (HIV)-infected patients but may be underreported due to its similarity with Candida albicans. Although most C. dubliniensis isolates are susceptible to fluconazole, the inducibility of azole resistance in vitro has been reported. Thus, the use of fluconazole prophylaxis in the treatment of these patients may have contributed to the increasing rates of isolation of C. dubliniensis. In this study, yeast strains were collected from the oral cavities of HIV-infected patients enrolled in a longitudinal study of OPC. Patients received fluconazole for the suppression or treatment of OPC, and isolates collected at both study entry and end of study were chosen for analysis. Samples were plated on CHROMagar Candida medium for initial isolation and further identified by Southern blot analysis with the species-specific probes Ca3 (for C. albicans) and Cd25 (for C. dubliniensis). Fluconazole MICs were determined by using NCCLS methods. At study entry, susceptible C. albicans isolates were recovered from oral samples in 42 patients who were followed longitudinally (1 to 36 months). C. albicans strains from 12 of these patients developed fluconazole resistance (fluconazole MIC, >/=64 micro g/ml). C. dubliniensis was not detected at end of study in any of these patients. Of the remaining 30 patients, eight (27%) demonstrated a replacement of C. albicans by C. dubliniensis when a comparison of isolates obtained at baseline and those from the last culture was done. For the 22 of these 30 patients in whom no switch in species was detected, the fluconazole MICs for initial and end-of-study C. albicans isolates ranged from 0.125 to 2.0 micro g/ml. For the eight patients in whom a switch to C. dubliniensis was detected, the fluconazole MICs for C. dubliniensis isolates at end of study ranged from 0.25 to 64 micro g/ml: the fluconazole MICs for isolates from six patients were 0.25 to 2.0 micro g/ml and those for the other two were 32 and 64 micro g/ml, respectively. In conclusion, a considerable number of patients initially infected with C. albicans strains that failed to develop fluconazole resistance demonstrated a switch to C. dubliniensis. C. dubliniensis in this setting may be underestimated due to lack of identification and may occur due to the impact of fluconazole on the ecology of oral yeast species.     

The AcrB multidrug transporter plays a major role in high-level fluoroquinolone resistance in Salmonella enterica serovar typhimurium phage type DT204

Salmonella enterica serovar Typhimurium phage type DT204 strains isolated from cattle and animal feed in Belgium were characterized for high-level fluoroquinolone resistance mechanisms [MICs to enrofloxacin (Enr) and ciprofloxacin (Cip), 64 and 32 microg/ml, respectively]. These strains isolated during the periods 1991-1994, and in 2000 were clonally related as shown by pulsed-field gel electrophoresis (PFGE). Selected strains studied carried several mutations in the quinolone target genes, i.e., a double mutation in the quinolone resistance-determining region (QRDR) of gyrA leading to amino acid changes Ser83Ala and Asp87Asn, a single mutation in the QRDR of gyrB leading to amino acid change Ser464Phe, and a single mutation in the QRDR of parC leading to amino acid change Ser80Ile. Moreover, Western blot analysis showed overproduction of the AcrA periplasmic protein belonging to the AcrAB-ToIC efflux system. This suggested active efflux as additional resistance mechanism resulting in a multiple antibiotic resistance (MAR) phenotype, which was measurable by an increased level of resistance to the structurally unrelated antibiotic florfenicol in the absence of the specific floR resistance gene. The importance of the AcrAB-TolC efflux system in high-level fluoroquinolone resistance was further confirmed by inactivating the acrB gene coding for the multidrug transporter. This resulted in a 32-fold reduction of resistance level to Enr (MIC = 2 microg/ml) and actually in a susceptible phenotype according to clinical breakpoints. Thus, AcrB plays a major role in high-level fluoroquinolone resistance, even when multiple target gene mutations are present. The same effect was obtained using the recently identified efflux pump inhibitor (EPI) Phe-Arg-naphthylamide also termed MC207,110. Among several fluoroquinolones tested in combination with EPI, the MIC of Enr was reduced most significantly. Thus, using EPI together with fluoroquinolones such as Enr may be promising in combination therapy against high-level fluoroquinolone-resistant S. enterica serovar Typhimurium.     

High prevalence and molecular analysis of macrolide-nonsusceptible Moraxella catarrhalis isolated from nasopharynx of healthy children in China

Three hundred eighty-three isolates of Moraxella catarrhalis were collected from healthy children aged less than 2 years in China and assessed for antimicrobial resistance. We found that 92.2% (n=353) produced a β-lactamase. Nonsusceptibility rates to erythromycin and azithromycin, determined using Clinical Laboratory Standards Institute (CLSI) breakpoints, were 40.3% and 22.5%, respectively; nonsusceptibility rates determined using pharmacokinetics/pharmacodynamics breakpoints, however, were 59% and 60.1%. The minimal inhibitory concentration (MIC)(90) values were >256 μg/ml. Nonsusceptibility rates varied by region from 9.7% in Dongguan to 75.9% in Jinan. Further, concomitant resistance to β-lactam antibiotics was also observed. Pulsed-field gel electrophoresis analysis of 27/37 high-level macrolide-resistant M. catarrhalis isolates showed that closely related pulsotypes dominated, with a total of 11 different pulsotypes being observed. The closely related pulsotypes were observed in isolates originating from all six Chinese cities investigated, possibly as a consequence of the mobility of the Chinese population. Sixteen patterns of 23S rRNA mutations were found among 97 selected isolates using polymerase chain reaction and sequencing, but no known ermA, ermB, mefA, or mefE genes could be detected. Mutations A2982T and A2796T in 23S rRNA were related to high-level macrolide resistance (MICs ranging from 24 to >256 μg/ml), while an A2983T mutation was associated with low-level macrolide resistance (MICs ranging from 0.19 to 16 μg/ml).     

Persistence of a clone of methicillin-resistant Staphylococcus aureus in a burns unit

A total of 128 MRSA isolates from a burns unit in 1992 and 1997 was studied by resistotyping, plasmid analysis and pulsed-field gel electrophoresis (PFGE) of SmaI-digested chromosomal DNA to ascertain whether a clone of MRSA had persisted in the unit or whether different clones had been introduced at different times. All the MRSA isolates produced beta-lactamase and had high MICs to methicillin (>256 mg/L). All were resistant to tetracycline, kanamycin, cadmium acetate and mercuric chloride. Most were resistant to gentamicin, neomycin, erythromycin, chloramphenicol, trimethoprim, ciprofloxacin, propamidine isethionate and ethidium bromide, and were susceptible to minocycline, vancomycin and teicoplanin. None of the 1992 isolates was resistant to mupirocin, but 56% and 19% of the 1997 isolates expressed high- and low-level mupirocin resistance, respectively. Many of the 1997 isolates had acquired a 38-kb plasmid encoding high-level mupirocin resistance. The 1992 isolates had two main PFGE patterns; 82% of them belonged to PFGE pattern 1. The 1997 isolates had PFGE pattern 1, the same as the majority of the 1992 isolates. All MRSA isolates from both years carried the mecA gene in the same SmaI fragment. These findings demonstrated that a clone of MRSA that was prevalentin the burns unit in 1992 had persisted and became the predominant clone in 1997.     

结核分枝杆菌耐利福平rpoB基因突变的序列分析

目的探讨结核分枝杆菌耐药基因rpoB突变与利福平耐药性的关系。方法采集81例临床标本,分离结核分枝杆菌菌株,PCR扩增rpoB基因及测序,并与利福平药物敏感试验结果比较。结果27例耐利福平菌株中,20株发生rpoB基因突变(74.1%),突变位点包括531和526在内的7个位点10种突变类型,并发现新的突变位点和突变形式;54株敏感株中,1株发生突变(1.9%)。结论利福平耐药rpoB基因突变有一定区域性,为发展快速的基因诊断技术检测耐多药结核病奠定了基础。     

Evaluation of the NCCLS extended-spectrum beta-lactamase confirmation methods for Escherichia coli with isolates collected during Project ICARE

To determine whether confirmatory tests for extended-spectrum beta-lactamase (ESBL) production in Escherichia coli are necessary, we selected 131 E. coli isolates that met the National Committee for Clinical Laboratory Standards (NCCLS) screening criteria for potential ESBL production from the Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) strain collection. For all 131 isolates, the broth microdilution (BMD) MIC of at least one extended-spectrum cephalosporin was >/=2 micro g/ml. For 21 of 131 (16%) isolates, the ESBL confirmatory test was positive; i.e., the BMD MICs of ceftazidime or cefotaxime decreased by >/=3 doubling dilutions in the presence of clavulanic acid (CA) or the disk diffusion zone diameters increased by >/=5 mm around ceftazidime or cefotaxime disks in the presence of CA. All 21 isolates were shown by PCR to contain at least one of the genes bla(TEM), bla(SHV), and bla(OXA), and in isoelectric focusing (IEF) tests, all isolates demonstrated at least one beta-lactamase band consistent with a TEM, SHV, or OXA enzyme. Of the 21 isolates, 3 showed a CA effect for cefotaxime by BMD but not by disk diffusion testing. A total of 59 (45%) of the 131 isolates demonstrated decreased susceptibility to cefpodoxime alone (MIC = 2 to 4 micro g/ml), and none had a positive ESBL confirmatory test result. These were classified as false positives according to ESBL screen test results. For the remaining 51 (39%) isolates, the cefpodoxime MICs ranged from 16 to >128 micro g/ml and the MICs for the other extended-spectrum cephalosporins were highly variable. All 51 isolates gave negative ESBL confirmatory test results. Most showed IEF profiles consistent with production of both a TEM and an AmpC beta-lactamase, and representative isolates of several phenotypic groups showed changes in porin profiles; these 51 isolates were considered true negatives. In all, only 16% of 131 E. coli isolates identified as potential ESBL producers by the current NCCLS screening criteria were confirmed as ESBL producers. Thus, changing the interpretation of extended-spectrum cephalosporins and aztreonam results from the susceptible to the resistant category without confirming the presence of an ESBL phenotype would lead to a large percentage of false resistance results and is not recommended. However, by increasing the cefpodoxime MIC screening breakpoint to >/=8 micro g/ml, 45% of the false-positive results could be eliminated. NCCLS has incorporated this change in the cefpodoxime screening breakpoint in its recent documents.     

Characterization of multidrug-resistant typhoid outbreaks in Kenya

We characterized by antibiotic susceptibility, plasmid analysis, incompatibility grouping, and pulsed-field gel electrophoresis (PFGE) of XbaI- and SpeI-digested DNA 102 Salmonella enterica serovar Typhi (serovar Typhi) isolated from recent outbreaks of typhoid in three different parts of Kenya. Only 13.7% were fully susceptible, whereas another 82.4% were resistant to each of the five commonly available drugs: ampicillin, chloramphenicol, and tetracycline (MICs of >256 microg/ml); streptomycin (MIC, >1,024 microg/ml); and cotrimoxazole (MIC of >32 microg/ml). Resistance to these antibiotics was encoded on a 110-kb self-transferable plasmid of IncHI1 incompatibility group. The MICs of nalidixic acid (MIC, 8 to 16 micro g/ml) and ciprofloxacin (MIC of 0.25 to 0.38 micro g/ml) for 41.7% of the 102 serovar Typhi isolates were 5- and 10-fold higher, respectively, than for sensitive strains. Amplification by PCR and sequencing of the genes coding for gyrase (gyrA and gyrB) and topoisomerase IV (parE and parC) within the quinolone resistance-determining region revealed that the increase in the MICs of the quinolones had not resulted from any significant mutation. Analysis of genomic DNA from both antimicrobial agent-sensitive and multidrug-resistant serovar Typhi by PFGE identified two distinct subtypes that were in circulation in the three different parts of Kenya. As the prevalence of multidrug-resistant serovar Typhi increases, newer, more expensive, and less readily available antimicrobial agents will be required for the treatment of typhoid in Kenya.     

Evaluation of a new system, VITEK 2, for identification and antimicrobial susceptibility testing of enterococci

We evaluated the new automated VITEK 2 system (bioMérieux) for the identification and antimicrobial susceptibility testing of enterococci. The results obtained with the VITEK 2 system were compared to those obtained by reference methods: standard identification by the scheme of Facklam and Sahm [R. R. Facklam and D. F. Sahm, p. 308-314, in P. R. Murray et al., ed., Manual of Clinical Microbiology, 6th ed., 1995] and with the API 20 STREP system and, for antimicrobial susceptibility testing, broth microdilution and agar dilution methods by the procedures of the National Committee for Clinical Laboratory Standards. The presence of vanA and vanB genes was determined by PCR. A total of 150 clinical isolates were studied, corresponding to 60 Enterococcus faecalis, 55 Enterococcus faecium, 26 Enterococcus gallinarum, 5 Enterococcus avium, 2 Enterococcus durans, and 2 Enterococcus raffinosus isolates. Among those isolates, 131 (87%) were correctly identified to the species level with the VITEK 2 system. Approximately half of the misidentifications were for E. faecium with low-level resistance to vancomycin, identified as E. gallinarum or E. casseliflavus; however, a motility test solved the discrepancies and increased the agreement to 94%. Among the strains studied, 66% were vancomycin resistant (57 VanA, 16 VanB, and 26 VanC strains), 23% were ampicillin resistant (MICs, >/=16 microgram/ml), 31% were high-level gentamicin resistant, and 45% were high-level streptomycin resistant. Percentages of agreement for susceptibility and resistance to ampicillin, vancomycin, and teicoplanin and for high-level gentamicin resistance and high-level streptomycin resistance were 93, 95, 97, 97, and 96%, respectively. The accuracy of identification and antimicrobial susceptibility testing of enterococci with the VITEK 2 system, together with the significant reduction in handling time, will have a positive impact on the work flow of the clinical microbiology laboratory.     

Comparison of four methods for testing high-level aminoglycoside resistance in enterococci

In a prospective study the prevalence of high-level aminoglycoside resistance (MIC 2,000 µg/ml) among 62 clinically significant enterococci was investigated. A total of 10⁵ organisms were inoculated a) onto a plate containing 2,000 µg/ml of gentamicin or streptomycin; b) into a microtube for dilution MIC determinations for gentamicin, amikacin, tobramycin and streptomycin; and c) into a single tube containing 500 µg/ml of gentamicin, amikacin, tobramycin or streptomycin in supplemented Mueller-Hinton broth. In addition, tubes containing 500 µg/ml of gentamicin, amikacin, tobramycin or streptomycin were inoculated with five enterococcal colonies (crude method). For 45 of the 62 isolates, MICs of gentamicin, amikacin and tobramycin were 500 µg/ml, while 17 (27 %) showed high-level resistance. The MICs of streptomycin were 500 µg/ml for 42 of 62 isolates, and 2,000 µg/ml for 20 (32.3 %). For 8 of the 17 (47 %) isolates showing high-level gentamicin resistance, MICs of streptomycin were 500 µg/ml. There was complete agreement between the results of the plate method, the microtube dilution MIC and the tube inoculated with 10⁵ CFU, but the crude method gave discordant results for two isolates. It is concluded that a tube containing 500 µg/ml of aminoglycoside is a simple, accurate and inexpensive method for determining high-level aminoglycoside resistance.