Development of a multiplex PCR and SHV melting-curve mutation detection system for detection of some SHV and CTX-M beta-lactamases of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae in Taiwan

Infection by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae has been increasing in Taiwan. Accurate identification of the ESBL genes is necessary for surveillance and for epidemiological studies of the mode of transmission in the hospital setting. We describe herein the development of a novel system, which consists of a multiplex PCR to identify bla(SHV), bla(CTX-M-3)-like, and bla(CTX-M-14)-like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent bla(SHV) genes (bla(SHV-1), bla(SHV-2), bla(SHV-2a), bla(SHV-5), bla(SHV-11), and bla(SHV-12)) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of the bla(SHV) and bla(CTX-M) genes, were identified by the system. The system was then used to genotype the ESBLs from 199 clinical isolates, including 40 Enterobacter cloacae, 68 Escherichia coli, and 91 Klebsiella pneumoniae, collected between August 2002 and March 2003. SHV-12 (80 isolates) was the most prevalent type of ESBL identified, followed in order of frequency by CTX-M-3 (65 isolates) and CTX-M-14 (36 isolates). Seventeen (9%) of the 199 clinical isolates harbored both SHV- and CTX-M-type ESBLs. In contrast to Enterobacter cloacae, the majority of which produced SHV-type ESBLs, E. coli and K. pneumoniae were more likely to possess CTX-M-type ESBLs. Three rare CTX-M types were identified through sequencing of the bla(CTX-M-3)-like (CTX-M-15) and bla(CTX-M-14)-like (CTX-M-9 and CTX-M-13) genes. The system appears to provide an efficient differentiation of ESBLs among E. coli, K. pneumoniae, and Enterobacter cloacae in Taiwan. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.     

Comparison of screening methods for detection of extended-spectrum beta-lactamases and their prevalence among blood isolates of Escherichia coli and Klebsiella spp. in a Belgian teaching hospital.

Using a set of 33 well-defined extended-spectrum beta-lactamase (ESBL)-producing strains of Escherichia coli and Klebsiella pneumoniae, we compared three screening methods for ESBL detection: (i) a double-disk synergy test, (ii) a three-dimensional test (both the double-disk synergy test and the three-dimensional test were performed with ceftriaxone, ceftazidime, aztreonam, and cefepime), and (iii) the Etest ESBL screen (AB Biodisk, Solna, Sweden), based on the recognition of a reduction in the ceftazidime MIC in the presence of clavulanic acid. In the double-disk test, all four indicator antibiotics scored equally and 31 of the 33 reference strains were recognized. In the three-dimensional test, ceftriaxone was the only satisfactory indicator and 30 ESBL-positive strains were detected by this antibiotic. Both systems produced two false-positive results with cefepime. With the Etest ESBL screen, 15 of 16 TEM-related and 11 of 16 SHV-related ESBL-producing strains scored positive. In 10 cases the clavulanic acid on one end of the strip interfered with the MIC determination for ceftazidime, which was read on the opposite end. This MIC had to be determined with an extra ceftazidime-only strip. No false-positive results were noted. Eighty-six blood isolates of E. coli and Klebsiella species were screened for ESBL expression by the double-disk and three-dimensional tests, both with ceftriaxone. Six strains with suspicious antibiogram phenotypes also gave positive results by the double-disk test. One E. coli strain remained undetected by the three-dimensional test. Identification of the enzymes suspected of being ESBLs by isoelectric focusing (all strains) and DNA sequencing (1 strain) confirmed the screening test results except for one Klebsiella oxytoca strain, which proved to be a hyperproducer of its chromosomal enzyme and which also had a negative Etest score. The five true ESBL producers were all confirmed by the Etest ESBL screen. Pulsed-field gel electrophoresis proved that the E. coli strains were unrelated, but that two of the three K. pneumoniae strains were closely related.     

Characterisation and molecular epidemiology of extended-spectrum β-lactamase-producing Enterobacter cloacae isolated from a district teaching hospital in Taiwan

Enterobacter cloacae (n = 110) isolates from a district hospital in Taiwan were screened for extended-spectrum beta-lactamases (ESBLs). In total, 17 ESBL-producers were identified, based on the combination-disk synergy test using cefotaxime and ceftazidime +/- clavulanic acid. Investigation of ESBL genes in 33 ceftazidime-resistant isolates revealed the SHV-12 gene in the same 17 ESBL-producers. In addition, one isolate also carried the CTX-M-3 gene, and two isolates also carried the CTX-M-9 gene. No major epidemic clone of ESBL-producers was identified by pulsed-field gel electrophoresis. Routine screening for the ESBL phenotype, focusing on ceftazidime-resistant E. cloacae, should be undertaken in this area.     

Effects of inoculum and beta-lactamase activity in AmpC- and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae clinical isolates tested by using NCCLS ESBL methodology

Escherichia coli and Klebsiella pneumoniae isolates with extended-spectrum beta-lactamases (ESBLs) or AmpC cephalosporinases generally respond as predicted to NCCLS tests for ESBL production. However, inoculum size may affect MICs. The effect of inoculum level in clinical isolates expressing beta-lactamases were studied at inocula within 0.5 log unit of the standard inoculum, using broth microdilution methodology with ceftazidime, cefotaxime, cefepime, cefpodoxime, and aztreonam. Strains with TEM-1 or no beta-lactamases gave consistent MIC results with inocula of 10(5) and 10(6) CFU/ml. When the bacteria were screened for ESBL production and the lower inoculum was used, several strains with ESBLs, including CTX-M-10, TEM-3, TEM-10, TEM-12, TEM-6, SHV-18, and K1, gave false-negative results for one or more antimicrobial agents (MICs below the NCCLS screening concentration for detecting suspected ESBLs). When the higher inoculum was used, MICs of at least one antimicrobial agent increased at least fourfold in strains producing TEM-3, TEM-10, TEM-28, TEM-43, SHV-5, SHV-18, and K1. All antimicrobial agents showed an inoculum effect with at least one ESBL producer. Confirmatory clavulanate effects were seen for both inocula for all ESBL-producing strains with all antimicrobial agents tested, except for the CTX-M-10-producing E. coli with ceftazidime and the SHV-18-producing K. pneumoniae with cefotaxime. In kinetic studies, cefpodoxime and cefepime were hydrolyzed by ESBLs in a manner similar to that of cefotaxime. When total beta-lactamase activity and hydrolysis parameters were evaluated, however, no single factor was predictive of inoculum effects. These results indicate that the NCCLS screening and confirmation tests are generally predictive of ESBL production, but false-negative results can arise when a lower inoculum is used in testing.     

Detection of Enterobacteriaceae producing CTX-M extended spectrum beta-lactamases from a tertiary care hospital in south India

A total of 23 clinical isolates (15 Escherichia coli and 8 Klebsiella pneumoniae), resistant to cefotaxime and ceftazidime recovered during 2002 and 2003, were investigated for production of CTX-M extended spectrum beta-lactamase (ESBL) by phenotypic and molecular methods. The presence of ESBL was tested by NCCLS phenotypic confirmatory test using cephalosporin/clavulanate combination discs and E-test ESBL strips. Determination of MIC of cefotaxime and ceftazidime was done with and without the presence of clavulanic acid by agar dilution technique. Polymerase chain reaction revealed the presence of CTX-M type ESBLs in 19 isolates. Further sequencing resulted in identification of CTX-M-15 ESBLs. This is the first report identifying CTX-M type ESBL from clinical isolates of E. coli and K. pneumoniae from a tertiary care hospital in south India.     

Extended-spectrum beta-lactamases in Taiwan: epidemiology, detection, treatment and infection control.

Extended-spectrum beta-lactamases (ESBLs) efficiently hydrolyze extended-spectrum beta-lactams such as cefotaxime, ceftriaxone, ceftazidime, and aztreonam. ESBLs are most often plasmid-mediated. In Taiwan, the prevalence of ESBLs in bacteria has risen, ranging from 8.5 to 29.8% in Klebsiella pneumoniae and 1.5 to 16.7% in Escherichia coli isolates. The most prevalent types of ESBLs are SHV-5, SHV-12, CTX-M-3, and CTX-M-14 in isolates of K. pneumoniae and E. coli, with differences between institutions. SHV-12 and CTX-M-3 have been reported as the most common ESBLs in isolates of Enterobacter cloacae and Serratia marcescens, respectively. Molecular epidemiology studies suggest that the ESBL-encoding genes have been disseminated either by proliferation of epidemic strains or by transfer of plasmids carrying the resistance traits. The current ESBL screen guidelines of the Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards) are issued for E. coli, Klebsiella spp., and Proteus mirabilis. Owing to the lack of standard methods, it remains difficult to assure the presence of ESBL in an isolate co-harboring an AmpC beta-lactamase, particularly in cases where the latter is produced in larger amounts than the former. Empirical therapy with piperacillin-tazobactam to replace third-generation cephalosporins may help to reduce the occurrence of ESBLs in an institution with a high prevalence of ESBL producers. Carbapenems remain the drugs of choice for serious infections caused by ESBL-producing organisms. To retard the selection for carbapenem-resistant bacteria, 7-alpha-methoxy beta-lactams or fourth-generation cephalosporins can be therapeutic alternatives for mild-to-moderate infections provided that the pharmacokinetic and pharmacodynamic target can be easily achieved.     

Survey of Extended-Spectrum β-Lactamases in Clinical Isolates of Escherichia coli and Klebsiella pneumoniae: Prevalence of TEM-52 in Korea

Two hundred ninety isolates of Escherichia coli were investigated for the production of extended-spectrum beta-lactamases (ESBLs). Fourteen (4.8%) of the 290 strains were found to produce ESBLs. Each of the 14 strains produced one or two ESBLs, as follows: 10 strains produced TEM-52, 1 strain produced SHV-2a, 1 strain produced SHV-12, 1 strain produced a CMY-1-like enzyme, and 1 strain expressed SHV-2a and a CMY-1-like enzyme. Another two strains for which the MICs of ceftazidime and cefoxitin were high, were probable AmpC enzyme hyperproducers. Because of the high prevalence of TEM-52 in E. coli isolates, we further investigated the TEM-type ESBLs produced by Klebsiella pneumoniae in order to observe the distribution of TEM-52 enzymes among Enterobacteriaceae in Korea. All TEM enzymes produced by 12 strains of K. pneumoniae were identified as TEM-52. To evaluate the genetic relatedness among the organisms, ribotyping of TEM-52-producing E. coli and K. pneumoniae was performed. The ribotyping profiles of the organisms showed similar but clearly different patterns. In conclusion, TEM-52 is the most prevalent TEM-type ESBL in Korea.     

Trends in production of extended-spectrum beta-lactamases among enterobacteria of medical interest: report of the second Italian nationwide survey

Results of a 2003 survey carried out in Italy to evaluate the prevalence of extended-spectrum beta-lactamase (ESBL)-producing enterobacteria are presented. Eleven Italian Microbiology Laboratories investigated 9,076 consecutive nonreplicate isolates (inpatients, 6,850; outpatients, 2,226). ESBL screening was performed by MIC data analysis. Confirmation was obtained using the double-disk synergy test and the combination disk test based on CLSI methodology. ESBL determinants were investigated by colony blot hybridization and confirmed by sequencing. Results were compared to those of the 1999 Italian survey (8,015 isolates). The prevalence of ESBL producers was 7.4% among isolates from inpatients (in 1999, 6.3%) and 3.5% among outpatients (no data were available for 1999). Among hospitalized patients, the most prevalent ESBL-positive species was Escherichia coli (Klebsiella pneumoniae in 1999). Proteus mirabilis was the most prevalent ESBL-positive species among outpatients. In both groups, most ESBL-positive pathogens were obtained from urinary tract infections. TEM-type ESBLs were the most prevalent enzymes (45.4%). Non-TEM, non-SHV determinants emerged: CTX-M-type in E. coli and K. pneumoniae, and PER-type in P. mirabilis, Providencia spp., and E. coli. With the exception of 3/163 P. mirabilis isolates and 1/44 Providencia stuartii isolate (all of which were intermediate for imipenem), carbapenems were active against all ESBL-positive enterobacteria. Susceptibility to other drugs was as follows: 84.7% for amikacin, 84.4% for piperacillin-tazobactam, 48.0% for gentamicin, and 32.8% for ciprofloxacin. Carbapenems appear to be the drug of choice. Amikacin and beta-lactam/beta-lactamase inhibitor combinations represent an alternative in non-life-threatening infections. The appearance of ESBL-positive enterobacteria in the community makes it mandatory that family physicians learn how to treat these pathogens.     

Clinical variables associated with the isolation of Klebsiella pneumoniae expressing different extended-spectrum β-lactamases

Clinical variables associated with the isolation of Klebsiella pneumoniae expressing different extended-spectrum beta-lactamases (ESBLs) were studied. Clinical records of patients with ESBL-positive K. pneumoniae isolates between 1989 and 2003 (n = 80) were reviewed retrospectively. Patients with SHV- and TEM-type ESBLs were identified more frequently in the intensive care units (67% and 78%, respectively), whereas those with CTX-M ESBLs were found in medical wards (52.2%) or were outpatients (17.4%) (p <0.01). The absence of urinary or central catheters was associated with CTX-M-10 (p 0.013 and p <0.01, respectively). Central catheter-related infections and secondary bacteraemia were associated more frequently with SHV- and TEM-type ESBLs, whereas urinary tract infections were associated with CTX-M-10. Previous aminoglycoside use was associated particularly with SHV-type ESBLs (p <0.01), whereas amoxycillin-clavulanate and oral cephalosporins were associated with CTX-M-10 (p <0.01 and p 0.050, respectively). The frequency of adequate empirical treatment was low (22%), and 61% of patients were treated according to the susceptibility testing results. Mortality (22%) and related mortality (14%) did not differ statistically according to the type of ESBL. Different ESBL types in K. pneumoniae were associated with different clinical variables, and this should be taken into account in current and future epidemiological scenarios.     

Antimicrobial susceptibility to parenteral and oral agents in a largely polyclonal collection of CTX-M-14 and CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae

Activity of oral and parenteral antimicrobials against consecutively isolated extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (n = 149) and Klebsiella pneumoniae (n = 20) was determined, and susceptibility test methods were compared for parenteral β-lactams. Polymerase chain reaction (PCR) targeting bla(CTX-M), bla(SHV) and bla(TEM), and DNA sequencing and epidemiological typing with pulsed-field gel electrophoresis were performed. PCR targeting pabB was screened for E. coli O25b-ST131. Minimum inhibitory concentrations (MICs) were determined using Etest and broth microdilution. Disc diffusion was performed according to European Committee on Antimicrobial Susceptibility Testing (EUCAST). Dominating genotypes were bla(CTX-M-15) (75%) and bla(CTX-M-14) (23%). Four E. coli clusters (7-18 isolates) were found. Forty-two per cent of E. coli belonged to O25b-ST131. Ciprofloxacin resistance was 72%, trimethoprim resistance was 70%. Among E. coli, resistance to mecillinam (13%), nitrofurantoin (7%) and fosfomycin (3%) was low, although resistance was high in K. pneumoniae (25%, 60%, 85%). Susceptibility to ertapenem was 99%, piperacillin-tazobactam 91%, tigecycline 96% and temocillin 76%. Susceptibility rates obtained with broth microdilution and Etest were in agreement for cefotaxime (2 vs 1%) and ceftazidime (9 vs 11%), but not for piperacillin-tazobactam (59 vs 91%). With disc diffusion major errors occurred with piperacillin-tazobactam (18/169). Several therapeutic alternatives exist for ESBL-producing E. coli, but few exist for K. pneumoniae. Disc diffusion and Etest can accurately predict susceptibility to cefotaxime and ceftazidime, but not to piperacillin-tazobactam with the present breakpoints.     

Using nucleic acid microarrays to perform molecular epidemiology and detect novel β-lactamases: a snapshot of extended-spectrum β-lactamases throughout the world

The worldwide dissemination of extended-spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae is a major concern in both hospital and community settings. Rapid identification of these resistant pathogens and the genetic determinants they possess is needed to assist in clinical practice and epidemiological studies. A collection of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis isolates, including phenotypically ESBL-positive (n = 1,093) and ESBL-negative isolates (n = 59), obtained in 2008-2009 from a longitudinal surveillance study (SMART) was examined using an in vitro nucleic acid-based microarray. This approach was used to detect and identify bla(ESBL) (bla(SHV), bla(TEM), and bla(CTX-M) genes of groups 1, 2, 9, and 8/25) and bla(KPC) genes and was combined with selective PCR amplification and DNA sequencing for complete characterization of the bla(ESBL) and bla(KPC) genes. Of the 1,093 phenotypically ESBL-positive isolates, 1,041 were identified as possessing at least one bla(ESBL) gene (95.2% concordance), and 59 phenotypically ESBL-negative isolates, used as negative controls, were negative. Several ESBL variants of bla(TEM) (n = 5), bla(SHV) (n = 11), bla(CTX-M) (n = 19), and bla(KPC) (n = 3) were detected. A new bla(SHV) variant, bla(SHV-129), and a new bla(KPC) variant, bla(KPC-11), were also identified. The most common bla genes found in this study were bla(CTX-M-15), bla(CTX-M-14), and bla(SHV-12). Using nucleic acid microarrays, we obtained a "molecular snapshot" of bla(ESBL) genes in a current global population; we report that CTX-M-15 is still the dominant ESBL and provide the first report of the new β-lactamase variants bla(SHV-129) and bla(KPC-11).     

Molecular characterization of extended-spectrum beta-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli from a Korean nationwide survey

To determine the prevalence and genotypes of extended-spectrum beta-lactamases (ESBLs) among clinical isolates of Klebsiella pneumoniae and Escherichia coli, we performed antibiotic susceptibility testing, pI determination, induction testing, transconjugation, and DNA sequencing analysis. Among the 509 isolates collected from 13 university hospitals in Korea, 39.2% produced ESBLs. ESBL-producing isolates were detected in every region in Korea. A total of 44.6% of the isolates produced both TEM- and SHV-type ESBLs, and 52% of ESBL-producing isolates transferred resistance to ceftazidime by transconjugation. The ESBLs were TEM-19, TEM-20, TEM-52, SHV-2a, SHV-12, and one new variant identified for the first time in Korea, namely, TEM-116. TEM-1 and SHV-12 were by far the most common variants. TEM-1, TEM-116, and SHV-12 showed a high prevalence in K. pneumoniae. Two isolates (E. coli SH16 and K. pneumoniae SV3) produced CMY-1-like beta-lactamases, which play a decisive role in resistance to cefoxitin and cefotetan, as well as TEM-type enzymes (TEM-20 and TEM-52, respectively). Using MIC patterns and DNA sequencing analysis, we postulated a possible evolution scheme among TEM-type beta-lactamases in Korea: from TEM-1 to TEM-19, from TEM-19 to TEM-20, and from TEM-20 to TEM-52.     

Detection of extended-spectrum beta-lactamase (ESBL)-producing strains by the Etest ESBL screen.

Resistance to contemporary broad-spectrum beta-lactams, mediated by extended-spectrum beta-lactamase (ESBL) enzymes, is an increasing problem worldwide. The Etest (AB Biodisk, Solna, Sweden) ESBL screen uses stable gradient technology to evaluate the MIC of ceftazidime alone compared with the MIC of ceftazidime with clavulanic acid (2 micrograms/ml) to facilitate the recognition of strains expressing inhibitable enzymes. In the present study, ESBL-producing strains (17 Escherichia coli transconjugants) were studied to define "sensitive" interpretive criteria for the Etest ESBL screen. These criteria (reduction of the ceftazidime MIC by > 2 log2 dilution steps in the presence of clavulanic acid) defined a group of 92 probable ESBL-positive organisms among the 225 tested strains of Klebsiella species and E. coli having suspicious antibiogram phenotypes. With a subset of 82 clinical strains, the Etest ESBL screen was more sensitive (100%) than the disk approximation test (87%) and was more convenient. The MICs of ciprofloxacin, gentamicin, and tobramycin at which 50% of isolates are inhibited were 16- to 128-fold higher (coresistance) for the ESBL screen-positive group of strains than for the ESBL screen-negative group of strains. Some strains for which cephalosporin MICs were elevated and which were Etest ESBL screen negative were also cefoxitin resistant, i.e., consistent with a chromosomally mediated AmpC resistance phenotype. The Etest ESBL screen test with the ceftazidime substrate appears to be a useful method for detecting or validating the presence of enteric bacilli potentially producing this type of beta-lactamase.     

Outbreak of infection caused by Enterobacter cloacae producing the novel VEB-3 beta-lactamase in China

Over a 4-month period from November 2002 to February 2003, 27 ceftazidime-resistant or cefotaxime-resistant nonrepetitive Enterobacter cloacae isolates were collected from 27 patients hospitalized at HuaShan Hospital, Shanghai, People's Republic of China. The Etest did not detect extended-spectrum beta-lactamases (ESBLs) in those 27 isolates; however, screening by the NCCLS ESBL disk test and confirmatory tests detected ESBLs in 4 of 27 isolates and PCR detected ESBLs in 23 of 27 isolates. The majority of ESBL producers exhibited the same repetitive extragenic palindromic PCR pattern but harbored different ESBL genes. CTX-M-3 was the most prevalent ESBL in our study. Interestingly, 12 clonally related E. cloacae isolates possessed a novel bla(VEB)-type beta-lactamase, bla(VEB-3). Bla(VEB-3) was encoded by the chromosome and was located in an integron. Nine of the 12 isolates harbored both the bla(VEB-3) and the bla(CTX-M-3)-like ESBLs. This is the first report of a VEB-1-like ESBL in China and the first report of the simultaneous presence of VEB-1 and CTX-M-3-like ESBLs in an isolate.     

Dissemination of extended-spectrum beta-lactamase-producing Enterobacteriaceae in intensive care units of a medical center in Taiwan

The prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in a tertiary hospital in Taiwan was assessed over a 16-month period. A total of 125 nonrepetitive ESBL-producing isolates of Enterobacter cloacae, Escherichia coli, and Klebsiella pneumoniae were available for investigation using molecular methods. Four predominant intensive care units (ICUs) were identified, and SHV-12 (59%), CTX-M- 3 (36%), and CTX-M-14 (14%) were the three most frequent ESBLs. SHV-12 was predominant among E. cloacae in the burn unit and K. pneumoniae in the other three chest medicine-related ICUs. CTX-M-3 was predominant among E. coli and K. pneumoniae in three other ICUs. The dissemination of ESBL-producing Enterobacteriaceae in four ICUs of a medical center in Taiwan is a consequence of the clonal dissemination of a few epidemic strains along with the horizontal transmission of resistance genes-carrying plasmids among bacterial organisms.     

Characterization of clinical isolates of Enterobacteriaceae from Italy by the BD Phoenix extended-spectrum beta-lactamase detection method

Production of extended-spectrum beta-lactamases (ESBLs) is an important mechanism of beta-lactam resistance in Enterobacteriaceae: Identification of ESBLs based on phenotypic tests is the strategy most commonly used in clinical microbiology laboratories. The Phoenix ESBL test (BD Diagnostic Systems, Sparks, Md.) is a recently developed automated system for detection of ESBL-producing gram-negative bacteria. An algorithm based on phenotypic responses to a panel of cephalosporins (ceftazidime plus clavulanic acid, ceftazidime, cefotaxime plus clavulanic acid, cefpodoxime, and ceftriaxone plus clavulanic acid) was used to test 510 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Providencia stuartii, Morganella morganii, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, Citrobacter freundii, and Citrobacter koseri. Of these isolates, 319 were identified as ESBL producers, and the remaining 191 were identified as non-ESBL producers based on the results of current phenotypic tests. Combined use of isoelectric focusing, PCR, and/or DNA sequencing demonstrated that 288 isolates possessed bla(TEM-1)- and/or bla(SHV-1)-derived genes, and 28 had a bla(CTX-M) gene. Among the 191 non-ESBL-producing isolates, 77 isolates produced an AmpC-type enzyme, 110 isolates possessed TEM-1, TEM-2, or SHV-1 beta-lactamases, and the remaining four isolates (all K. oxytoca strains) hyperproduced K1 chromosomal beta-lactamase. The Phoenix ESBL test system gave positive results for all the 319 ESBL-producing isolates and also for two of the four K1-hyperproducing isolates of K. oxytoca. Compared with the phenotypic tests and molecular analyses, the Phoenix system displayed 100% sensitivity and 98.9% specificity. These findings suggest that the Phoenix ESBL test can be a rapid and reliable method for laboratory detection of ESBL resistance in gram-negative bacteria.     

Dramatic increase in prevalence of fecal carriage of extended-spectrum beta-lactamase-producing Enterobacteriaceae during nonoutbreak situations in Spain

The occurrence of extended-spectrum beta-lactamase (ESBL)-producing isolates has increased worldwide. Fecal carriage of ESBL-producing isolates has mainly been detected in nosocomial outbreaks, and few studies have evaluated fecal carriage during nonoutbreak situations and among patients in the community. We have studied the prevalence of ESBLs in 1,239 fecal samples from 849 patients (64.1% of whom were ambulatory) in 1991 and have compared the prevalence data with those obtained in 2003 for 400 fecal samples from 386 patients (75.9% of whom were ambulatory) and 108 samples from independent healthy volunteers. Samples were diluted in saline and cultured in two MacConkey agar plates supplemented with ceftazidime (1 microg/ml) and cefotaxime (1 microg/ml), respectively. Colonies were screened (by the double-disk synergy test) for ESBL production. The clonal relatedness of all ESBL-producing isolates was determined by pulsed-field gel electrophoresis with XbaI digestion; and the ESBLs of all ESBL-producing isolates were characterized by isoelectric focusing, PCR, and sequencing. The rates of fecal carriage of ESBL-producing isolates increased significantly (P < 0.001) in both hospitalized patients and outpatients, from 0.3 and 0.7%, respectively, in 1991, to 11.8 and 5.5%, respectively, in 2003. The rate of occurrence of ESBL-producing isolates among healthy volunteers was 3.7%. All ESBL-producing isolates recovered in 2003 were nonepidemic clones of Escherichia coli. ESBL characterization revealed an increasing diversity of ESBL types: TEM-4 and CTX-M-10 were the only enzymes detected in 1991, whereas TEM-4, TEM-52, SHV-12, CTX-M-9, CTX-M-10, CTX-M-14, and a CTX-M-2-like enzyme were recovered in 2003. The ESBL-producing isolates recovered from outpatients in 2003 corresponded to a CTX-M-9-type cluster (62.5%) and SHV-12 (31.2%), whereas TEM-4 was detected only in hospitalized patients. The frequencies of coresistance in isolates recovered in 2003 were as follows: sulfonamide, 75%; tetracycline, 64.3%; streptomycin, 57.1%; quinolones, 53.5%; and trimethoprim, 50%. The increased prevalence of fecal carriage of ESBL-producing isolates during nonoutbreak situations in hospitalized patients and the establishment of these isolates in the community with coresistance to non-beta-lactam antibiotics, including quinolones, represent an opportunity for these isolates to become endemic.     

Prevalence of extended-spectrum beta-lactamases among Escherichia coli and Klebsiella pneumoniae isolates in a tertiary care hospital

Extended-spectrum beta-lactamases (ESBLs) continue to be a major problem in clinical setups the world over, conferring resistance to the expanded-spectrum cephalosporins. Knowledge about their prevalence is essential to guide towards appropriate antibiotic treatment. The aim of the present study is to determine the prevalence of ESBL producers among Escherichia coli and Klebsiella pneumoniae isolates at a tertiary care institution. A total of 357 clinical isolates comprising E. coli (n = 181) and K. pneumoniae (n = 176) were recovered from various clinical samples over a period of six months from April to September 2006. Antibiogram profile of these isolates was determined to commonly used antibiotics, along with screening for ESBL production by the screening test as recommended by the Clinical Laboratory Standards Institute (CLSI). Isolates which showed positive results with screening test were shortlisted for confirmatory tests of ESBL production. Two tests were performed: phenotypic confirmatory test with combination disk and the minimum inhibitory concentration (MIC) reduction test. Out of 357 isolates of E. coli and K. pneumoniae screened for ESBL production, 120 were found to be potential ESBL producers. Of these, 80 isolates were confirmed to be ESBL producers. Thus the prevalence of ESBL-producing isolates of E. coli and K. pneumoniae was found to be 22% (80 out of 357). This was significantly lower than the data available from other hospitals.     

The first major extended-spectrum beta-lactamase outbreak in Scandinavia was caused by clonal spread of a multiresistant Klebsiella pneumoniae producing CTX-M-15

Between May and December 2005, 64 multidrug-resistant isolates of Klebsiella pneumoniae were detected from patients admitted to Uppsala University Hospital. This represented a dramatic increase in ESBL-producing K. pneumoniae compared to previous years. To investigate the epidemiology and to characterize the resistance mechanisms of the isolates, a study was initiated. Antibiotic susceptibility was determined by means of the Etest and the disc diffusion method. Extended-spectrum beta-lactamase (ESBL) production was identified by clavulanic acid synergy test and confirmed with PCR amplification followed by DNA sequencing. DNA profiles of the isolates were examined with pulsed-field gel electrophoresis (PFGE). All isolates were resistant or exhibited reduced susceptibility to cefadroxil, cefuroxime, cefotaxime, ceftazidime, aztreonam, piperacillin/tazobactam, ciprofloxacin, tobramycin, and trimethoprim-sulfamethoxazole. They produced ESBL of the CTX-M-15 type, and the involvement of a single K. pneumoniae clone was shown. This is the first major clonal outbreak of multiresistant ESBL-producing K. pneumoniae in Scandinavia. The outbreak demonstrates the epidemic potential of enterobacteria containing ESBLs of the CTX-M type, even in a country with a relatively low selective pressure and a low prevalence of multiresistant bacteria.