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.     

Prevalence of plasmid-mediated AmpC beta-lactamases in Escherichia coli and Klebsiella pneumoniae in Korea

Cefoxitin-resistant Escherichia coli and Klebsiella pneumoniae are relatively prevalent in Korea, suggesting dissemination of plasmid-mediated AmpC beta-lactamases. In this study, 238 isolates of cefoxitin-resistant E. coli and K. pneumoniae (not including subspecies ozaenae and rhinoscleromatis) were collected in 2003 from 16 Korean hospitals. The prevalence of plasmid-mediated AmpC beta-lactamases was determined by PCR. The AmpC gene alleles detected in E. coli and K. pneumoniae were bla(DHA-1), 10 (8.6%) and 93 (76.2%); bla(CMY-1)-like, 14 (12.1%) and 2 (1.6%); and bla(CMY-2)-like, 38 (32.7%) and 1 (0.8%) isolates, respectively. The genes identified were bla(DHA-1), bla(CMY10)-like, and bla(CMY-2)-like, and a new variant, bla(CMY-18). Plasmidmediated AmpC gene allele-positive isolates were present both in large city and in small province hospitals, as well as in isolates from outpatients. The proportions of plasmid-mediated AmpC gene-positive isolates were similar in both expanded spectrum beta-lactamase (ESBL)-producing and -nonproducing isolates. In conclusion, DHA-1, CMY-2-like, and CMY-10-like plasmid-mediated AmpC beta-lactamase-producing K. pneumoniae and E. coli isolates are widely disseminated in both large city and small province hospitals. Absence of bla(CMY-1) and detection of a novel variant of bla(CMY-2), bla(CMY-18), indicate continued evolution of the prototype genes. Similar proportions of plasmid-mediated AmpC gene-positive isolates in both ESBL-producing and -nonproducing isolates suggest unhindered future spread of these resistances.     

Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the National Committee for Clinical Laboratory Standards extended-spectrum beta-lactamase detection methods

Extended-spectrum beta-lactamases (ESBLs) are enzymes found in gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. In 1999, the National Committee for Clinical Laboratory Standards (NCCLS) published methods for screening and confirming the presence of ESBLs in Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli. To evaluate the confirmation protocol, we tested 139 isolates of K. pneumoniae that were sent to Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) from 19 hospitals in 11 U.S. states. Each isolate met the NCCLS screening criteria for potential ESBL producers (ceftazidime [CAZ] or cefotaxime [CTX] MICs were > or =2 microg/ml for all isolates). Initially, 117 (84%) isolates demonstrated a clavulanic acid (CA) effect by disk diffusion (i.e., an increase in CAZ or CTX zone diameters of > or =5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by > or =3 dilutions). For five isolates, a CA effect could not be determined initially by broth microdilution because of off-scale CAZ results. However, a CA effect was observed in two of these isolates by testing cefepime and cefepime plus CA. The cefoxitin MICs for 23 isolates that failed to show a CA effect by broth microdilution were > or =32 microg/ml, suggesting either the presence of an AmpC-type beta-lactamase or porin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a beta-lactamase with a pI of > or =8.3 suggestive of an AmpC-type beta-lactamase; 6 of the 7 isolates were shown by PCR to contain both ampC-type and bla(OXA) genes. The IEF profiles of the remaining 16 isolates showed a variety of beta-lactamase bands, all of which had pIs of < or =7.5. All 16 isolates were negative by PCR with multiple primer sets for ampC-type, bla(OXA), and bla(CTX-M) genes. In summary, 83.5% of the K. pneumoniae isolates that were identified initially as presumptive ESBL producers were positive for a CA effect, while 5.0% contained beta-lactamases that likely masked the CA effect. The remaining 11.5% of the isolates studied contained beta-lactamases that did not demonstrate a CA effect. An algorithm based on phenotypic analyses is suggested for evaluation of such isolates.     

Prevalence of plasmid-mediated AmpC beta-lactamases in a Chinese university hospital from 2003 to 2005: first report of CMY-2-Type AmpC beta-lactamase resistance in China

The aim of this study was to investigate the prevalences of plasmid-mediated AmpC beta-lactamases (PABLs) in isolates of Escherichia coli and Klebsiella spp. from a university hospital in China. A total of 1,935 consecutive nonrepeat clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca were collected between January 2003 and July 2005. The isolates with cefoxitin zone diameters less than 18 mm (screen positive) were selected for PCR of the bla(AmpC) genes and sequencing. Fifty-four (2.79%) isolates harbored PABLs, as demonstrated by PCR and isoelectric focusing. Sequence analysis revealed the presence of bla(DHA-1) and bla(CMY-2) genes. The Southern blot hybridization studies confirmed that bla(CMY-2) and bla(DHA-1) were located on plasmids. Based on species, PABLs were detected in 4.29% (29 isolates of DHA-1 and 1 isolate of CMY-2) of K. pneumoniae, 1.91% (11 isolates of DHA-1 and 12 isolates of CMY-2) of E. coli, and 3.03% (1 isolate of DHA-1) of K. oxytoca isolates. In contrast to our anticipation, the occurrence rate of DHA-1-producing K. pneumonia significantly decreased (P < 0.01), from 7.54% in 2003 to 2.72% in 2004. The results of random amplified polymorphic DNA analysis indicate that the prevalences of DHA-1-producing K. pneumoniae and CMY-2-producing E. coli strains were not due to epidemic strains. In conclusion, DHA-1 was the most prevalent acquired AmpC beta-lactamase in this collection of isolates from a medical center in China, and DHA-1-producing K. pneumoniae was the most prevalent bacterium harboring a PABL. To the best of our knowledge, this is the first report of CMY-2-type AmpC beta-lactamases in the Chinese mainland.     

Involvement of SHV-12 and SHV-2a encoding plasmids in outbreaks of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a Tunisian neonatal ward

Previous genotypic investigations of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae recovered in a Tunisian neonatal ward revealed the spread of two epidemic strains and a high number of genetically unrelated isolates. The aim of the present study was to determine the role of the dissemination of self-transferrable plasmids harboring bla genes in the outbreaks experienced by the ward. The 49 previously identified clinical isolates of ESBL-producing K. pneumoniae were examined for relationships between their enzymes and plasmids. Analysis of crude extracts by isoelectric focusing showed four beta-lactamase-activities at pI 8.2, 7.6, 6, and 5.4. Clinical isolates contained large plasmids that could be transferred by conjugation and transformation conferring resistance to expanded-spectrum cephalosporins. DNA amplification and sequencing were performed to confirm the identities of transferred beta-lactamases. Nucleotide sequence analysis of SHV-specific PCR products from six isolates identified two bla(SHV) genes corresponding to SHV derived ESBLs, SHV-12 and SHV-2a. PstI digestion of plasmid DNA from transformants revealed six restriction patterns. The occurrence of the prevalent plasmid pattern in both epidemic strains and unrelated isolates indicated that diffusion and endemic persistence of the bla(SHV-ESBL) genes in the ward were due to concomitant spread of epidemic strains and plasmid dissemination among unrelated strains.     

First outbreak of multidrug-resistant Klebsiella pneumoniae producing both SHV-12-type extended-spectrum beta-lactamase and DHA-1-type AmpC beta-lactamase at a Korean hospital

PURPOSE: Coexistence of different classes of beta-lactamases in a single bacterial isolate may pose diagnostic and therapeutic challenges. We investigated a spread of Klebsiella pneumoniae isolates co-producing an AmpC beta-lactamase and an extended-spectrum beta-lactamase (ESBL) in a university hospital. MATERIALS AND METHODS: Over a three-month period, a total of 11 K. pneumoniae isolates, which exhibited resistance to cefotaxime, aztreonam, and cefoxitin, were isolated. These isolates showed positive to ESBLs by double disk tests. Minimal inhibitory concentrations (MICs) were determined by broth microdilution testing. All isolates were examined by isoelectric focusing, PCR and sequence analysis to identify bla(SHV) and bla(DHA), and molecular typing by pulsed-field gel electrophoresis (PFGE). RESULTS: All 11 isolates were highly resistant (MIC, > or = 128microg/ml) to ceftazidime, aztreonam, and cefoxitin, while they were susceptible (MIC, < or = 2microg/ml) to imipenem. The bla(SHV-12) and bla(DHA-1) genes were detected by PCR and sequence analysis. PFGE revealed a similar pattern in 10 of the 11 strains tested. CONCLUSION: This is the first outbreak report of K. pneumoniae in Korea which co-produced SHV-12 and DHA-1 beta-lactamase, and we suggest a clonal spread of multidrug-resistant K. pneumoniae at a hospital.     

Characterization of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from hospital environments in Algeria

For detecting extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in the hospital environment, sedimentation plates were placed in the rooms of two hospitals. Three environmental isolates, two Klebsiella pneumoniae, and one Escherichia coli resistant to extended-spectrum cephalosporins with a phenotype indicating CTX-M enzymes production (the minimum inhibitory concentration [MIC] of cefotaxime was higher than the MIC of ceftazidime) were recovered. By PCR and sequencing, the three isolates were found to produce CTX-M-15. The bla(CTX-M-15) genes in the three isolates were transferred by conjugation. One K. pneumoniae environmental isolate showed an identical and unique RAPD profile with two other K. pneumoniae clinical isolates recovered from urinary tract infection from patients hospitalized in two different wards of another hospital.     

Variety of TEM-, SHV-, and CTX-M-type beta-lactamases present in recent clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae from Taiwan

A total of 171 hospitals' isolates of E. coli, K. pneumoniae, and E. cloacae with a minimum inhibitory concentration (MIC) of > or =2 microg/ml for ceftazidime or cefotaxime were evaluated for the production of beta-lactamases. PCR amplification with specific primers for the bla (SHV), bla (TEM), and bla (CTX) genes revealed that a total of 53, 81, and 43 of these genes were amplified, respectively. Sequencing results confirmed that TEM-1, CTX-M-3 and -14, SHV-1, -5, -11, -12, and -33, OXY-1a, and LEN-1 were presented among these isolates. No specific large cluster of isolates carried the same beta-lactamases, indicating the wide diversity of the collected strains. Plasmid spread between E. coli and K. pneumoniae was identified in few isolates. Combinations of TEM, SHV, and CTX beta-lactamase genes, including extended-spectrum beta-lactamase genes, were observed in all three species.     

Extended-spectrum beta-lactamase genes of Klebsiella pneumoniae strains in Taiwan: recharacterization of shv-27, shv-41, and tem-116

Klebsiella pneumoniae causing primary liver abscess (PLA) is emerging. This study identified the beta-lactamases genes of K. pneumoniae isolates in Taiwan. The susceptibilities of beta-lactam antibiotics of 30 K. pneumoniae strains associated with primary liver abscess and 30 noninvasive strains were analyzed. The beta-lactamase genes of randomly selected 24 strains from community-acquired infection and 7 extended-spectrum beta-lactamases (ESBL) strains were identified by PCR and DNA sequencing. Protein expression and the ESBL phenotype of beta-lactamase were determined. All 60 strains were ampicillin resistant and cefotaxime susceptible, whereas no strain was ESBL producing. In the 24 selected strains, shv-1a was found in 14, shv-1 in 7; shv-26, shv-27, and shv-41 were detected in one. However, all of these 24 strains had the tem-116 gene. In 7 ESBL-producing K. pneumoniae strains, shv-5a was found in 5, whereas shv-5 and ctx-m-9 group were detected in 1 strain. Two previously reported ESBL genes, shv-27 and tem-116, as well as a suspected ESBL gene, shv-41, were found in non-ESBL-producing strains. Transformation of these genes conferred ampicillin resistance but not the ESBL-producing phenotype in Escherichia coli. Beta-lactamase protein expression of these strains was further confirmed by western blotting. In conclusion, ESBL is rare in community-acquired K. pneumoniae infection and is not associated with PLA in Taiwan. The shv-5a, shv-5, and ctx-m-9 groups are present in ESBL-producing strains in Taiwan, but shv-27, shv-41, and tem-116 are not ESBL genes.     

Characterization of beta-lactam-resistant Klebsiella oxytoca isolated in a neonatal intensive care unit

The occurrence of Klebsiella oxytoca resistant to ampicillin, piperacillin, aztreonam and cefuroxime in a neonatal intensive care unit, including two cases of septicemia, was shown to consist of a spread on three consecutive occasions caused by three different biochemical Klebsiella oxytoca phenotypes. All isolates, except six surface isolates from one infant belonging to phenotype 1, were sensitive to cefotaxime (MIC 0.5-4 mg/l) and ceftazidime (MIC 0.25-1 mg/l). Isolates of phenotypes 1 and 2 produced a beta-lactamase with an isoelectric point of 5.5 and isolates of phenotype 3, a beta-lactamase with an isoelectric point of 7.9. The beta-lactamases of all three phenotypes hydrolysed benzylpenicillin and more slowly cephalothin. All phenotype 1 isolates carried a 2.9 Md plasmid and most isolates also a 36 Md plasmid. All phenotype 2 isolates carried a 4.8 Md plasmid and one isolate also a 30 Md plasmid. The phenotype 3 isolates carried only one 85 Md plasmid.     

Utility of NCCLS guidelines for identifying extended-spectrum beta-lactamases in non-Escherichia coli and Non-Klebsiella spp. of Enterobacteriaceae

NCCLS screening and confirmation methods for detecting extended-spectrum beta-lactamases (ESBLs) apply only to Escherichia coli and Klebsiella spp., yet ESBLs have been found in other members of the family Enterobacteriaceae. We evaluated the effectiveness of NCCLS methods for detecting ESBLs in 690 gram-negative isolates of Enterobacteriaceae that excluded E. coli, Klebsiella pneumoniae, and Klebsiella oxytoca. Isolates were collected between January 1996 and June 1999 from 53 U.S. hospitals participating in Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology). The antimicrobial susceptibility patterns of the isolates were determined by using the NCCLS broth microdilution method (BMD), and those isolates for which the MIC of ceftazidime, cefotaxime, ceftriaxone, or aztreonam was >or=2 microg/ml or the MIC of cefpodoxime was >or=8 microg/ml (positive ESBL screen test) were further tested for a clavulanic acid (CA) effect by BMD and the disk diffusion method (confirmation tests). Although 355 (51.4%) of the isolates were ESBL screen test positive, only 15 (2.2%) showed a CA effect. Since 3 of the 15 isolates were already highly resistant to the five NCCLS indicator drugs, ESBL detection would have an impact on the reporting of only 1.7% of the isolates in the study. Only 6 of the 15 isolates that showed a CA effect contained a bla(TEM), bla(SHV), bla(CTX-M), or bla(OXA) beta-lactamase gene as determined by PCR (with a corresponding isoelectric focusing pattern). Extension of the NCCLS guidelines for ESBL detection to Enterobacteriaceae other than E. coli and Klebsiella spp. does not appear to be warranted in the United States at present, since the test has poor specificity for this population and would result in changes in categorical interpretations for only 1.7% of Enterobacteriaceae tested.     

Mechanisms of resistance in Enterobacteriaceae towards beta-lactamase antibiotics

Except for Salmonella spp., all Enterobacteriaceae produce intrinsic chromosomal encoded beta-lactamases which, beside their physiologic role in cell-wall synthesis and natural beta-lactam protection, are responsible for intrinsic resistance of individual species among Enterobacteriaceae. E. coli and Shigella spp. produce a small amount of AmpC beta-lactamases and are susceptible to ampicillin and other beta-lactam antibiotic agents. Enterobacter spp, C. freundii, Serratia spp., M. morganii, P. stuarti and P. rettgeri produce small amounts of inducible AmpC beta-lactamases which are not inhibited by beta-lactamases inhibitor, causing intrinsic resistance to ampicillin, co-amoxiclav and first-generation cephalosporins. K. pneumoniae produces small amounts of SHV-1 beta-lactamases, and K. oxytoca chromosomal K1 beta-lactamase, causing resistance to ampicillin, carbencillin, ticarcillin and attenuated zone of inhibition to piperacillin, compared to piperacillin with tazobactam. They are susceptible to beta-lactamase inhibitors. Whereas P. mirabilis shows a minor chromosomal expression of beta-lactamases, P. vulgaris produces chromosomal beta-lactamases of class A (cefuroximases), causing resistance to ampicillin, ticarcillin, and first- and second-generation cephalosporins. Antibiotics have caused the appearance of acquired or secondary beta-lactamases, with the sole function of protecting bacteria from antibiotics. The production of broad-spectrum beta-lactamases (TEM-1, TEM-2, SHV-1, OXA-1) results in resistance to ampicillin, ticarcillin, first-generation cephalosporins and piperacillin. A high level of beta-lactamases leads to resistance to their inhibitors. The plasmid-mediated extended-spectrum beta-lactamases (ESBLs) are of increasing concern. Most are mutants of classic TEM- and SHV-beta-lactamases types. Unlike these parent enzymes, ESBLs hydrolyze oxymino-cephalosporins such as cefuroxime, cefotaxime, ceftriaxone, ceftizoxime, ceftazidime, cefpirome and cefepime, aztreonam, as well as penicillins and other cephalosporins, except for cephamycin (cefoxitin and cefotetan). They are inhibited by beta-lactamase inhibitors. AmpC beta-lactamases are chromosomal and inducible in most Enterobacter spp., C. freundii, Serratia spp., M. morganii and Providentia spp. They are resistant to almost all penicillins and cephalosporins, to beta-lactamase inhibitors and aztreonam, and are susceptible to cefepime and carbapenems as well. Plasmid-mediated AmpC beta-lactamases have arisen through the transfer of chromosomal genes for the inducible AmpC beta-lactamase onto plasmids. All plasmid-mediated AmpC beta-lactamases have similar substrate profiles to the parental enzymes from which they appear to be derived. With one exception, plasmid-mediated AmpCs differ from chromosomal AmpCs in being uninducible. The National Committee for Clinical Laboratory Standards (NCCLS) has issued recommendations for ESBL screening and confirmation for isolates of E. coli, K. pneumoniae and K. oxytoca. No NCCLS recommendations exist for ESBLs detection and reporting for other organisms or for detecting plasmid-mediated AmpC beta-lactamases. High-level expression of AmpC may prevent recognition of an ESBL in species that produce a chromosomally encoded inducible AmpC beta-lactamase. AmpC-inducible species (e. g. Enterobacter spp. and C. freundii) can be recognized by cefoxitin/cefotaxime disk antagonism tests. Since clinical laboratories are first to encounter bacteria with new forms of antibiotic resistance, they need appropriate tools to recognize these bacteria, including trained staff with sufficient time and equipment to follow up important observations. Because bacterial pathogenes are constantly changing, training must be an ongoing process.     

Multiplex PCR amplification assay for the detection of blaSHV, blaTEM and blaCTX-M genes in Enterobacteriaceae

Extended-spectrum beta-lactamases (ESBLs) are often mediated by (bla-)SHV, (bla)TEM and (bla)CTX-M genes in Enterobacteriaceae and other Gram-negative bacteria. Numerous molecular typing methods, including PCR-based assays, have been developed for their identification. To reduce the number of PCR amplifications needed we have developed a multiplex PCR assay which detects and discriminates between (bla-)SHV, (bla)TEM and (bla)CTX-M PCR amplicons of 747, 445 and 593 bp, respectively. This multiplex PCR assay allowed the identification of (bla-)SHV, (bla)TEM and (bla)CTX-M genes in a series of clinical isolates of Enterobacteriaceae with previously characterised ESBL phenotype. The presence of (bla)SHV, (bla)TEM and (bla)CTX-M genes was confirmed by partial DNA sequence analysis. Apparently, the universal well-established CTX-M primer pair used here to reveal plasmid-encoded (bla)CTX-M genes would also amplify the chromosomally located K-1 enzyme gene in all Klebsiella oxytoca strains included in the study.     

Cross-class resistance to non-beta-lactam antimicrobials in extended-spectrum beta-lactamase-producing Klebsiella pneumoniae

Extended spectrum beta-lactamases are modified beta-lactamase enzymes that impart resistance to third-generation cephalosporins and make all beta-lactam antibiotics and cephalosporins useless for therapy. We compared the antimicrobial susceptibility profiles of extended-spectrum beta-lactamase (ESBL)-producing and non-ESBL-producing isolates of Klebsiella pneumoniae. The ESBL producers had significantly diminished susceptibility compared with the non-ESBL producers for gentamicin (P < .001), tobramycin (P < .001), amikacin (P < .005), trimethoprim-sulfamethoxazole (P < .01), ciprofloxacin (P < .001), and nitrofurantoin (P < .001). All isolates were susceptible to imipenem. ESBL-producing K pneumoniae may also be resistant to non-beta-lactam antibiotics. Therefore, susceptibility testing of these isolates is critical for guiding therapy.     

Occurrence and diversity of integrons and beta-lactamase genes among ampicillin-resistant isolates from estuarine waters

The aim of the present study was to assess the occurrence and molecular diversity of beta-lactamase genes and integrons among Gram-negative ampicillin-resistant bacteria from Ria de Aveiro. Ampicillin-resistant isolates were selected and subjected to genotyping using REP-PCR. Representatives from each REP pattern were affiliated with the following taxa by sequencing the 16S rRNA gene: Aeromonas caviae, A. hydrophila, A. media, A. molluscorum, A. veronii, A. salmonicida, Aeromonas sp., Pseudomonas putida, Pseudomonas sp., Escherichia coli, Escherichia sp., Shigella sonnei, Shigella sp., Klebsiella pneumoniae, K. oxytoca, Raoultella ornithinolytica, R. terrigena, R. planticola, Citrobacter freundii, Morganella morganii and Enterobacter sp. Isolates affiliated with genera Escherichia or Shigella were identified as Escherichia coli using phenotypic-based tests. PCR was used to assess beta-lactamase encoding sequences (bla(TEM), bla(SHV), bla(CARB), bla(CTX-M), bla(IMP), bla(VIM), bla(CphA/IMIS), bla(OXA-A), bla(OXA-B), bla(OXA-C)), class 1 and class 2 integrases, and integron variable regions. Sequence analysis of PCR products was performed. beta-Lactamase genes were detected in 77.8% of the Enterobacteriaceae and in 10.5% of the Aeromonas. The most frequently detected gene was bla(TEM), followed by bla(SHV,)bla(OXA-B), bla(CphA/IMIS) and bla(CARB). Retrieved sequences shared high homology with previously described beta-lactamases. The intI1 gene was present in 29.6% of the Enterobacteriaceae and in 21% of the Aeromonas isolates. The intI2 gene was present in 4 isolates. A total of 13 cassettes included in 12 different cassette arrays were identified. The most frequently found resistance gene cassettes were aadA variants. Previous investigations based on cultivation-independent approaches revealed higher molecular diversity among beta-lactamase-encoding sequences in this estuary. This fact reinforces the hypothesis that cultivation-dependent approaches may underestimate the prevalence of antibiotic resistance genes in environmental samples and may introduce bias in the recovery of their molecular variants.     

Molecular epidemiology of Klebsiella pneumoniae producing SHV-5 beta- lactamase: parallel outbreaks due to multiple plasmid transfer.

Over a period of 22 months, 32 patients treated in three independent intensive care units of the Innsbruck University Hospital were infected with extended-spectrum beta-lactamase-producing members of the family Enterobacteriaceae (30 Klebsiella pneumoniae isolates, 1 Klebsiella oxytoca isolate, and 1 Escherichia coli isolate). As confirmed by sequencing of a bla gene PCR fragment, all isolates expressed the SHV-5-type beta-lactamase. Genomic fingerprinting of epidemic strains with XbaI and pulsed-field gel electrophoresis grouped 20 of 21 isolates from ward A into two consecutive clusters which included 1 of 3 ward B isolates. All six K. pneumoniae isolates from ward C formed a third cluster. Stool isolates of asymptomatic patients and environmental isolates belonged to these clusters as well. Additionally, 2,600 routine K. pneumoniae isolates from the surrounding provinces (population, 900,000) were screened for SHV-5 production. Only one of six nonepidemic isolates producing SHV-5 beta-lactamase was matched with the outbreak strains by genomic fingerprinting. Plasmid fingerprinting, however, revealed the epidemic spread of a predominant R-plasmid, with a size of approximately 80 kb, associated with 29 of the 30 K. pneumoniae isolates. This plasmid was also present in the single K. oxytoca and E. coli isolates from ward C and in three nonepidemic isolates producing SHV-5. Our results underline that strain typing exclusively on the genomic level can be misleading in the epidemiological investigation of plasmid-encoded extended-spectrum beta-lactamases. Our evidence for multiple events of R-plasmid transfer between species of the family Enterobacteriaceae in this nosocomial outbreak stresses the need for plasmid typing, especially because SHV-5 beta-lactamase seems to be regionally spread predominantly via plasmid transfer.     

Outbreak of OXY-2-Producing Klebsiella oxytoca in a renal transplant unit

We describe a Klebsiella oxytoca infection outbreak in a renal transplant unit that involved seven patients. All strains belonged to a single pulsed-field gel electrophoresis pattern and were resistant to amoxicillin-clavulanate, cefuroxime, piperacillin-tazobactam, and aztreonam but susceptible to ceftriaxone, ceftazidime, cefepime, and imipenem. Chromosomal beta-lactamase hyperproduction was caused by a point mutation in the bla(OXY-2) gene promoter region.     

Molecular epidemiology and characterization of plasmid-encoded beta-lactamases produced by Tunisian clinical isolates of Salmonella enterica serotype Mbandaka resistant to broad-spectrum cephalosporins

We studied 31 clinical isolates of Salmonella enterica serotype Mbandaka resistant to broad-spectrum cephalosporins and recovered in Tunisia over a 5-year period. The transferability of this resistance was demonstrated by conjugation experiments. Thirty of the 31 isolates were positive in the double-disk synergy test. By isoelectric focusing analysis, all of the isolates were found to produce a band of beta-lactamase activity with a pI of 5.9. Three of these isolates produced an additional band with a pI of 7.6. PCR and DNA sequencing identified these beta-lactamases as TEM-4 and SHV-2a, respectively. The remaining isolate, highly resistant to ceftazidime but susceptible to cefepime, produced a beta-lactamase that focused at pI 7.8. No synergy was detected by the double-disk synergy test. Sequence analysis of the bla gene amplified by PCR showed that the plasmid-mediated AmpC-type enzyme was ACC-1a. Fingerprinting analysis by repetitive-element PCR and enterobacterial repeat intergenic consensus-PCR suggested that 29 of the 31 Salmonella serotype Mbandaka isolates belonged to the same clonal population.     

Occurrence of extended-spectrum and AmpC beta-lactamases in bloodstream isolates of Klebsiella pneumoniae: isolates harbor plasmid-mediated FOX-5 and ACT-1 AmpC beta-lactamases

We tested 190 Klebsiella pneumoniae bloodstream isolates recovered from 189 patients in 30 U.S. hospitals in 23 states to determine the occurrence of extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase producers. Based on growth inhibition by clavulanic acid by disk and MIC test methods, 18 (9.5%) of the isolates produced ESBLs. Although the disk diffusion method with standard breakpoints identified 28 cefoxitin-nonsusceptible isolates, only 5 (18%) of these were confirmed as AmpC producers. Of two AmpC confirmatory tests, the three-dimensional extract test was easier to perform than was the double-disk approximation test using a novel inhibitor, Syn2190. Three of the five AmpC producers carried the bla(FOX-5) gene, while the other two isolates harbored the bla(ACT-1) gene. All AmpC genes were transferable. In vitro susceptibility testing with standard inocula showed that all five AmpC-producing strains were susceptible to cefepime, imipenem, and ertapenem but that with a high inoculum, more of these strains were susceptible to the carbapenems than to cefepime. All but 1 of 14 screen-positive AmpC nonproducers (and ESBL nonproducers) were susceptible to ceftriaxone and cefepime at the standard inoculum as were 6 of 6 isolates that were randomly selected and tested with a high inoculum. These results indicate that (i). a significant number of K. pneumoniae bloodstream isolates harbor ESBL or AmpC beta-lactamases, (ii). confirmatory tests are necessary to identify true AmpC producers, and (iii). in vitro, carbapenems are active against AmpC-producing strains of K. pneumoniae.