Identification of SHV-type extended spectrum beta-lactamase genes in Pseudomonas aeruginosa by PCR-restriction fragment length polymorphism and insertion site restriction-PCR

We propose a simple and rapid method to discriminate SHV-type extended spectrum beta-lactamase (ESBL) genes in P. aeruginosa based on PCR techniques (PCR-RFLP and RSI-PCR). We studied 22 producing ESBL P. aeruginosa strains isolated from seven immunocompromised patients (19 isolates) and from environmental swabs (three isolates) at the Bone Marrow Transplantation Center of Tunis. Screening PCR with primer pairs designed to detect gene encoding TEM, SHV, OXA group I, OXA group II, OXA-18 and PER-1 ESBL was positive for bla(OXA18) and bla(SHV) genes in all isolates. Pulsed field gel electrophoresis using SpeI endonuclease defined five genotypic groups. For at least one isolate corresponding to each genotype observed, restriction of PCR products by DdeI and BsrI revealed the same restriction pattern that the bla(SHV-1) negative control; in the same way, RSI-PCR products digestion by NruI, thus excluding 35, 238 and 240 mutations characterizing reported ESBL in P. aeruginosa (SHV-2a, SHV5 et SHV12), and suggesting that studied bla(SHV) genes were not ESBL ones. Genomic DNA hybridization by southern blot with probe consisting in bla(SHV-1) gene was positive in these isolates. Sequencing the full-length open reading frame revealed nucleotide sequence of the bla(SHV-1). PCR-RFLP and RSI-PCR results were then confirmed. This approach is effective for screening P. aeruginosa for ESBL genes carriage in epidemiological studies and for detecting new variants.     

Detection of SHV-1 beta-lactamase in Pseudomonas aeruginosa strains by genetic methods

Twelve multidrug-resistant Pseudomonas aeruginosa (MDRPA) isolates were recovered over a period of two years in the National Bone Marrow Transplant Centre of Tunisia. MDRPA isolates were isolated from seven patients and from three environmental samples. Isoelectric focusing revealed pIs of 8.2, 5.5 and 7.6 in all MDRPA isolates. These strains produced the OXA-18 extended spectrum beta-lactamase and an SHV type beta-lactamase as shown by screening PCR analysis. DNA hybridization confirmed this inference, detecting bla(SHV) gene in these isolates. Pulsed-field gel electrophoresis (PFGE) defined one predominant genomic group; group A (seven isolates) and four different genotypes containing one to two isolates. Clonally related isolates were recovered from three patients and from two washbasins. Sequencing DNA of cluster representative strains identified the classical bla(SHV-1) gene. For these strains, the nucleotide sequence of the structural bla(SHV-1) gene was nearly identical to those previously described. Such enzyme has not been reported from P. aeruginosa. This is the first report of the SHV-1 penicillinase in epidemic P. aeruginosa strain.     

Diversity of β-lactamases in Pseudomonas aeruginosa isolates producing metallo-β-lactamase in two Tunisian hospitals

This study was conducted to identify the β-lactamase content of 30 metallo-β-lactamase-producing Pseudomonas aeruginosa isolated in 2007 from two Tunisian hospitals and to investigate their genetic relatedness. All these isolates produced VIM-2. bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) were identified in 17, 5, 21, and 1 isolates, respectively. These enzymes were often associated in the same isolate: 26 isolates had at least two β-lactamases. The predominant serotype was O12. Pulsed-field gel electrophoresis revealed genetic diversity among the metallo-β-lactamase-producing P. aeruginosa isolates. This is the first report on the existence of bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) in Tunisia.     

Identification of blaOXA-128 and blaOXA-129, two novel OXA-type extended-spectrum-β-lactamases in Pseudomonas aeruginosa, in Hunan Province, China

We collected 97 non-repetitive carbapenemases-sensitive clinical isolates of Pseudomonas aeruginosa in Human Province, China, during the period of October 2006 to January 2007. From these isolates, we identified two novel oxacillin-hydrolysing (OXA) type extended-spectrum-β-lactamases (ESBLs): bla OXA-128 and bla OXA-129, which contain the mutations of I89V from bla OXA-56 and K134N from bla OXA-10, respectively. Clinical isolates containing either bla OXA-128 or bla OXA-129 show resistance to cephamycin-class antibiotics but sensitive to carbapenem-class antibiotics. The occurrence of novel OXA-type lactamases suggests a regional prevalent pattern of ESBLs Pseudomonas aeruginosa in this area.     

VIM and IMP metallo-β-lactamases and other extended-spectrum β-lactamases in Escherichia coli and Klebsiella pneumoniae from environmental samples in a Tunisian hospital

An extremely drug-resistant Enterobacteriaceae species emerged in Kasserine Hospital, Tunisia between 2009 and 2010 causing a local outbreak. We aimed to characterize extended-spectrum β-lactamase (ESBL) and metallo-β-lactamase (MBL)-producing Enterobacteriaceae from the hospital environment. Swabs were collected from ten different wards from Kasserine Hospital, Tunisia. A total of 46 isolates were cultured onto MacConkey agar supplemented with ceftazidime to select for ESBL-producing Enterobacteriaceae. Identification and susceptibility patterns were performed using Phoenix-automated phenotypic identification criteria. Extended spectrum β-lactamases (ESBLs) were detected using cefepime ESBL E-test. Colony blotting was first used to detect the occurrence of bla(SHV) , bla(CTX-M) , bla(CMY) , bla(IMP) , and bla(VIM) genes. PCR was used to amplify these genes, and the amplicons were sequenced and analyzed. Total DNA was digested with XbaI, and PFGE was used to type the major isolates that produced IMP-1. Among the 46 isolates, 63% were Klebsiella pneumoniae, 13% were Escherichia coli, 8.7% were Proteus mirabilis, 6% were Enterobacter cloaceae, 4.3% were Providencia rettgeri, 2.5% were Serratia marcescens, and 2.5% were Pantoea agglomerans. PCR amplification and DNA sequencing showed that hospital environment isolates produced SHV-125, CTX-M-15, CMY-2 ESBLs, and IMP-1 and VIM-2 MBLs. PFGE typing showed the emergence of IMP-1 MBL-producing K. pneumoniae isolates that were not clonal. In this study, we report the first characterization of IMP-1 and VIM-2 MBL-producing K. pneumoniae and E. coli isolates collected from Kasserine Hospital, Tunisia.     

Rapid identification of gram-negative bacteria with and without CTX-M extended-spectrum β-lactamase from positive blood culture bottles by PCR followed by microchip gel electrophoresis

We evaluated the usefulness of PCR analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) and the CTX-M extended-spectrum β-lactamase (ESBL) followed by microchip gel electrophoresis (MGE) for direct identification and CTX-M detection of Gram-negative bacteria (GNB) from positive blood culture bottles. Of 251 GNB isolated from blood cultures containing a single bacterium, 225 (90%) were correctly identified at the species level directly from positive blood culture bottles by comparing the ITS-PCR patterns of the sample strain with those of the control strains. There were no cases of incorrect identification. Limitations encountered included the inability to detect mixed cultures (four bottles) as well as some species (Enterobacter species and Klebsiella oxytoca) demonstrating identical ITS-PCR patterns. A total of 109 ESBL-producing isolates from various clinical materials obtained between January 2005 and December 2008 were examined for bla(CTX-M), bla(SHV), and bla(TEM) genes by PCR and sequences of PCR products. CTX-M ESBL was detected in 105 isolates, and SHV ESBL was detected in two isolates. The remaining two isolates (K. oxytoca) were shown to harbor bla(OXY.) Twenty (19%) of 104 Escherichia coli isolates from blood cultures were suspected to produce ESBL by the combination disk method, and these isolates were shown to harbor CTX-M ESBL by PCR-MGE. The results were obtained within 1.5 h at a calculated cost of $6.50 per specimen. In conclusion, simultaneous detection of ITS length polymorphisms and bla(CTX)-(M) by single PCR followed by MGE is useful for rapid, cost-effective, and reliable species-level identification of CTX-M ESBL-producing GNB responsible for bloodstream infections.     

Identification of clinical isolates of indole-positive Klebsiella spp., including Klebsiella planticola, and a genetic and molecular analysis of their beta-lactamases.

In a collection of 43 indole-positive Klebsiella clinical isolates, which were initially identified as Klebsiella oxytoca, there were 18 isolates which exhibited a pattern characteristic of extended-spectrum beta-lactamase (ESBL) resistance. This study aimed to confirm their identity by biochemical tests and by PCR and to determine the genetic basis for their resistance to the beta-lactams and broad-spectrum cephalosporins. Chromosomal beta-lactamase genes were analyzed by PCR, and plasmid-mediated beta-lactamase genes were analyzed by conjugation and transformation. There were 39 isolates which grew on melezitose but failed to grow on 3-hydroxybutyrate, confirming them as K. oxytoca. PCR analysis of their beta-lactamase genes divided these isolates into two groups, the bla(OXY-1) group and the bla(OXY-2) group. Each group had beta-lactamases with different isoelectric points; the bla(OXY-1) group had beta-lactamases with isoelectric points at 7.2, 7.8, 8.2, and 8.8, and the more common bla(OXY-2) group had beta-lactamases with pIs at 5.2, 5.4 (TEM-1), 5.7, 5.9, 6.4, and 6.8. A pI of 5.2 was the most frequently detected and accounted for 59% of all the bla(OXY-2) beta-lactamases. Hyperproduction of clavulanate-inhibited chromosomal beta-lactamases was detected in 17 K. oxytoca isolates, resulting in an ESBL phenotype. K. oxytoca isolates having a plasmid-mediated genetic basis for their ESBL phenotype were not found, confirming that, in K. oxytoca, plasmids are rarely involved in conferring resistance to the newer cephalosporins. Four isolates proved to be isolates of K. planticola in which the beta-lactamase genes failed to react with the primers used in the PCR. One K. planticola isolate contained a transferable plasmid harboring the SHV-5 beta-lactamase gene and showed an ESBL phenotype, while the other non-ESBL K. planticola isolates contained chromosomal beta-lactamases with isoelectric points at 7.2, 7.7, and 7.9 plus 7.2.     

Molecular characterisation of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp. isolates at a tertiary-care centre in Lebanon.

The prevalence of bla CTX-M, bla TEM and bla SHV genes among extended-spectrum beta-lactamase (ESBL)-producing clinical isolates of Escherichia coli (n = 50) and Klebsiella spp. (n = 50) from Lebanon was 96%, 57% and 67%, and 40%, 82% and 84%, respectively. Genotyping revealed that the clonal diversity was unrelated to the presence of bla genes. Sequence analysis of 16 selected isolates identified the bla CTX-M-15, bla TEM-1, bla OXA-1 and six bla SHV genes, as well as the gene encoding the quinolone-modifying enzyme AAC(6')-Ib-cr. The genes encoding CTX-M-15 and AAC(6')-Ib-cr were carried on a 90-kb plasmid of the pC15-1a or pCTX-15 type, which transferred both ESBL production and quinolone resistance from donors to transconjugants.     

Identification and characterization of OXA-48 producing, carbapenem-resistant Enterobacteriaceae isolates in Turkey

Carbapenem resistance in Enterobacteriaceae isolates has been reported from Turkey and is most often mediated by OXA-48 type carbapenemases. We report the identification and characterization of four carbapenem-resistant isolates (three Klebsiella pneumoniae and one Escherichia coli) among 515 clinical Enterobacteriaceae isolates collected during a 7-month study period in Ankara, Turkey. The four isolates were recovered from blood and urine specimens in patients with varied clinical manifestations. They had distinct pulsed-field gel electrophoresis patterns and harbored a variety of β-lactamases including bla(TEM-1), bla(SHV-12) genes, bla(SHV-11), and/or bla(CTX-M-15). PCR and sequencing analysis revealed that the bla(OXA-48) gene was present in all four isolates. Our data indicated that the OXA-48-type carbapenemase was the only mechanism for carbapenem resistance in our hospital.     

Prolonged outbreak of infection due to TEM-21-producing strains of Pseudomonas aeruginosa and enterobacteria in a nursing home

Over a 6-year period, 24 extended-spectrum beta-lactamase (ESBL)-producing isolates of Pseudomonas aeruginosa were collected from 18 patients living in a nursing home. These isolates had a delayed development of a red pigment and exhibited a similar antibiotype (resistance to all beta-lactams except for imipenem and to gentamicin, tobramycin, netilmicin, ciprofloxacin, and rifampin) associated with the production of the TEM-21 beta-lactamase and a type II 3'-N-aminoglycoside acetyltransferase [AAC(3)-II] enzyme. Surprisingly, serotyping showed that these isolates belonged to four successive serotypes (P2, P16, P1, and PME), although molecular typing by PCR methods and pulsed-field gel electrophoresis yielded identical or similar profiles. Moreover, in all isolates the bla(TEM-21) gene was part of a chromosomally located Tn801 transposon truncated by an IS6100 element inserted within the resolvase gene, and the aac(3)-II gene was adjacent to this structure. During the same period, 17 ESBL-producing isolates of enterobacteria were also collected from 10 of these patients. These isolates harbored a similar large plasmid that contained the bla(TEM-21) and the aac(3)-II genes and that conferred additional resistance to sulfonamides and chloramphenicol, as well as to kanamycin, tobramycin, netilmicin, and amikacin, conveyed by an AAC(6')-I enzyme. The bla(TEM-21) gene was part of the Tn801 transposon disrupted by IS4321. Thus, a single clone of P. aeruginosa that had undergone a progressive genetic drift associated with a change in serotype appeared to be responsible for an outbreak of nosocomial infections in a nursing home. This strain has probably acquired the bla(TEM-21)-encoding plasmid that was epidemic among the enterobacteria at the institution, followed by chromosomal integration and genomic reorganization.     

Presence of OXA-23-producing isolates of Acinetobacter baumannii in wastewater from hospitals in southern Brazil

The aim of the study was to evaluate the dissemination of multiresistant isolates of Acinetobacter baumannii carrying resistance genes, by samples of wastewater from hospitals in Porto Alegre, Rio Grande do Sul, Brazil. We obtained 303 bacterial isolates from the wastewater of three hospitals in Porto Alegre, Rio Grande do Sul. For each isolate, we determined the profile of susceptibility to antimicrobials and the presence of the genes bla(OXA-23), bla(OXA-24), bla(OXA-51), bla(OXA-58), bla(SPM-1), bla(IMP), and bla(VIM.) The bla(OXA-51) gene was found in 56% of the isolates, indicating the presence of A. baumannii in this environment. Of these, three multiresistant isolates were positive for the bla(OXA-23) gene, in wastewater from two of the hospitals. The results obtained in this study indicate that isolates of A. baumannii which are multiresistant and carry resistance genes such as bla(OXA-51) and bla(OXA-23) are being released into the environment in the wastewater from the hospitals analyzed. Multiresistant Acinetobacter junii, the newly emerging pathogen, were also found among the multiresistant isolates. Hospital wastewater may be crucial to the development and dispersal of multiresistant bacteria, making waterbodies reservoirs of bacterial resistance.     

Outbreak of Pseudomonas aeruginosa infections with PER-1 extended-spectrum beta-lactamase in Warsaw, Poland: further evidence for an international clonal complex

Forty-one Pseudomonas aeruginosa isolates with extended-spectrum beta-lactamases (ESBLs) from a hospital in Warsaw, Poland, were analyzed. Thirty-seven isolates from several wards were collected over 9 months in 2003 and 2004. The isolates were recovered from patients with multiple types of infections, mostly respiratory tract and postoperative wound infections. All 41 isolates produced the PER-1 ESBL, originally observed in Turkey but recently also identified in several countries in Europe and the Far East. The bla(PER-1) gene resided within the Tn1213 composite transposon, which was chromosomally located. Pulsed-field gel electrophoresis and multilocus sequence typing (MLST) revealed the presence of three separate clones among the isolates. Two of these, corresponding to sequence types (STs) ST244 and ST235, were responsible for parallel outbreaks. Apart from PER-1, all the isolates produced OXA-2 oxacillinase. ST235 isolates additionally expressed a novel enzyme, OXA-74, differing by one amino acid from the OXA-17 ESBL identified originally in PER-1- and OXA-2-positive P. aeruginosa isolates from Ankara, Turkey, in 1992. These earlier Ankara isolates with PER-1, OXA-2, and OXA-17 were also classified into ST235, which is a single-locus variant of two other STs, ST227 and ST230. ST227, ST230, and ST235 all correspond to the recently described clonal complex BG11, which seems to be internationally distributed, having spread in Turkey, Greece, Italy, Hungary, Poland, Sweden, and much of Russia. It is associated with various beta-lactamases, including PER-1 and VIM metalloenzymes. This work further demonstrates the value of MLST of P. aeruginosa.     

Identification of the naturally occurring genes encoding carbapenem-hydrolysing oxacillinases from Acinetobacter haemolyticus, Acinetobacter johnsonii, and Acinetobacter calcoaceticus

Carbapenem resistance is increasingly being reported among Acinetobacter species, and results mostly from the expression of acquired carbapenem-hydrolysing oxacillinases (CHDLs). Several Acinetobacter species intrinsically possess chromosomal CHDL genes: Acinetobacter baumannii (bla(OXA-51) ), Acinetobacter radioresistens (bla(OXA-23) ), and Acinetobacter lwoffii (bla(OXA-134) ). We aimed to identify the progenitors of novel CHDL-encoding genes for identification of potential reservoirs. We performed PCR screening using degenerated internal primers designed from a sequence alignment of the known CHDLs (OXA-23, OXA-40, OXA-51, OXA-58, OXA-134, and OXA-143) applied to a collection of 50 Acinetobacter strains belonging to 23 different species. Two strains of Acinetobacter johnsonii, one strain of Acinetobacter calcoaceticus and two strains of Acinetobacter haemolyticus were found to harbour, respectively, the totally novel bla(OXA-211) -like, bla(OXA-213) -like and bla(OXA-214) -like genes. In addition, the complete genomes of those three species available in GenBank, i.e. one A. johnsonii genome, four A. calcoaceticus genomes, and one A. haemolyticus genome, were analysed and found to be positive for the presence of bla(OXA211) -like, bla(OXA-213) -like and bla(OXA-214) -like genes, respectively. The β-lactamases OXA-211, OXA-213 and OXA-214 are diverse, with amino acid identities ranging from 53% to 76%, as compared with the naturally occurring OXA-51-like CHDL from A. baumannii. These β-lactamases showed a peculiar hydrolysis profile, including mostly penicillins and carbapenems. Regarding bla(OXA-23) in A. radioresistens and bla(OXA-134) in A. lwoffii, these genes were not expressed (or expressed at a non-significant level) in their host. Detection of these β-lactamase genes might be used as a useful tool for accurate identification of these Acinetobacter species.     

OXA-type beta-lactamases among extended-spectrum cephalosporin-resistant Pseudomonas aeruginosa isolates in a university hospital in southern Taiwan.

BACKGROUND AND PURPOSE: Data on the epidemiology of OXA-type extended-spectrum beta (beta)-lactamases (ESBLs) are limited due to difficulty of identification by routine microbiology laboratories. We determined the prevalence rate of OXA-type beta-lactamases among extended-spectrum cephalosporin (ESC)-non-susceptible Pseudomonas aeruginosa isolates at a university hospital in southern Taiwan. METHODS: A total of 1,294 ESC-non-susceptible P. aeruginosa isolates collected between 1989 and 1996 (n = 42) and between December 1999 and December 2002 (n = 1,252) were analyzed by polymerase chain reaction assays with primers specific for bla(OXA) genes and isoelectric focusing. RESULTS: Forty five isolates (3.5%) were found to produce an OXA-type beta-lactamase. Overall, 2 OXA-type ESBLs, OXA-14 (n = 2) and OXA-17 (n = 35), were detected in 37 (2.9%) isolates, and the OXA-10-type narrow-spectrum beta-lactamase was found in 8 (0.6%) isolates. OXA-10 and the 2 OXA-type ESBLs were detected in 6 (14.3%) and 4 (9.5%) of 42 ESC-non-susceptible isolates collected between 1989 and 1996. OXA-10 and OXA-17 were detected in 2 (0.2%) and 33 (2.6%) of 1,252 ESC-non-susceptible isolates collected between December 1999 and December 2002. CONCLUSIONS: These data indicate that OXA-17 was the most common OXA-type ESBL and that OXA-type beta-lactamases have decreased in ESC-non-susceptible P. aeruginosa at this hospital in recent years. Pulsed-field gel electrophoresis revealed clonal diversity among the OXA-producing isolates.     

Identification of Acinetobacter baumannii by detection of the blaOXA-51-like carbapenemase gene intrinsic to this species

bla(OXA-51-like) was sought in clinical isolates of Acinetobacter species in a multiplex PCR, which also detects bla(OXA-23-like) and class 1 integrase genes. All isolates that gave a band for bla(OXA-51-like) identified as A. baumannii. This gene was detected in each of 141 isolates of A. baumannii but not in those of 22 other Acinetobacter species.     

A diversity of OXA-carbapenemases and class 1 integrons among carbapenem-resistant Acinetobacter baumannii clinical isolates from Sweden belonging to different international clonal lineages

This study was aimed to investigate the molecular epidemiology, mechanism of carbapenem resistance, and occurrence of class 1 integrons among 13 carbapenem-resistant clinical isolates of Acinetobacter baumannii obtained between 2004 and 2007 from Sweden. Nine isolates were linked with hospitalization abroad. Molecular epidemiology was investigated by multilocus sequence typing, multiplex PCRs for major international clones/sequence groups (SGs), and pulsed-field gel electrophoresis. OXA-carbapenemase genes/genetic surroundings and class 1 integrons were examined by PCRs and sequencing. The isolates belonged to sequence type (ST) 2/international clone II (n=6), ST23/SG5 (n=2), ST25 (n=2), ST5/SG7 (n=1), and ST109 (n=2). OXA-58, OXA-23, and OXA-24 were detected in seven, five, and one isolate, respectively. Different genetic structures surrounded the bla(OXA-58-like) and bla(OXA-23-like) genes. Interestingly, ISAba825 was detected upstream bla(OXA-58-like) in two isolates. Class 1 integrons with three different variable regions (VR) were detected. VR1 (aacA4-orfO-bla(OXA-20)) was found in four isolates from ST2/international clone II, with three of them imported from Greece. VR2 (aadB-aadA1-IS) was detected in one ST5 isolate imported from Poland, and VR3 (bla(GES-11)-aacA4-dfrA7) was present in a nonimport ST25 isolate. In conclusion, a variety of clonal lineages, OXA-carbapenemases genes and genetic structures, and class 1 integrons were detected among carbapenem-resistant A. baumannii from Sweden.     

High prevalence of carbapenem-hydrolysing oxacillinases in epidemiologically related and unrelated Acinetobacter baumannii clinical isolates in Spain

Carbapenem-hydrolysing oxacillinases are reported increasingly in Acinetobacter baumannii. This study investigated the role of these beta-lactamases in causing resistance to carbapenems in 83 epidemiologically related and unrelated imipenem-resistant A. baumannii clinical isolates. The isolates were also analysed for the presence of ISAba1 in the promoter region of the bla(OXA-51)-like gene in order to investigate the role of ISAba1 in OXA-51 expression. All clinical isolates contained a bla(OXA-51)-like gene, 20% contained a bla(OXA-58)-like gene, and 42% contained a bla(OXA-40)-like gene; bla(OXA-23)-like, bla(IMP) and bla(VIM) genes were not detected in any of the isolates investigated. ISAba1 was found in 24 (82.7%) of 28 pulsetypes, and was located in the promoter region of the bla(OXA-51)-like gene in five (20.8%) of these pulsetypes. Expression of bla(OXA-51) was detected in the five isolates with ISAba1 located in the promoter region, but was not detected in an isogenic imipenem-susceptible A. baumannii isolate that did not have ISAba1 located in the promoter region. It was concluded that there is a high prevalence of oxacillinases with activity against carbapenems among genetically unrelated A. baumannii clinical isolates from Spain, and that concomitant expression of two carbapenemases (OXA-51-like and either OXA-40-like or OXA-58-like) may take place. Insertion of an ISAba1-like element in the promoter of the bla(OXA-51)-like gene promotes the expression of this gene, although this did not seem to play a major role in carbapenem resistance.     

Phenotypic and molecular detection of CTX-M-beta-lactamases produced by Escherichia coli and Klebsiella spp

Organisms producing CTX-M-beta-lactamases are emerging around the world as a source of resistance to oxyiminocephalosporins such as cefotaxime (CTX). However, the laboratory detection of these strains is not well defined. In this study, a molecular detection assay for the identification of CTX-M-beta-lactamase genes was developed and used to investigate the prevalence of these enzymes among clinical isolates of Escherichia coli and Klebsiella species in the Calgary Health Region during 2000 to 2002. In addition, National Committee for Clinical Laboratory Standards (NCCLS) recommendations were evaluated for the ability to detect isolates with CTX-M extended-spectrum beta-lactamases (ESBLs). The PCR assay consisted of four primer sets and demonstrated 100% specificity and sensitivity for detecting different groups of CTX-M-beta-lactamases in control strains producing well-characterized ESBLs. Using these primer sets, 175 clinical strains producing ESBLs were examined for the presence of CTX-M enzymes; 24 (14%) were positive for bla(CTX-M-1-like) genes, 95 (54%) were positive for bla(CTX-M-14-like) genes, and the remaining 56 (32%) were negative for bla(CTX-M) genes. Following the NCCLS recommendations for ESBL testing, all of the control and clinical strains were detected when screened with cefpodoxime and when both cefotaxime and ceftazidime with clavulanate were used as confirmation tests.     

Two different extended-spectrum beta-lactamases (ESBLs) in one of the first ESBL-producing salmonella isolates in Poland

Two extended-spectrum beta-lactamase (ESBL)-producing salmonella isolates, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium, were analyzed. Both isolates produced the CTX-M-3 ESBL; however, their bla(CTX-M-3) genes were located on different plasmids. The serovar Typhimurium isolate also expressed another ESBL, SHV-2a, and probably the two ESBL genes had been acquired independently by the strain.