Updated Sequence Information for TEM β-Lactamase Genes

The sequences of the promoter regions and of the structural genes for 13 penicillinase, extended-spectrum, and inhibitor-resistant TEM-type β-lactamases have been determined, and an updated bla_TEM gene nomenclature is proposed.     

β-Lactamases in amoxicillin-clavulanate–resistant Escherichia coli strains isolated from a Chinese tertiary hospital

A total of 52 strains were resistant to amoxicillin-clavulanate by disk diffusion method in a Chinese tertiary hospital from July 2011 to December 2011. Among these isolates, 2 isolates possessed a phenotype consistent with production of inhibitor-resistant temoniera (TEM) (IRT) β-lactamase, and the TEM-type gene was cloned into strains of Escherichia coli JM109 cells. Both had no bla_TEM mutations and were identified as TEM-1 β-lactamase producers. As a result, no IRT β-lactamase was detected. Multiplex PCR detected most of these strains produced TEM-1 enzymes, and plasmid-mediated AmpC β-lactamase and oxacillinase-1 β-lactamases are important mechanisms of resistance as well.     

Identification of 50 Class D β-Lactamases and 65 Acinetobacter-Derived Cephalosporinases in Acinetobacter spp.

Whole-genome sequencing of a collection of 103 Acinetobacter strains belonging to 22 validly named species and another 16 putative species allowed detection of genes for 50 new class D β-lactamases and 65 new Acinetobacter-derived cephalosporinases (ADC). All oxacillinases (OXA) contained the three typical motifs of class D β-lactamases, STFK, (F/Y)GN, and K(S/T)G. The phylogenetic tree drawn from the OXA sequences led to an increase in the number of OXA groups from 7 to 18. The topologies of the OXA and RpoB phylogenetic trees were similar, supporting the ancient acquisition of bla_OXA genes by Acinetobacter species. The class D β-lactamase genes appeared to be intrinsic to several species, such as Acinetobacter baumannii, Acinetobacter pittii, Acinetobacter calcoaceticus, and Acinetobacter lwoffii. Neither bla_OXA-40/143- nor bla_OXA-58-like genes were detected, and their origin remains therefore unknown. The phylogenetic tree analysis based on the alignment of the sequences deduced from bla_ADC revealed five main clusters, one containing ADC belonging to species closely related to A. baumannii and the others composed of cephalosporinases from the remaining species. No indication of bla_OXA or bla_ADC transfer was observed between distantly related species, except for bla_OXA-279, possibly transferred from Acinetobacter genomic species 6 to Acinetobacter parvus. Analysis of β-lactam susceptibility of seven strains harboring new oxacillinases and cloning of the corresponding genes in Escherichia coli and in a susceptible A. baumannii strain indicated very weak hydrolysis of carbapenems. Overall, this study reveals a large pool of β-lactamases in different Acinetobacter spp., potentially transferable to pathogenic strains of the genus.     

Prevalence of β-Lactamase-Encoding Genes among Enterobacteriaceae Bacteremia Isolates Collected in 26 U.S. Hospitals: Report from the SENTRY Antimicrobial Surveillance Program (2010)

Enterobacteriaceae bacteremia isolates (n = 195; 6.4% overall) collected from 26 U.S. hospitals located in 20 states were screened for various β-lactamase classes. A total of 175 isolates carried one to eight acquired β-lactamase genes of 44 types that were detected in 55 combinations. Eighty-five (43.6%) strains carried bla_CTX-M, and bla_CTX-M-15 was the most prevalent (33.8%). Genes encoding OXA-1/30 (often associated with bla_CTX-M-15), CMY-2, SHV extended-spectrum β-lactamase (ESBLs), and TEM-1 were also prevalent. Among 33 carbapenem-resistant strains, 28 carried bla_KPC-2 or bla_KPC-3 (17 and 11 strains, respectively), and those were recovered mostly in the New York City area (16 strains) and Houston, TX (9 strains). Fourteen new SHV variants were identified among Klebsiella pneumoniae isolates carrying one or multiple SHV alleles, three carrying G238S and/or E240K amino acid alterations that confer ESBL activity. Only two of eight K. oxytoca isolates carried acquired β-lactamases, but most had mutations on the bla_OXY promoter region, and three new OXY-encoding genes were characterized. Concordance between a commercial nucleic acid-based microarray (Check-MDR CT101) and reference methods was noted for 105/109 (97.2%) strains. Thirty-two strains having genes that are not targeted by the commercial system were detected (OXA ESBLs, PER, PSE, or intrinsic genes). Overall, a great variety of enzymes were observed, with numerous strains carrying multiple genes. Rates of CTX-M-producing strains appear to be increasing in U.S. hospitals (26.6% in 2007 to 43.8% for 2010) participating in the SENTRY Program. Furthermore, the Check-Points system seems to be a reliable, robust, and user-friendly assay for detection of enzyme-mediated resistance.     

Ceftazidime-Resistant Enterobacteriaceae Isolates from Three Polish Hospitals: Identification of Three Novel TEM- and SHV-5-Type Extended-Spectrum β-Lactamases

Twelve ceftazidime-resistant isolates of the family Enterobacteriaceae (11 Klebsiella pneumoniae isolates and 1 Escherichia coli isolate) were collected in 1995 from three Polish hospitals located in different cities. All were identified as producers of extended-spectrum β-lactamases (ESBLs). Detailed analysis of their β-lactamase contents revealed that six of them expressed SHV-5-like ESBLs. The remaining six were found to produce three different TEM enzymes, each characterized by a pI value of 6.0 and specified by new combinations of amino acid substitutions. The amino acid substitutions compared to the TEM-1 β-lactamase sequence were Gly238Ser, Glu240Lys, and Thr265Met for TEM-47; Leu21Phe, Gly238Ser, Glu240Lys, and Thr265Met for TEM-48; and Leu21Phe, Gly238Ser, Glu240Lys, Thr265Met, and Ser268Gly for TEM-49. The new TEM β-lactamases, TEM-47, TEM-48, and TEM-49, belong to a subfamily of TEM-2-related enzymes. Genes coding for TEM-47 and TEM-49 could have originated from the TEM-48-encoding sequence by various single genetic events. The new TEM derivatives probably document the already advanced microevolution of ESBLs ongoing in Polish hospitals, in a majority of which no monitoring of ESBL producers was performed before 1996.     

Characterization and Sequence Analysis of Extended-Spectrum-β-Lactamase-Encoding Genes from Escherichia coli,Klebsiella pneumoniae,and Proteus mirabilis Isolates Collected during Tigecycline Phase 3 Clinical Trials

In concert with the development of novel β-lactams and broad-spectrum cephalosporins, bacterially encoded β-lactamases have evolved to accommodate the new agents. This study was designed to identify, at the sequence level, the genes responsible for the extended-spectrum-β-lactamase (ESBL) phenotypes of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates collected during the global tigecycline phase 3 clinical trials. PCR assays were developed to identify and clone the bla_TEM, bla_SHV, bla_OXA, and bla_CTX genes from clinical strains. Isolates were also screened for AmpC genes of the bla_CMY, bla_ACT, bla_FOX, and bla_DHA families as well as the bla_KPC genes encoding class A carbapenemases. E. coli, K. pneumoniae, and P. mirabilis isolates with ceftazidime MICs of ≥2 μg/ml were designated possible ESBL-producing pathogens and were then subjected to a confirmatory test for ESBLs by use of Etest. Of 272 unique patient isolates, 239 were confirmed by PCR and sequencing to carry the genes for at least one ESBL, with 44% of the positive isolates harboring the genes for multiple ESBLs. In agreement with current trends for ESBL distribution, bla_CTX-M-type β-lactamase genes were found in 83% and 71% of the ESBL-positive E. coli and K. pneumoniae isolates, respectively, whereas bla_SHV genes were found in 41% and 28% of the ESBL-positive K. pneumoniae and E. coli isolates, respectively. Ninety-seven percent of the E. coli and K. pneumoniae isolates were tigecycline susceptible (MIC₉₀ = 2 μg/ml), warranting further studies to define the therapeutic utility of tigecycline against strains producing ESBLs in a clinical setting.Throughout the 60-year clinical experience with β-lactams and broad-spectrum cephalosporins, bacterial pathogens have kept pace with the introduction of novel agents through the evolution of new β-lactamases that efficiently conform to the new antibacterials (5, 10, 20, 33). One of the most clinically important groups of β-lactamases is the extended-spectrum β-lactamases (ESBLs). Numerous studies have revealed increased mortality and, in general, poorer clinical outcomes for patients infected with bacteria encoding ESBLs (35, 45). According to Bush et al. (11), ESBLs are defined as oxyimino-cephalosporin-hydrolyzing β-lactamases that are inhibited by clavulanic acid.The current rising threat in the clinic is the CTX-M family of ESBLs; these enzymes have appeared in both community and nosocomial settings (3, 12, 38). CTX-M, a class A enzyme, was originally defined by the preferential cleavage of cefotaxime (CTX) versus ceftazidime (CAZ), although several recent derivatives that cleave both agents equivalently have been described (4, 39). In most European countries, Latin America, and East Asia, CTX-M variants have displaced TEM and SHV enzymes as the predominant β-lactamases produced by gram-negative pathogens, especially Klebsiella pneumoniae and Escherichia coli (3, 12, 26). The Lahey website (http://www.lahey.org/Studies/) reports that 82 CTX-M derivatives have been described.TEM-1, the first plasmid-mediated β-lactamase identified in gram-negative organisms, initially described in E. coli in the 1960s (16), is now present in a number of members of the family Enterobacteriaceae, including Klebsiella pneumoniae and Proteus mirabilis, and has spread into additional gram-negative pathogens (5). At last count (http://www.lahey.org/Studies/), more than 160 TEM derivatives have been reported, although not all have an expanded spectrum. In a similar fashion, SHV-type ESBLs have evolved from the SHV-1 enzyme common to K. pneumoniae. They are found on both the chromosome and plasmids and have migrated into Citrobacter spp., E. coli, Pseudomonas aeruginosa, and other genera. Approximately 112 SHV derivatives have been reported; however, not all of the SHV derivatives described are ESBLs (http://www.lahey.org/Studies/). Lastly, OXA, a diverse family of enzymes belonging to the class D family, was initially characterized by rates of hydrolysis of oxacillin that were faster than the rates of hydrolysis of benzylpenicillin and relatively less sensitivity to inhibition by clavulanic acid (3, 5, 11). Over 130 OXA derivatives have been reported; however, not all of the OXA derivatives described have an extended spectrum (http://www.lahey.org/Studies/).The current study was designed to identify, at the sequence level, the genetic determinants responsible for the ESBL phenotype of E. coli, K. pneumoniae, and P. mirabilis isolates collected during the global tigecycline phase 3 clinical trials. In addition, isolates were screened for several AmpC determinants and the carbapenemase-encoding bla_KPC genes.     

Detection and Molecular Characterization of Escherichia coli CTX-M-15 and Klebsiella pneumoniae SHV-12 β-Lactamases from Bovine Mastitis Isolates in the United Kingdom

Recent reports raised concerns about the role that farm stock may play in the dissemination of extended-spectrum β-lactamase (ESBL)-producing bacteria. This study characterized the ESBLs in two Escherichia coli and three Klebsiella pneumoniae subsp. pneumoniae isolates from cases of clinical bovine mastitis in the United Kingdom. Bacterial culture and sensitivity testing of bovine mastitic milk samples identified Gram-negative cefpodoxime-resistant isolates, which were assessed for their ESBL phenotypes. Conjugation experiments and PCR-based replicon typing (PBRT) were used for characterization of transferable plasmids. E. coli isolates belonged to sequence type 88 (ST88; determined by multilocus sequence typing) and carried bla_CTX-M-15 and bla_TEM-1, while K. pneumoniae subsp. pneumoniae isolates carried bla_SHV-12 and bla_TEM-1. Conjugation experiments demonstrated that bla_CTX-M-15 and bla_TEM-1 were carried on a conjugative plasmid in E. coli, and PBRT identified this to be an IncI1 plasmid. The resistance genes were nontransferable in K. pneumoniae subsp. pneumoniae isolates. Moreover, in the E. coli isolates, an association of ISEcp1 and IS26 with bla_CTX-M-15 was found where the IS26 element was inserted upstream of both ISEcp1 and the bla_CTX-M promoter, a genetic arrangement highly similar to that described in some United Kingdom human isolates. We report the first cases in Europe of bovine mastitis due to E. coli CTX-M-15 and also of bovine mastitis due to K. pneumoniae subsp. pneumoniae SHV-12 β-lactamases in the United Kingdom. We also describe the genetic environment of bla_CTX-M-15 and highlight the role that IncI1 plasmids may play in the spread and dissemination of ESBL genes, which have been described in both human and cattle isolates.     

Ceftazidime-Resistant Klebsiella pneumoniae and Escherichia coli Isolates Producing TEM-10 and TEM-43 β-Lactamases from St. Louis, Missouri

Ceftazidime-resistant Escherichia coli and Klebsiella pneumoniae (49 and 102 isolates, respectively) were collected from Barnes-Jewish Hospital, St. Louis, Mo., from 1992 to 1996. They were uniformly resistant to ceftazidime, generally resistant to aztreonam, and variably susceptible to cefotaxime. Four representative E. coli strains and 15 Klebsiella strains were examined. From one to four β-lactamases were produced per strain, with three possible enzymes related to ceftazidime resistance: enzymes with pI values of 5.6, 6.1, or 7.6. By pulsed-field gel electrophoresis there were at least 13 different Klebsiella strain types and 3 different E. coli strain types, indicating that the outbreak was not clonal. After cloning and sequencing of the β-lactamase-encoding genes, the enzyme with a pI of 5.6 was identified as TEM-10. The enzyme with a pI of 6.1 was a novel TEM variant (TEM-43) with Lys at 104, His at 164, and Thr at 182. TEM-43 showed broad-spectrum hydrolytic activity against all penicillins, with the highest hydrolysis rate for ceftazidime compared to those for the other expanded-spectrum cephalosporins. Aztreonam was also a good substrate for TEM-43, with hydrolytic activity similar to that of ceftazidime and affinity higher than that of ceftazidime. The TEM-43 β-lactamase was well inhibited by clavulanic acid and tazobactam at concentrations of <10 nM. Sulbactam was less effective than the other inhibitors. The Thr182 mutation previously reported in an inhibitor-resistant β-lactamase did not cause the TEM-43 enzyme to become resistant to any of the inhibitors.     

Characterization of an IncA/C Multidrug Resistance Plasmid in Vibrio alginolyticus

Cephalosporin-resistant Vibrio alginolyticus was first isolated from food products, with β-lactamases encoded by bla_PER-1, bla_VEB-1, and bla_CMY-2 being the major mechanisms mediating their cephalosporin resistance. The complete sequence of a multidrug resistance plasmid, pVAS3-1, harboring the bla_CMY-2 and qnrVC4 genes was decoded in this study. Its backbone exhibited genetic homology to known IncA/C plasmids recoverable from members of the family Enterobacteriaceae, suggesting its possible origin in Enterobacteriaceae.     

Identification of TEM-135 β-Lactamase in Neisseria gonorrhoeae Strains Carrying African and Toronto Plasmids in Argentina

One hundred forty-three penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates obtained in Argentina from 2008 and 2012 were examined to detect bla_TEM-135 genes and to investigate plasmid profiles and multiantigen sequence types. Forty-two PPNG isolates were found to carry TEM-135, and two contained a new TEM derivative characterized as TEM-220. The bla_TEM-135 allele was carried by the Toronto/Rio and African plasmids. Molecular epidemiology revealed that two bla_TEM-135 isolates were related to previously described isolates from Thailand and China, indicating a common evolutionary origin.     

Molecular characterization and epidemiology of extended-spectrum-beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates causing health care-associated infection in Thailand, where the CTX-M family is endemic

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae have rapidly spread worldwide and pose a serious threat for health care-associated (HA) infection. We conducted molecular detection and characterization of ESBL-related bla genes, including bla_TEM, bla_SHV, bla_CTX-M, bla_VEB, bla_OXA, bla_PER, and bla_GES, among 362 isolates of ESBL-producing E. coli (n = 235) and ESBL-producing K. pneumoniae (n = 127) collected from patients who met the definition of HA infection at two major university hospitals in Thailand from December 2004 to May 2005. The prevalence of ESBL-producing E. coli and ESBL-producing K. pneumoniae, patient demographics and the susceptibilities of these bacteria to various antimicrobial agents were described. A total of 87.3% of isolates carried several bla genes. The prevalence of bla_CTX-M was strikingly high: 99.6% for ESBL-producing E. coli (CTX-M-14, -15, -27, -40, and -55) and 99.2% for ESBL-producing K. pneumoniae (CTX-M-3, -14, -15, -27, and -55). ISEcp1 was found in the upstream region of bla_CTX-M in most isolates. Up to 77.0% and 71.7% of ESBL-producing E. coli and ESBL-producing K. pneumoniae, respectively, carried bla_TEM; all of them encoded TEM-1. ESBL-producing K. pneumoniae carried bla_SHV at 87.4% (SHV-1, -2a, -11, -12, -27, -71, and -75) but only at 3.8% for ESBL-producing E. coli (SHV-11 and -12). bla genes encoding VEB-1 and OXA-10 were found in both ESBL-producing E. coli (8.5% and 8.1%, respectively) and ESBL-producing K. pneumoniae (10.2% and 11.8%, respectively). None of the isolates were positive for bla_PER and bla_GES. Pulsed-field gel electrophoresis analysis demonstrated that there was no major clonal relationship among these ESBL producers. This is the first study to report CTX-M-3, CTX-M-27, CTX-M-40, SHV-27, SHV-71, and SHV-75 in Thailand and to show that CTX-M ESBL is highly endemic in the country.     

Chromosomally Encoded AmpC-Type β-Lactamase in a Clinical Isolate of Proteus mirabilis

A clinical strain of Proteus mirabilis (CF09) isolated from urine specimens of a patient displayed resistance to amoxicillin (MIC >4,096 μg/ml), ticarcillin (4,096 μg/ml), cefoxitin (64 μg/ml), cefotaxime (256 μg/ml), and ceftazidime (128 μg/ml) and required an elevated MIC of aztreonam (4 μg/ml). Clavulanic acid did not act synergistically with cephalosporins. Two β-lactamases with apparent pIs of 5.6 and 9.0 were identified by isoelectric focusing on a gel. Substrate and inhibition profiles were characteristic of an AmpC-type β-lactamase with a pI of 9.0. Amplification by PCR with primers for ampC genes (Escherichia coli, Enterobacter cloacae, and Citrobacter freundii) of a 756-bp DNA fragment from strain CF09 was obtained only with C. freundii-specific primers. Hybridization results showed that the ampC gene is only chromosomally located while the TEM gene is plasmid located. After cloning of the gene, analysis of the complete nucleotide sequence (1,146 bp) showed that this ampC gene is close to bla_CMY-2, from which it differs by three point mutations leading to amino acid substitutions Glu → Gly at position 22, Trp → Arg at position 201, and Ser → Asn at position 343. AmpC β-lactamases derived from that of C. freundii (LAT-1, LAT-2, BIL-1, and CMY-2) have been found in Klebsiella pneumoniae, E. coli, and Enterobacter aerogenes and have been reported to be plasmid borne. This is the first example of a chromosomally encoded AmpC-type β-lactamase observed in P. mirabilis. We suggest that it be designated CMY-3.     

Development of a Novel Real-Time PCR Assay with High-Resolution Melt Analysis To Detect and Differentiate OXA-48-Like β-Lactamases in Carbapenem-Resistant Enterobacteriaceae

The rapid global spread of carbapenem-resistant Enterobacteriaceae (CRE) poses an urgent threat to public health. More than 250 class D β-lactamases (OXAs) have been described in recent years, with variations in hydrolytic activity for β-lactams. The plasmid-borne OXA-48 β-lactamase and its variants are identified only sporadically in the United States but are common in Europe. Recognition of these OXA-48-like carbapenemases is vital in order to control their dissemination. We developed a real-time PCR assay based on high-resolution melt analysis, using bla_OXA-48-like-specific primers coupled with an unlabeled 3′-phosphorylated oligonucleotide probe (LunaProbe) homologous to OXA-48-like carbapenemase genes. The assay was validated using genomic DNA from 48 clinical isolates carrying a variety of carbapenemase genes, including bla_KPC, bla_SME, bla_IMP, bla_NDM-1, bla_VIM, bla_OXA-48, bla_OXA-162, bla_OXA-181, bla_OXA-204, bla_OXA-244, bla_OXA-245, and bla_OXA-232. Our assay identified the presence of bla_OXA-48-like β-lactamase genes and clearly distinguished between bla_OXA-48 and its variants in control strains, including between bla_OXA-181 and bla_OXA-232, which differ by only a single base pair in the assay target region. This approach has potential for use in epidemiological investigations and continuous surveillance to help control the spread of CRE strains producing OXA-48-like enzymes.     

Complete Sequence of a Conjugative IncN Plasmid Harboring blaKPC-2, blaSHV-12, and qnrS1 from an Escherichia coli Sequence Type 648 Strain

We sequenced a novel conjugative bla_KPC-2-harboring IncN plasmid, pYD626E, from an Escherichia coli sequence type 648 strain previously identified in Pittsburgh, Pennsylvania. pYD626E was 72,800 bp long and carried four β-lactamase genes, bla_KPC-2, bla_SHV-12, bla_LAP-1, and bla_TEM-1. In addition, it harbored qnrS1 (fluoroquinolone resistance) and dfrA14 (trimethoprim resistance). The plasmid profile and clinical history supported the in vivo transfer of this plasmid between Klebsiella pneumoniae and Escherichia coli.     

Complete Sequence of a KPC-Producing IncN Multidrug-Resistant Plasmid from an Epidemic Escherichia coli Sequence Type 131 Strain in China

We report here the nucleotide sequence of a novel bla_KPC-2-harboring incompatibility group N (IncN) plasmid, pECN580, from a multidrug-resistant Escherichia coli sequence type 131 (ST131) isolate recovered from Beijing, China. pECN580 harbors β-lactam resistance genes bla_KPC-2, bla_CTX-M-3, and bla_TEM-1; aminoglycoside acetyltransferase gene aac(6′)-Ib-cr; quinolone resistance gene qnrS1; rifampin resistance gene arr-3; and trimethoprim resistance gene dfrA14. The emergence of a bla_KPC-2-harboring multidrug-resistant plasmid in an epidemic E. coli ST131 clone poses a significant potential threat in community and hospital settings.     

NDM-5 Carbapenemase-Encoding Gene in Multidrug-Resistant Clinical Isolates of Escherichia coli from Algeria

Here, we report the first autochthonous cases of infections caused by bla_NDM-5 New Delhi metallo-β-lactamase-producing Escherichia coli strains recovered from urine and blood specimens of three patients from Algeria between January 2012 and February 2013. The three isolates belong to sequence type 2659 and they coexpress bla_CTX-M-15 with the bla_TEM-1 and bla_aadA2 genes.     

Nationwide Investigation of Extended-Spectrum β-Lactamases,Metallo-β-Lactamases,and Extended-Spectrum Oxacillinases Produced by Ceftazidime-Resistant Pseudomonas aeruginosa Strains in France

A nationwide study aimed to identify the extended-spectrum β-lactamases (ESBLs), metallo-β-lactamases (MBLs), and extended-spectrum oxacillinases (ES-OXAs) in a French collection of 140 clinical Pseudomonas aeruginosa isolates highly resistant to ceftazidime. Six ESBLs (PER-1, n = 3; SHV-2a, n = 2; VEB-1a, n = 1), four MBLs (VIM-2, n = 3; IMP-18, n = 1), and five ES-OXAs (OXA-19, n = 4; OXA-28, n = 1) were identified in 13 isolates (9.3% of the collection). The prevalence of these enzymes is still low in French clinical P. aeruginosa isolates but deserves to be closely monitored.Pseudomonas aeruginosa could potentially become resistant to any of the antibiotics used to treat Gram-negative nosocomial infections. The development of resistance to β-lactams in this opportunistic pathogen results from mutations leading to stable overexpression of intrinsic cephalosporinase AmpC, overproduction of efflux systems, reduced permeability, acquisition of transferable genes coding for a variety of secondary β-lactamases, or a combination of these mechanisms (21). A growing number of Ambler class A extended-spectrum β-lactamases (ESBLs), class B carbapenemases (metallo-β-lactamases [MBLs]), and class D extended-spectrum oxacillinases (ES-OXAs) have been reported in clinical strains of P. aeruginosa (14, 18, 19, 34, 40). The present multicenter study gave a snapshot of these acquired enzymes in a French collection of 140 P. aeruginosa isolates highly resistant to ceftazidime.During a 1-month period (June 2007), 85 hospital laboratories participating in the surveillance networks affiliated with ONERBA (Observatoire National de l''Epidémiologie de la Résistance Bactérienne aux Antibiotiques) collected nonredundant strains of P. aeruginosa resistant to ceftazidime (Caz^r) (as defined by the Comité de l''Antibiogramme de la Société Française de Microbiologie [CA-SFM] in 2006 [12]), except those obtained from screening samples and cystic fibrosis patients. The susceptibility tests were performed in each laboratory according to their routine testing methods. All isolates showing an inhibition zone of <15 mm around the ceftazidime-containing disk (30 μg) or with a MIC of ceftazidime of >32 μg/ml were sent to a central laboratory for further investigation. In addition, the total number of patients with at least one clinical specimen positive for P. aeruginosa as well as the number of hospitalization days was recorded in each participating center during the study period. The central laboratory confirmed bacterial identification by using API32GN strips (bioMérieux, Craponnes, France) and determined the MICs of eight antipseudomonal antibiotics by the conventional 2-fold dilution method in agar (26). The β-lactamase contents of the strains were first analyzed by isoelectric focusing (IEF) (23) and then confirmed by gene sequencing with consensus primers targeting the bla_TEM, bla_PSE, bla_SHV, bla_PER, bla_VEB, bla_GES, bla_BEL, bla_CTX-M, bla_VIM, bla_SPM, bla_OXA-I group, bla_OXA-II group, bla_OXA-III group, and bla_OXA-18 genes (1, 3, 5, 6, 24, 25, 28, 30-32, 35, 38). Genes bla_IMP, bla_GIM, and bla_OXA-9, respectively, were also specifically amplified with primers IMP2004-A and IMP2004-B (5′-ACAYGGYTTGGTTGTTCTTG-3′ and 5′-GGTTTAAYAAAACAACCACC-3′, respectively), GIM-A and GIM-B (5′-GGAGTATATCTTCATACCTCC-3′ and 5′-TTCCAACTTTGCCATGCCCC-3′, respectively), and OXA9A and OXA9B (5′-CCGAGAGATCGCACATACAA-3′ and 5′-CCCATCAACACGGGTAATTC-3′, respectively). Class 1 integrons were amplified in the isolates producing ESBLs, MBLs, and ES-OXAs with consensus primers (20) for content analysis and bla_ESBL, bla_MBL, and bla_ES-OXA localization. Purified amplicons were sequenced on both strands, and their nucleotide sequences were compared and aligned with reference sequences using the NCBI BLAST program (2). Clonality of the Caz^r isolates was investigated by pulsed-field gel electrophoresis (PFGE) of DraI macrorestricted genomic DNA (36, 37).     

Inhibitor-Resistant OXY-2-Derived β-Lactamase Produced by Klebsiella oxytoca

Klebsiella oxytoca strains are generally moderately resistant to amoxicillin and ticarcillin due to the activities of the chromosomally encoded OXY-1 and OXY-2 class A β-lactamase families. These enzymes have the ability to hydrolyze not only penicillins but also cephalosporins, including cefuroxime, ceftriaxone, and aztreonam, and are inhibited by clavulanic acid. A Klebsiella oxytoca strain was isolated from a culture of blood from a patient who had been treated with amoxicillin-clavulanate (3 g/day) for 10 days 1 month earlier. This strain harbored an unusual phenotype characterized by resistance to amoxicillin-clavulanate. It produced an OXY-2-type β-lactamase (pI 6.3), as confirmed by PCR amplification with primers specific for the OXY-2-encoding gene. Gene sequencing revealed a point mutation (A→G) corresponding to the amino acid substitution Ser→Gly at position 130. This mutant enzyme was poorly inhibited by inhibitors, and its kinetic constants compared to those of the parent enzyme were characterized by an increased K_m value for ticarcillin, with a drastically reduced activity against cephalosporins, as is observed with inhibitor-resistant TEM enzymes. The substitution Ser→Gly-130 was previously described in the inhibitor-resistant β-lactamase SHV-10 derived from an SHV-5 variant, but this is the first report of such a mutant in OXY enzymes from K. oxytoca.Klebsiella oxytoca, like Klebsiella spp., carries a chromosomally encoded β-lactamase belonging to class A. Recently, the β-lactamase genes of K. oxytoca were divided into two main groups: bla_oxy-1 and bla_oxy-2 (9). These chromosomal enzymes preferentially inactivate penicillins, conferring resistance on amino- and carboxypenicillins. The ability of the K. oxytoca β-lactamases to hydrolyze extended-spectrum β-lactams could confer resistance to these antibiotics when the β-lactamase is overproduced from a modified promoter (5, 6, 8). The synergistic effects of the three β-lactamase inhibitors clavulanate, sulbactam, and tazobactam were generally poor and varied according to the type of OXY enzymes, the combination of drugs examined, and the type of inhibitor (7). Clavulanate was effective against the overproducing strains, even though it had a relatively high 50% inhibitory concentration (IC₅₀).We report here on a clinical strain of K. oxytoca harboring an unusual resistance phenotype characterized by high-level resistance to amoxicillin, amoxicillin-clavulanate, piperacillin, piperacillin-tazobactam, and, to a lesser extent, ticarcillin and ticarcillin-clavulanate; these resistances were associated with susceptibility to all cephalosporins including cephalothin and aztreonam. This resistance phenotype was very similar to that observed when an inhibitor-resistant TEM (IRT) enzyme was produced in Escherichia coli (19).     

Antibiotic-Resistant Klebsiella pneumoniae and Escherichia coli High-Risk Clones and an IncFIIk Mosaic Plasmid Hosting Tn1 (blaTEM-4) in Isolates from 1990 to 2004

We describe the genetic background of bla_TEM-4 and the complete sequence of pRYC11::bla_TEM-4, a mosaic plasmid that is highly similar to pKpQIL-like variants, predominant among TEM-4 producers in a Spanish hospital (1990 to 2004), which belong to Klebsiella pneumoniae and Escherichia coli high-risk clones responsible for the current spread of different antibiotic resistance genes. Predominant populations of plasmids and host adapted clonal lineages seem to have greatly contributed to the spread of resistance to extended-spectrum cephalosporins.     

A Novel Extended-Spectrum TEM-Type β-Lactamase (TEM-52) Associated with Decreased Susceptibility to Moxalactam in Klebsiella pneumoniae

Klebsiella pneumoniae NEM865 was isolated from the culture of a stool sample from a patient previously treated with ceftazidime (CAZ). Analysis of this strain by the disk diffusion test revealed synergies between amoxicillin-clavulanate (AMX-CA) and CAZ, AMX-CA and cefotaxime (CTX), AMX-CA and aztreonam (ATM), and more surprisingly, AMX-CA and moxalactam (MOX). Clavulanic acid (CA) decreased the MICs of CAZ, CTX, and MOX, which suggested that NEM865 produced a novel extended-spectrum β-lactamase. Genetic, restriction endonuclease, and Southern blot analyses revealed that the resistance phenotype was due to the presence in NEM865 of a 13.5-kb mobilizable plasmid, designated pNEC865, harboring a Tn3-like element. Sequence analysis revealed that the blaT gene of pNEC865 differed from bla_TEM-1 by three mutations leading to the following amino acid substitutions: Glu₁₀₄→Lys, Met₁₈₂→Thr, and Gly₂₃₈→Ser (Ambler numbering). The association of these three mutations has thus far never been described, and the blaT gene carried by pNEC865 was therefore designated bla_TEM-52. The enzymatic parameters of TEM-52 and TEM-3 were found to be very similar except for those for MOX, for which the affinity of TEM-52 (K_i, 0.16 μM) was 10-fold higher than that of TEM-3 (K_i, 1.9 μM). Allelic replacement analysis revealed that the combination of Lys₁₀₄, Thr₁₈₂, and Ser₂₃₈ was responsible for the increase in the MICs of MOX for the TEM-52 producers.