The spread of OXA-24/OXA-40 (OXA-24/40)-producing Acinetobacter spp. in the Iberian Peninsula has been strongly influenced by clonal expansion, but the role of horizontal gene transfer has scarcely been explored. blaOXA-24/40-carrying plasmids and genetic environments were characterized in representative (n = 15) Acinetobacter species clinical isolates (obtained between 2001 and 2007) by Acinetobacter baumannii PCR-based replicon typing, sequencing, hybridization, and restriction fragment length polymorphism. Besides the identification of blaOXA-24/40 within the chromosomes of some isolates, the circulation of common blaOXA-24/40-carrying plasmids (30-kb repA_AB; 10-kb aci2) and genetic backbones among Acinetobacter spp. was demonstrated.

Minibacterium massiliensis strain CIP107820 is a recently discovered waterborne Gram-negative rod isolated from hospital water samples. It harbors a chromosomally located gene encoding an Ambler class A extended-spectrum β-lactamase termed MIN-1, sharing 56%, 54%, and 51% amino acid identities with β-lactamases LUT-1, KPC-2, and CTX-M-2, respectively. β-Lactamase MIN-1 hydrolyzes penicillins, narrow-spectrum cephalosporins, cefotaxime, and, less efficiently, cefepime, while ceftazidime and carbapenems are very poor substrates, and cephamycins and aztreonam are not hydrolyzed.

Acinetobacter bereziniae (formerly Acinetobacter genomospecies 10) isolate Nec was recovered from a skin sample of a patient hospitalized in Paris, France. It was resistant to penicillins, penicillin-inhibitor combinations, and carbapenems. Cloning and expression in Escherichia coli identified the carbapenem-hydrolyzing class D β-lactamase OXA-229, which is weakly related to other oxacillinases (66% amino acid identity with the closest oxacillinase, OXA-58). It hydrolyzed penicillins, oxacillin, and imipenem but not expanded-spectrum cephalosporins. Sequencing of the genetic context of the blaOXA-229 gene did not identify an insertion sequence but did identify mutations in the promoter sequences in comparison to the fully susceptible A. bereziniae reference strain. The overexpression of blaOXA-229 in A. bereziniae Nec as a source of carbapenem resistance was identified by quantitative real-time PCR.

A high rate of broad-spectrum-β-lactamase-producing Escherichia coli isolates was identified from seagull and pelican feces collected in the Miami Beach, Florida, area. The most commonly identified resistance determinants were CMY-2 and CTX-M-15. Those wild birds might be therefore considered vehicles for wide dissemination of multidrug-resistant Enterobacteriaceae in the United States.

A lethal peritonitis model was induced in mice with a Klebsiella pneumoniae isolate producing the carbapenemase OXA-48. Administration of a single dose (up to 100 mg/kg) of the antibiotic piperacillin-tazobactam, imipenem-cilastatin, ertapenem, or cefotaxime had little or no impact on lethality. Ceftazidime had the highest efficacy in vivo, which mirrored its in vitro activity; this was not the case for carbapenems. Therefore, ceftazidime may be recommended for the treatment of infections due to OXA-48 producers if they do not coproduce an extended-spectrum β-lactamase or a plasmid-mediated AmpC cephalosporinase.

Nine carbapenem-resistant Enterobacteriaceae isolates collected from eight patients in five German hospitals were investigated. Six isolates produced the OXA-48 carbapenemase, and three isolates produced OXA-162, which is a point mutant form of OXA-48. Both carbapenemase genes were located on IncL/M-type conjugative plasmids. Insertion sequence IS1999 (truncated or not by IS1R) was located upstream of the blaOXA-48 and blaOXA-162 genes in all of the isolates. Pulsed-field gel electrophoresis typing indicated the clonal transmission of an OXA-48-producing Klebsiella pneumoniae strain in two hospitals.

The carbapenemase NDM-1 has been identified recently in Enterobacteriaceae and Acinetobacter baumannii as a source of multidrug resistance, including resistance to carbapenems. By analyzing the immediate genetic environment of the blaNDM-1 carbapenemase gene among a series of NDM-1-producing enterobacterial isolates, a novel gene (bleMBL, for ble gene associated with the metallo-β-lactamase NDM-1) was identified. The bleMBL gene encodes a novel bleomycin resistance protein (BRP), named BRPMBL, that shares weak similarities with known BRPs (less than 60% amino acid identity). The expression of BRPMBL conferred resistance to bleomycin and to bleomycin-like molecules in Enterobacteriaceae and A. baumannii. The blaNDM-1 and bleMBL genes were coexpressed under the control of the same promoter, located upstream of the blaNDM-1 gene and at the extremity of the insertion sequence ISAba125. Most of the NDM producers possessed the bleMBL gene. Although BRPMBL did not modify the growth or death rates of Escherichia coli under experimental conditions, it suppressed the mutation rate of hypermutable E. coli and therefore may stabilize the plasmid-borne blaNDM-1 gene. This study suggests that the emerging carbapenemase NDM-1 is selected by bleomycin-like molecules, and that BRPMBL producers (and consequently NDM producers) are better suited to various environments.

Analysis of two clonally related multiresistant Pseudomonas aeruginosa isolates led to the identification of a novel IMP-type metallo-β-lactamase. IMP-29 was significantly different from the other IMP variants (the closest variant being IMP-5 with 93% amino acid identity). The blaIMP-29 gene cassette was carried by a class 1 integron in strain 10.298, while in strain 10.266 it was located in a rearranged DNA region on a 30-kb conjugative plasmid. Biochemical analysis confirmed that IMP-29 efficiently hydrolyzed carbapenems.

A clinical Escherichia coli isolate resistant to all β-lactams, including carbapenems, expressed a novel metallo-β-lactamase (MBL), NDM-4, differing from NDM-1 by a single amino acid substitution (Met154Leu). NDM-4 possessed increased hydrolytic activity toward carbapenems and several cephalosporins compared to that of NDM-1. This amino acid substitution was not located in the known active sites of NDM-1, indicating that remote amino acid substitutions might also play a role in the extended activity of this MBL.

A chromosomally located β-lactamase gene, cloned and expressed in Escherichia coli from a reference strain of the enterobacterial species Kluyvera cryocrescens, encoded a clavulanic acid-inhibited Ambler class A enzyme, KLUC-1, with a pI value of 7.4. KLUC-1 shared 86% amino acid identity with a subgroup of plasmid-mediated CTX-M-type extended-spectrum β-lactamases (CTX-M-1, -3, -10, -11, and -12), the most closely related enzymes, and 77% amino acid identity with KLUA-1 from Kluyvera ascorbata. The substrate profile of KLUC-1 corresponded to that of CTX-M-type enzymes.

Escherichia coli ILT-1, Klebsiella pneumoniae ILT-2, and K. pneumoniae ILT-3 were isolated in May 1999 in Paris, France, from a rectal swab of a hospitalized 5-month-old girl. These isolates had a clavulanic acid-inhibited substrate profile that included expanded-spectrum cephalosporins. The MICs of cefotaxime were higher for E. coli ILT-1 and K. pneumoniae ILT-2 than for K. pneumoniae ILT-3, while the opposite was found for the MICs of ceftazidime. Genetic and biochemical analyses revealed that E. coli ILT-1 and K. pneumoniae ILT-2 produced the CTX-M-18 β-lactamase, while K. pneumoniae ILT-3 produced the CTX-M-19 β-lactamase. The amino acid sequence of the CTX-M-18 β-lactamase differed from that of the CTX-M-9 β-lactamase by an Ala-to-Val change at position 231, while CTX-M-19 possessed an additional Pro-to-Ser change at position 167 in the omega loop of Ambler class A enzymes. The latter amino acid substitution may explain the CTX-M-19-mediated hydrolysis of ceftazidime, which has not been reported for other CTX-M-type enzymes. The blaCTX-M-18 and blaCTX-M-19 genes were located on transferable plasmids that varied in size (ca. 60 and 50 kb, respectively) but that showed similar restriction patterns.

From whole-cell DNA of a clinical isolate of the enterobacterial species Rahnella aquatilis, a β-lactamase gene was cloned that encoded a chromosomally encoded Ambler class A enzyme, RAHN-1. RAHN-1, with a pI of 7.2, shares 76, 73, and 71% amino acid identity with the extended-spectrum β-lactamase of chromosomal origin from Serratia fonticola and with the plasmid-mediated β-lactamases CTX-M-2 and CTX-M-1, respectively. The hydrolysis spectrum of the clavulanic acid-inhibited RAHN-1 was expanded to cephalosporins such as cefuroxime, cefotaxime, and ceftriaxone, but not to ceftazidime. Its expression was not inducible.

Pseudomonas aeruginosa GW-1 was isolated in 2000 in South Africa from blood cultures of a 38-year-old female who developed nosocomial pneumonia. This isolate harbored a self-transferable ca. 100-kb plasmid that conferred an expanded-spectrum cephalosporin resistance profile associated with an intermediate susceptibility to imipenem. A β-lactamase gene, blaGES-2, was cloned from whole-cell DNA of P. aeruginosa GW-1 and expressed in Escherichia coli. GES-2, with a pI value of 5.8, hydrolyzed expanded-spectrum cephalosporins, and its substrate profile was extended to include imipenem compared to that of GES-1, identified previously in Klebsiella pneumoniae. GES-2 activity was less inhibited by clavulanic acid, tazobactam and imipenem than GES-1. The GES-2 amino acid sequence differs from that of GES-1 by a glycine-to-asparagine substitution in position 170 located in the omega loop of Ambler class A enzymes. This amino acid change may explain the extension of the substrate profile of the plasmid-encoded β-lactamase GES-2.

Pseudomonas aeruginosa clinical isolate SOF-1 was resistant to cefepime and susceptible to ceftazidime. This resistance phenotype was explained by the expression of OXA-31, which shared 98% amino acid identity with a class D β-lactamase, OXA-1. The oxa-31 gene was located on a ca. 300-kb nonconjugative plasmid and on a class 1 integron. No additional efflux mechanism for cefepime was detected in P. aeruginosa SOF-1. Resistance to cefepime and susceptibility to ceftazidime in P. aeruginosa were conferred by OXA-1 as well.

A β-lactamase gene was cloned from a Nocardia asteroides sensu stricto clinical isolate. A recombinant plasmid, pAST-1, expressed the β-lactamase AST-1 in Escherichia coli JM109. Its pI was 4.8, and its relative molecular mass was 31 kDa. E. coli JM109(pAST-1) was resistant to penicillins and narrow-spectrum cephalosporins. The β-lactamase AST-1 had a restricted hydrolytic activity spectrum. Its activity was partially inhibited by clavulanic acid but not by sulbactam and tazobactam. AST-1 is an Ambler class A β-lactamase sharing 65% amino acid identity with β-lactamase FAR-1, the most closely related enzyme.

Two clonally unrelated Pseudomonas aeruginosa clinical strains, RON-1 and RON-2, were isolated in 1997 and 1998 from patients hospitalized in a suburb of Paris, France. Both isolates expressed the class B carbapenem-hydrolyzing β-lactamase VIM-2 previously identified in Marseilles in the French Riviera. In both isolates, the blaVIM-2 cassette was part of a class 1 integron that also encoded aminoglycoside-modifying enzymes. In one case, two novel aminoglycoside resistance gene cassettes, aacA29a and aacA29b, were located at the 5′ and 3′ end of the blaVIM-2 gene cassette, respectively. The aacA29a and aacA29b gene cassettes were fused upstream with a 101-bp part of the 5′ end of the qacE cassette. The deduced amino acid sequence AAC(6′)-29a protein shared 96% identity with AAC(6′)-29b but only 34% identity with the aacA7-encoded AAC(6′)-I1, the closest relative of the AAC(6′)-I family enzymes. These aminoglycoside acetyltransferases had amino acid sequences much shorter (131 amino acids) than the other AAC(6′)-I enzymes (144 to 153 amino acids). They conferred resistance to amikacin, isepamicin, kanamycin, and tobramycin but not to gentamicin, netilmicin, and sisomicin.

Pseudomonas aeruginosa ED-1, isolated from a pulmonary brush of a patient hospitalized in a suburb of Paris, France, was resistant to ceftazidime and of intermediate susceptibility to ureidopenicillins and to cefotaxime. Cloning and expression of the β-lactamase gene content of this isolate in Escherichia coli DH10B identified a novel OXA-10 variant, OXA-28, with a pI value of 8.1 and a molecular mass of 29 kDa. It differed from OXA-10 by 10 amino acid changes and from OXA-13 and OXA-19 by 2 amino acid changes, including a glycine instead of tryptophan at position 164, which is likely involved in its resistance to ceftazidime. Like OXA-11, -14, -16, and -19 and as opposed to OXA-17, OXA-28 predominantly compromised ceftazidime and had only marginal effect on the MICs of aztreonam and cefotaxime in P. aeruginosa. Once expressed in E. coli, OXA-28 raised the MIC of ceftazidime to a much higher level than those of amoxicillin, cephalothin, and cefotaxime (128, 16, 8, and 4 μg/ml, respectively). OXA-28 β-lactamase had a broad spectrum of activity, including ceftazidime. Its activity was partially antagonized by clavulanic acid (50% inhibitory concentration, 10 μM) and NaCl addition. The oxa28 gene cassette was inserted in the variable region of a class 1 integron, In57, immediately downstream of an amino 6′-N-acetyltransferase gene cassette, aac(6′)Ib. The structures of the integrons carrying either oxa28, oxa13, or oxa19 gene cassettes were almost identical, suggesting that they may have derived from a common ancestor as a result of the common European origin of the P. aeruginosa isolates. In57 was located on a self-transferable plasmid of ca. 150 kb that was transferred from P. aeruginosa to P. aeruginosa.

Ten unrelated Hafnia alvei clinical isolates were grouped according to either their low-level and inducible cephalosporinase production or their high-level and constitutive cephalosporinase production phenotype. Their AmpC sequences shared 85 to 100% amino acid identity. The immediate genetic environment of ampC genes was conserved in H. alvei isolates but was different from that found in other ampC-possessing enterobacterial species.

The class B carbapenem-hydrolyzing β-lactamase IND-1 has been characterized for Chryseobacterium indologenes strain 001. With internal primers for the bla gene for IND-1 (blaIND-1) and an internal blaIND-1 probe, PCR amplifications failed, while hybridization results were positive when DNA from another C. indologenes isolate, strain CIP101026, was used as a template. Thus, a blaIND-related gene was cloned from this C. indologenes reference strain. Sequencing of the insert of a recombinant plasmid conferring resistance to carbapenems revealed an open reading frame with a G + C content of 39.9% and coding for a 243-amino-acid preprotein named IND-2. IND-2 shared 80% amino acid identity with IND-1 and had a similar broad-spectrum resistance profile, including resistance to carbapenems. It was classified in functional subgroup 3a of class B carbapenem-hydrolyzing β-lactamases. IND-1 and IND-2, despite their genetic diversity, possessed similar kinetic parameters, except that ceftazidime was hydrolyzed less by IND-2. To obtain the entire blaIND-related gene sequences of eight other C. indologenes isolates, PCR was performed using internal and external primers, followed by inverse PCR techniques. The likely chromosome-mediated metallo-β-lactamases of the 10 C. indologenes isolates were divided into several groups and subgroups. IND-1, IND-2, IND-2a, IND-3, and IND-4 shared 77 to 99% amino acid identity.

From genomic DNA of Ralstonia pickettii isolate PIC-1, a β-lactamase gene was cloned that encodes the oxacillinase OXA-22. It differs from known oxacillinases, being most closely related to OXA-9 (38% amino acid identity). The hydrolytic spectrum of OXA-22 is limited mostly to benzylpenicillin, cloxacillin, and restricted-spectrum cephalosporins. OXA-22-like genes were identified as single chromosomal copies in five other R. pickettii clinical isolates. The expression of OXA-22-like β-lactamases was inducible in R. pickettii.

A naturally occurring AmpC β-lactamase (cephalosporinase) gene was cloned from the Hafnia alvei 1 clinical isolate and expressed in Escherichia coli. The deduced AmpC β-lactamase (ACC-2) had a pI of 8 and a relative molecular mass of 37 kDa and showed 50 and 47% amino acid identity with the chromosome-encoded AmpCs from Serratia marcescens and Providentia stuartii, respectively. It had 94% amino acid identity with the recently described plasmid-borne cephalosporinase ACC-1 from Klebsiella pneumoniae, suggesting the chromosomal origin of ACC-1. The hydrolysis constants (kcat and Km) showed that ACC-2 was a peculiar cephalosporinase, since it significantly hydrolyzed cefpirome. Once its gene was cloned and expressed in E. coli (pDEL-1), ACC-2 conferred resistance to ceftazidime and cefotaxime but also an uncommon reduced susceptibility to cefpirome. A divergently transcribed ampR gene with an overlapping promoter compared with ampC (blaACC-2) was identified in H. alvei 1, encoding an AmpR protein that shared 64% amino acid identity with the closest AmpR protein from P. stuartii. β-Lactamase induction experiments showed that the ampC gene was repressed in the absence of ampR and was activated when cefoxitin or imipenem was added as an inducer. From H. alvei 1 cultures that expressed an inducible-cephalosporinase phenotype, several ceftazidime- and cefpirome-cross-resistant H. alvei 1 mutants were obtained upon selection on cefpirome- or ceftazidime-containing plates, and H. alvei 1 DER, a ceftazidime-resistant mutant, stably overproduced cephalosporinase. Transformation of H. alvei 1 DER or E. coli JRG582 (ampDE mutant) harboring ampC and ampR from H. alvei 1 with a recombinant plasmid containing ampD from E. coli resulted in a decrease in the MIC of β-lactam and recovery of an inducible phenotype for H. alvei 1 DER. Thus, AmpR and AmpD proteins may regulate biosynthesis of the H. alvei cephalosporinase similarly to other enterobacterial cephalosporinases.

Pseudomonas aeruginosa COL-1 was identified in a blood culture of a 39-year-old-woman treated with imipenem in Marseilles, France, in 1996. This strain was resistant to β-lactams, including ureidopenicillins, ticarcillin-clavulanic acid, cefepime, ceftazidime, imipenem, and meropenem, but remained susceptible to the monobactam aztreonam. The carbapenem-hydrolyzing β-lactamase gene of P. aeruginosa COL-1 was cloned, sequenced, and expressed in Escherichia coli DH10B. The deduced 266-amino-acid protein was an Ambler class B β-lactamase, with amino acid identities of 32% with B-II from Bacillus cereus; 31% with IMP-1 from several gram-negative rods in Japan, including P. aeruginosa; 27% with CcrA from Bacteroides fragilis; 24% with BlaB from Chryseobacterium meningosepticum; 24% with IND-1 from Chryseobacterium indologenes; 21% with CphA-1 from Aeromonas hydrophila; and 11% with L-1 from Stenotrophomonas maltophilia. It was most closely related to VIM-1 β-lactamase recently reported from Italian P. aeruginosa clinical isolates (90% amino acid identity). Purified VIM-2 β-lactamase had a pI of 5.6, a relative molecular mass of 29.7 kDa, and a broad substrate hydrolysis range, including penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems, but not monobactams. As a metallo-β-lactamase, its activity was zinc dependent and inhibited by EDTA (50% inhibitory concentration, 50 μM). VIM-2 conferred a resistance pattern to β-lactams in E. coli DH10B that paralleled its in vitro hydrolytic properties, except for susceptibility to ureidopenicillins, carbapenems, and cefepime. blaVIM-2 was located on a ca. 45-kb plasmid that in addition conferred resistance to sulfamides and that was not self-transmissible either from P. aeruginosa to E. coli or from E. coli to E. coli. blaVIM-2 was the only gene cassette located within the variable region of a novel class 1 integron, In56, that was weakly related to the blaVIM-1-containing integron. VIM-2 is the second carbapenem-hydrolyzing metalloenzyme characterized from a P. aeruginosa isolate outside Japan.

Klebsiella pneumoniae ORI-1 was isolated in 1998 in France from a rectal swab of a 1-month-old girl who was previously hospitalized in Cayenne Hospital, Cayenne, French Guiana. This strain harbored a ca. 140-kb nontransferable plasmid, pTK1, that conferred an extended-spectrum cephalosporin resistance profile antagonized by the addition of clavulanic acid, tazobactam, or imipenem. The gene for GES-1 (Guiana extended-spectrum β-lactamase) was cloned, and its protein was expressed in Escherichia coli DH10B, where this pI-5.8 β-lactamase of a ca. 31-kDa molecular mass conferred resistance to oxyimino cephalosporins (mostly to ceftazidime). GES-1 is weakly related to the other plasmid-located Ambler class A extended-spectrum β-lactamases (ESBLs). The highest percentage of amino acid identity was obtained with the carbenicillinase GN79 from Proteus mirabilis; with YENT, a chromosome-borne penicillinase from Yersinia enterocolitica; and with L-2, a chromosome-borne class A cephalosporinase from Stenotrophomonas maltophilia (36% amino acid identity each). However, a dendrogram analysis showed that GES-1 clustered within a class A ESBL subgroup together with ESBLs VEB-1 and PER-1. Sequencing of a 7,098-bp DNA fragment from plasmid pTK1 revealed that the GES-1 gene was located on a novel class 1 integron named In52 that was characterized by (i) a 5′ conserved segment containing an intI1 gene possessing two putative promoters, P1 and P2, for coordinated expression of the downstream antibiotic resistance genes and an attI1 recombination site; (ii) five antibiotic gene cassettes, blaGES-1, aac(6′)Ib′ (gentamicin resistance and amikacin susceptibility), dfrXVb (trimethoprim resistance), a novel chloramphenicol resistance gene (cmlA4), and aadA2 (streptomycin-spectinomycin resistance); and (iii) a 3′ conserved segment consisting of qacEΔ1 and sulI. The blaGES-1 and aadA2 gene cassettes were peculiar, since they lacked a typical 59-base element. This work identified the second class A ESBL gene of a non-TEM, non-SHV series which was located in the plasmid and integron, thus providing it additional means for its spread and its expression.

In vitro synergy between extended-spectrum cephalosporins and either clavulanic acid or cefoxitin was found for Chryseobacterium meningosepticum isolates during a double-disk assay on an agar plate. An extended-spectrum β-lactamase (ESBL) gene from a C. meningosepticum clinical isolate was cloned and expressed in Escherichia coli DH10B. Its protein conferred resistance to most β-lactams including extended-spectrum cephalosporins but not to cephamycins or to imipenem. Its activity was strongly inhibited by clavulanic acid, sulbactam, and tazobactam, as well as by cephamycins and imipenem. Sequence analysis of the cloned DNA fragment revealed an open reading frame (ORF) of 891 bp with a G+C content of 33.9%, which lies close to the expected range of G+C contents of members of the Chryseobacterium genus. The ORF encoded a precursor protein of 297 amino acids, giving a mature protein with a molecular mass of 31 kDa and a pI value of 9.2 in E. coli. This gene was very likely chromosomally located. Amino acid sequence comparison showed that this β-lactamase, named CME-2 (C. meningosepticum ESBL), is a novel ESBL of the Ambler class A group (Bush functional group 2be), being weakly related to other class A β-lactamases. It shares only 39 and 35% identities with the ESBLs VEB-1 from E. coli MG-1 and CBL-A from Bacteroides uniformis, respectively. The distribution of blaCME-2 among unrelated C. meningosepticum species isolates showed that blaCME-2-like genes were found in the C. meningosepticum strains studied but were absent from strains of other C. meningosepticum-related species. Each C. meningosepticum strain produced at least two β-lactamases, with one of them being a noninducible serine ESBL with variable pIs ranging from 7.0 to 8.5.