Genetic and Biochemical Characterization of the First Extended-Spectrum CARB-Type ?-Lactamase,RTG-4, from Acinetobacter baumannii

Acinetobacter baumannii isolate KAR was uncommonly more resistant to cefepime and cefpirome than to ceftazidime and cefotaxime. Cloning and expression of the β-lactamase gene content of this isolate into Escherichia coli TOP10 identified ß-lactamase RTG-4 (or CARB-10), which corresponds to the first reported extended-spectrum CARB-type enzyme. RTG-4 is a plasmid-encoded Ambler class A β-lactamase whose sequence differs by 4 amino acid substitutions from the narrow-spectrum β-lactamase RTG-3. RTG-4 hydrolyzes cefepime and cefpirome and weakly hydrolyzes ceftazidime due to the single Ser-to-Thr substitution at Ambler position 69. RTG-4 is less susceptible to inhibition by tazobactam and sulbactam than RTG-3. Expression of β-lactamase RTG-4 in a wild-type A. baumannii reference strain showed that it conferred resistance to cefepime and cefpirome. The genetic environment of the bla_RTG-4 gene was made of a peculiar transposon located on a ca. 50-kb plasmid. ISAba9, located upstream of bla_RTG-4, may be responsible for its acquisition by recognizing a secondary right inverted repeat sequence, thus acting by a one-ended transposition process.Acinetobacter baumannii is an opportunistic pathogen that is an important source of nosocomial infections such as pneumonia, septicemia, urinary tract infections, and wound infections (2). Treatment of infections due to this microorganism is becoming a serious clinical concern since A. baumannii is frequently resistant to multiple classes of antibiotics (23, 37). The main mechanism of resistance to β-lactam molecules in A. baumannii is the production of β-lactamases. Resistance to carbapenems is mostly related to the production of metallo-β-lactamases or carbapenem-hydrolyzing oxacillinases (33), whereas resistance to expanded-spectrum cephalosporins mostly results from the overexpression of the natural AmpC-type enzyme (3) or from the acquisition of extended-spectrum β-lactamases (ESBLs). Those ESBLs may correspond to TEM or SHV derivatives but mostly correspond to β-lactamases of the VEB or PER type in A. baumannii (21). Carbenicillin-hydrolyzing β-lactamases (also named CARB enzymes) are narrow-spectrum class A penicillinases that share less than 50% amino acid identity with SHV and TEM β-lactamases (7). The 10 ß-lactamase variants of this family show similar hydrolytic properties but are divided into two subgroups, named the CARB and RTG subgroups, according to their amino acid sequences. The CARB subgroup includes CARB-1 (formerly PSE-4) (18), CARB-2 (formerly PSE-1) (18), CARB-3 (14), CARB-4 (26), Proteus mirabilis N29 β-lactamase (11), CARB-6 (6), CARB-7 (20), and CARB-9 (25). The RTG subgroup includes RTG-1 (P. mirabilis GN79 enzyme) (34), RTG-2 (CARB-5; described in Acinetobacter calcoaceticus var. anitratus [24]), and RTG-3 (CARB-8; identified from an Oligella urethralis clinical isolate [17]). Although those three RTG-type ß-lactamases possess low levels of amino acid identity with members of the CARB family (44%), they might be considered the ancestors of that enzymatic group, as proposed by Choury et al. (7).The carbenicillin-hydrolyzing enzymes belonging to the CARB group were first identified from Pseudomonas aeruginosa clinical isolates (38). The bla_CARB genes are now widespread among distantly related bacteria, including Vibrio spp. (19), because of their location on mobile genetic structures. Indeed, bla_CARB genes such as bla_CARB-1 (22), bla_CARB-2 (28), and bla_CARB-4 (36) were found as a form of gene cassettes of class 1 integrons. In contrast, little is known about the genetic environment of the bla_RTG genes.Analysis of the β-lactamase content of an A. baumannii clinical isolate exhibiting an atypical higher level of resistance to cefepime and cefpirome than to ceftazidime and cefotaxime allowed us to identify a novel plasmid-encoded ESBL-encoding gene belonging to the bla_RTG group.     

High Prevalence of Extended-Spectrum β-Lactamase,Plasmid-Mediated AmpC,and Carbapenemase Genes in Pet Food

We evaluated the pet food contained in 30 packages as a potential origin of extended-spectrum cephalosporin-resistant Gram-negative organisms and β-lactamase genes (bla). Live bacteria were not detected by selective culture. However, PCR investigations on food DNA extracts indicated that samples harbored the bla_CTX-M-15 (53.3%), bla_CMY-4 (20%), and bla_VEB-4-like (6.7%) genes. Particularly worrisome was the presence of bla_OXA-48-like carbapenemases (13.3%). The original pet food ingredients and/or the production processes were highly contaminated with bacteria carrying clinically relevant acquired bla genes.     

Genetic and Biochemical Characterization of FRI-1, a Carbapenem-Hydrolyzing Class A β-Lactamase from Enterobacter cloacae

An Enterobacter cloacae isolate was recovered from a rectal swab from a patient hospitalized in France with previous travel to Switzerland. It was resistant to penicillins, narrow- and broad-spectrum cephalosporins, aztreonam, and carbapenems but remained susceptible to expanded-spectrum cephalosporins. Whereas PCR-based identification of the most common carbapenemase genes failed, the biochemical Carba NP test II identified an Ambler class A carbapenemase. Cloning experiments followed by sequencing identified a gene encoding a totally novel class A carbapenemase, FRI-1, sharing 51 to 55% amino acid sequence identity with the closest carbapenemase sequences. However, it shared conserved residues as a source of carbapenemase activity. Purified β-lactamase FRI-1 hydrolyzed penicillins, aztreonam, and carbapenems but spared expanded-spectrum cephalosporins. The 50% inhibitory concentrations (IC₅₀s) of clavulanic acid and tazobactam were 10-fold higher than those found for Klebsiella pneumoniae carbapenemase (KPC), IMI, and SME, leading to lower sensitivity of FRI-1 activity to β-lactamase inhibitors. The bla_FRI-1 gene was located on a ca. 110-kb untypeable, transferable, and non-self-conjugative plasmid. A putative LysR family regulator-encoding gene at the 5′ end of the β-lactamase gene was identified, leading to inducible expression of the bla_FRI-1 gene.     

Genetic and Biochemical Characterization of TRU-1, the Endogenous Class C β-Lactamase from Aeromonas enteropelogenes

Aeromonas enteropelogenes (formerly A. tructi) was described to be an ampicillin-susceptible and cephalothin-resistant Aeromonas species, which suggests the production of a cephalosporinase. Strain ATCC 49803 was susceptible to amoxicillin, cefotaxime, and imipenem but resistant to cefazolin (MICs of 2, 0.032, 0.125, and >256 μg/ml, respectively) and produced an inducible β-lactamase. Cefotaxime-resistant mutants (MIC, 32 μg/ml) that showed constitutive β-lactamase production could be selected in vitro. The gene coding for the cephalosporinase of A. enteropelogenes ATCC 49803 was cloned, and its biochemical properties were investigated. Escherichia coli transformants showing resistance to various β-lactams carried a 3.5-kb plasmid insert whose sequence revealed a 1,146-bp open reading frame (ORF) encoding a class C β-lactamase, named TRU-1, showing the highest identity scores with A. punctata CAV-1 (75%), A. salmonicida AmpC (75%), and A. hydrophila CepH (71%). The bla_TRU-1 locus includes open reading frames (ORFs) showing significant homology with genes found in the genomes of other Aeromonas species, although it exhibits a different organization, as reflected by the presence of additional ORFs located downstream of the β-lactamase gene in the A. hydrophila and A. salmonicida genomes. Specific PCR assays were negative for cphA-like and bla_OXA-12-like genes in three A. enteropelogenes ATCC strains. Purified TRU-1 showed a broad substrate profile, efficiently hydrolyzing benzylpenicillin, cephalothin, cefoxitin, and, although with significantly lower turnover rates, oxyiminocephalosporins. Cephaloridine and cefepime were poorly recognized by the enzyme, as reflected by the high K_m values observed with these substrates. Thus far, A. enteropelogenes represents the only known example of an Aeromonas species that produces only one β-lactamase belonging to molecular class C.Resistance to β-lactam antibiotics in Gram-negative bacilli is mediated mainly by the production of β-lactamases, which are divided into four major molecular classes, classes A, B, C, and D (3, 11). Genes encoding AmpC (class C) β-lactamases are generally found on the chromosome of many Gram-negative, aerobic, rod-shaped bacteria and are usually inducible (38). About 20 years ago, plasmid-borne class C enzymes were identified in important clinical bacterial species that do not naturally produce significant amounts of these types of β-lactamase (such as Klebsiella pneumoniae or Escherichia coli) or do not possess an ampC gene (e.g., Salmonella spp.) (10, 34) and actually bear an increasing clinical importance. From a functional standpoint, class C β-lactamases are typically characterized by a very efficient hydrolysis of narrow-spectrum cephalosporins but are also able to degrade more recently found extended-spectrum cephalosporins and cephamycins and, thus, can confer resistance to these agents, especially in strains where the β-lactamase is derepressed or plasmid encoded (9, 11, 34).Aeromonads are ubiquitous waterborne organisms, and some Aeromonas species are known to cause human gastroenteritis or serious opportunistic infections, such as septicemia and cellulitis (20, 27). Since these species are present in both the environment and the clinical setting, aeromonads have been identified as a reservoir of antibiotic resistance genes, including β-lactamase determinants such as the bla_FOX-like and bla_CMY-1-like genes (19, 24, 34), which have been successfully transferred to more important opportunistic human pathogens, in particular members of the Enterobacteriaceae.Aeromonas spp. also show an important diversity in terms of β-lactamase content, and at least two different situations have been reported, consisting of the coordinated production of two or three different naturally occurring β-lactamases. Known Aeromonas β-lactamases include the narrow-spectrum class B metallocarbapenemase, a class C cephalosporinase, and a class D oxacillinase, which provide resistance to carbapenems, cephalosporins, and penicillins, respectively (2, 6, 18, 26).Aeromonas enteropelogenes (formerly A. trota or A. tructi) recently appeared as a distinct species (12, 25, 33) that presents the same pathogenicity factors featured by A. hydrophila or A. veronii, such as the production of enterotoxins (1). This species was previously described as being susceptible to penicillins and carbapenems, while it exhibits resistance to narrow-spectrum cephalosporins (12, 33), strongly suggesting the production of a cephalosporinase. This study describes detailed genetic and biochemical properties of the endogenous chromosome-encoded class C β-lactamase from A. enteropelogenes type strain ATCC 49803.     

PER-6, an Extended-Spectrum β-Lactamase from Aeromonas allosaccharophila

An Aeromonas allosaccharophila environmental isolate recovered from the Seine River (Paris, France) produced a novel extended-spectrum β-lactamase, PER-6, that shared 92% amino acid identity with the closest ß-lactamase, PER-2. The kinetic properties of PER-6 showed a slightly increased affinity for carbapenems. The bla_PER-6 gene was chromosomally located and bracketed by non-transposon-related structures.Aeromonas species are ubiquitous in aquatic environments. Formerly considered strict fish pathogens, Aeromonas spp. are reported increasingly as emerging human pathogens for both immunocompetent and immunocompromised patients as a source of gastroenteritis, bacteremia, meningitis, and skin and soft tissue infections (10).Extended-spectrum β-lactamases (ESBLs) have rarely been reported to occur in Aeromonas species, with TEM-24 and CTX-M-1 identified in Aeromonas hydrophila (6, 24) and PER-1 in Aeromonas media (18). Although β-lactamases of the PER type are not the most common ESBLs (15) identified, they have been reported increasingly, with PER-1 being detected mainly in Europe, especially in Turkey (20) and in Italy (17). It has been also identified in Korea in Providencia spp. (12) and more recently in Algeria in Proteus vulgaris and Providencia stuartii (8). β-Lactamase PER-2, which is distantly related to PER-1 (86% amino acid identity), has been reported to occur in Enterobacteriaceae in South America, mostly in Argentina (2, 23). The PER-3, -4, and -5 β-lactamases, which are closely related to PER-1 (99% amino acid identity), have been identified in Aeromonas punctata, P. vulgaris, and Acinetobacter baumannii, according to data available in GenBank (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY740681","term_id":"52858431","term_text":"AY740681"}}AY740681, {"type":"entrez-nucleotide","attrs":{"text":"EU748544","term_id":"190364828","term_text":"EU748544"}}EU748544, and {"type":"entrez-nucleotide","attrs":{"text":"EU687473","term_id":"188988410","term_text":"EU687473"}}EU687473, respectively).Aeromonas spp. are Gram-negative, mostly environmental species frequently containing plasmids and integrons with multiple genes for antibiotic resistance (9). Aeromonas spp. have been shown to be the source of plasmid-mediated resistance to quinolones of the Qnr type associated with novel genetic elements (3, 19).The bla_PER-1 gene has been identified mostly as part of a Tn1213 composite transposon (20). The bla_PER-2 gene has been identified on a large self-conjugative plasmid downstream of the ISPa12 tnpA gene in Citrobacter freundii (23).The present study was initiated by isolation of an Aeromonas allosaccharophila isolate from a water sample of the Seine River in Paris in January 2009, as a result of screening for multidrug-resistant isolates. The sampling procedure consisted of filtrating 100 ml of water through nitrocellulose membranes (0.45 μm; Millipore, Molsheim, France), resuspending the filters in 1 ml of sterile water, and plating 100-μl aliquots on ceftazidime (2 μg/ml)-containing MacConkey agar plates. Isolate AL-1 was identified by the API 32GN system (bioMérieux, Marcy-l''Etoile, France) and by sequencing of the 16S rRNA genes. MICs were determined by Etest (AB Biodisk, Solna, Sweden) and by the agar dilution method with Mueller-Hinton agar plates for imipenem and imipenem clavulanate and were interpreted according to the CLSI guidelines (4). A. allosaccharophila AL-1 was resistant or reduced in susceptibility to amoxicillin, ticarcillin, ceftazidime, and cefotaxime (Table ​(Table1).1). A synergy image between aztreonam or ceftazidime and clavulanic acid-containing disks suggested expression of an ESBL gene. Standard PCR amplification experiments performed with primers specific for the genes encoding β-lactamases TEM, SHV, PER-1, VEB, and GES (7) failed. Shotgun cloning using EcoRI-restricted genomic DNA and EcoRI-restricted pBKCMV plasmid (Invitrogen, Life Technologies, Cergy- Pontoise, France) was performed as previously described (16). Selection on amoxicillin (100 μg/ml)- and kanamycin (30 μg/ml)-agar plates yielded a recombinant Escherichia coli DH10B(pEco-1) clone expressing an ESBL phenotype. The cloned insert of 4,716 bp was sequenced by using universal primers T3 and T7 and the primers listed in Table ​Table2.2. Recombinant E. coli(pEco-1) expressed a novel β-lactamase, PER-6 (www.lahey.org/Studies/). PER-6 shares 92% amino acid identity with PER-2, with the bla_PER-6 gene sharing 79% nucleotide identity with the bla_PER-1 gene, explaining the lack of PCR detection with the use of the bla_PER-1-specific primers. The G+C content of the bla_PER-6 gene (45.5%) was much lower than that of the A. allosaccharophila genes (56.9%) (5), suggesting that the bla_PER-6 gene in A. allosaccharophila Al-1 originated from another bacterial species. Transfer of the β-lactam resistance marker by conjugation or by transformation as described previously (21) from A. allosaccharophila AL-1 to an E. coli reference strain failed. Plasmid extraction performed as described previously (11) identified four plasmids of ca. 5, 12, 22, and 160 kb. None of these plasmids gave a hybridization signal with a bla_PER-6-specific probe after Southern transfer (Hybond N⁺; GE Healthcare), suggesting a chromosomal location for the bla_PER-6 gene (data not shown). This location was confirmed by restricting total DNA with the I-CeuI enzyme (New England Biolabs, Saint-Quentin-en-Yvelines, France), followed by pulsed-field gel electrophoresis and hybridization with the bla_PER-6 and rRNA probes, as described previously (13) (data not shown).

TABLE 1.

MICs of β-lactams for A. allosaccharophila AL-1 and E. coli DH10B harboring recombinant plasmids pPER-1, pPER-2, and pPER-6 and the E. coli DH10B reference strain

Characterization of OXA-204, a Carbapenem-Hydrolyzing Class D β-Lactamase from Klebsiella pneumoniae

A Klebsiella pneumoniae clinical isolate recovered in Tunisia showed resistance to all β-lactams and decreased susceptibility to carbapenems. K. pneumoniae 204 expressed the carbapenem-hydrolyzing β-lactamase OXA-204, differing from OXA-48 by two amino acid substitutions (Gln98His and Thr99Arg) (class D β-lactamase [DBL] numbering). OXA-48 and OXA-204 shared similar resistance profiles, hydrolyzing carbapenems but sparing broad-spectrum cephalosporins. The bla_OXA-204 gene was located on a ca. 150-kb IncA/C-type plasmid, which also carried the bla_CMY-4 gene. The bla_OXA-204 gene was associated with an ISEcp1 element, whereas the bla_OXA-48 genes are usually associated with IS1999.     

BEL-2, an Extended-Spectrum β-Lactamase with Increased Activity toward Expanded-Spectrum Cephalosporins in Pseudomonas aeruginosa

A Pseudomonas aeruginosa isolate recovered in Belgium produced a novel extended-spectrum ß-lactamase, BEL-2, differing from BEL-1 by a single Leu162Phe substitution. That modification significantly altered the kinetic properties of the enzyme, increasing its affinity for expanded-spectrum cephalosporins. The bla_BEL-2 gene was identified from a P. aeruginosa isolate clonally related to another bla_BEL-1-positive isolate.Extended-spectrum ß-lactamases (ESBLs), such as TEM, SHV, PER, VEB, GES, and more recently, CTX-M variants, are reported increasingly to be found in Pseudomonas aeruginosa in various areas (1, 2, 7, 8, 10-12, 15, 17, 21, 23, 27, 28, 30). The BEL-1 ß-lactamase, distantly related to other ESBLs, was identified from a P. aeruginosa isolate from Roeselare, Belgium, which interestingly shows resistance to ticarcillin and ceftazidime but only reduced susceptibility to piperacillin, cefepime, cefpirome, and aztreonam (24). The bla_BEL-1 determinant was found as a gene cassette in the chromosome-borne class 1 integron, In120, that includes other resistance genes (aacA4, aadA5, and smr2) and that was part of a Tn1404-type transposon structure (24). Very recently, Bogaerts et al. (5) reported on the diffusion of BEL-1-producing isolates in various hospital centers of Belgium and also found that BEL-1 could be associated with other relevant β-lactamases, such as the VIM-1 metallo-β-lactamase (5).P. aeruginosa isolate 531 (this study) was recovered from a urine sample of a patient hospitalized in Roeselare, Belgium, in February 2007 for pneumonia and was resistant to all β-lactams but imipenem (Table ​(Table1).1). A synergy between aztreonam or ceftazidime and clavulanic acid-containing disks suggested the synthesis of an ESBL (19). PCR followed by sequencing using ESBL gene-specific primers (24) identified a novel gene encoding BEL-2, which differs from BEL-1 by a single amino acid substitution (Leu to Phe at Ambler position 162) (3). Transfer of a ß-lactam resistance marker from P. aeruginosa 531 to Escherichia coli or to P. aeruginosa reference strains was unsuccessful by either conjugation or transformation (25). Plasmid extraction performed as described previously (14) did not identify any plasmid, suggesting a chromosomal location of the bla_BEL-2-like gene in P. aeruginosa 531. A pulsed-field gel electrophoresis (PFGE) analysis (4) showed that isolates 531 (BEL-2 positive) and 51170 (BEL-1 positive), recovered from the same geographical area, were clonally related. A PCR mapping approach confirmed the presence of a class 1 integron whose structure was identical to that of In120 of P. aeruginosa 51170 (24) and identified an identical structure in P. aeruginosa 531 (data not shown). Overall, these data suggest that the bla_BEL-2 sequence likely resulted from a mutational event that had occurred in In120-carrying P. aeruginosa strains.

TABLE 1.

MICs of β-lactams^a

Genetic and Biochemical Characterization of an Acquired Subgroup B3 Metallo-β-Lactamase Gene,blaAIM-1, and Its Unique Genetic Context in Pseudomonas aeruginosa from Australia

Three clinical Pseudomonas aeruginosa isolates (WCH2677, WCH2813, and WCH2837) isolated from the Women''s and Children''s Hospital, Adelaide, Australia, produced a metallo-β-lactamase (MBL)-positive Etest result. All isolates were PCR negative for known MBL genes. A gene bank was created, and an MBL gene, designated bla_AIM-1, was cloned and fully characterized. The encoded enzyme, AIM-1, is a group B3 MBL that has the highest level of identity to THIN-B and L1. It is chromosomal and flanked by two copies (one intact and one truncated) of an ISCR element, ISCR15. Southern hybridization studies indicated the movement of both ISCR15 and bla_AIM-1 within the three different clinical isolates. AIM-1 hydrolyzes most β-lactams, with the exception of aztreonam and, to a lesser extent, ceftazidime; however, it possesses significantly higher k_cat values for cefepime and carbapenems than most other MBLs. AIM-1 was the first mobile group B3 enzyme detected and signals further problems for already beleaguered antimicrobial regimes to treat serious P. aeruginosa and other Gram-negative infections.     

OXA-16, a Further Extended-Spectrum Variant of OXA-10 β-Lactamase,from Two Pseudomonas aeruginosa Isolates

Two extended-spectrum mutants of the class D β-lactamase OXA-10 (PSE-2) from Pseudomonas aeruginosa isolates obtained in Ankara, Turkey, were described previously and were designated OXA-11 and -14. P. aeruginosa 906 and 961, isolated at the same hospital, were highly resistant to ceftazidime (MIC ≥ 128 μg/ml) and produced a β-lactamase with a pI of 6.2. The MICs of ceftriaxone, cefoperazone, cefsulodin, and cefepime were 4- to 16-fold above the typical values for P. aeruginosa, whereas the MICs of penicillins and cefotaxime were raised only marginally. Ceftazidime MICs were not significantly reduced by clavulanate or tazobactam at 4 μg/ml. Ceftazidime resistance did not transfer conjugatively but was mobilized to P. aeruginosa PU21 by plasmid pUZ8. Both isolates gave similar DNA restriction patterns, suggesting that they were replicates; moreover, they yielded identically sized BamHI fragments that hybridized with a bla_OXA-10 probe. DNA sequencing revealed that both isolates had the same new β-lactamase, designated OXA-16, which differed from OXA-10 in having threonine instead of alanine at position 124 and aspartate instead of glycine at position 157. The latter change is also present in OXA-11 and -14 and seems critical to ceftazidime resistance. Kinetic parameters showed that OXA-16 enzyme was very active against penicillins, cephaloridine, cefotaxime, and ceftriaxone, but hydrolysis of ceftazidime was not detected despite the ability of the enzyme to confer resistance.Resistance to oxyimino cephalosporins in enterobacteria is often associated with extended-spectrum β-lactamases (ESBLs), most of which are mutants of molecular class A β-lactamases, specifically TEM-1 and SHV-1 (2, 19). In Pseudomonas aeruginosa, on the other hand, the most frequent mechanisms of resistance to the oxyimino cephalosporins are derepression of the AmpC chromosomal enzyme and up-regulation of multi-drug efflux (3, 4), and only one extended-spectrum TEM mutant (TEM-42) has been reported (24). Nevertheless, P. aeruginosa has been a major source of unusual ESBLs. Examples include IMP-I, the first plasmidic zinc β-lactamase (32); PER-1, a class A enzyme now widespread in Turkey, though not elsewhere (6, 26, 27, 30); OXA-15, an ESBL mutant of OXA-2 (7); and OXA-11 and -14, which are ESBL mutants of OXA-10 enzyme (5, 11). OXA-11 β-lactamase has two mutations compared with OXA-10, whereas OXA-14 has only one of them. OXA-11 and -14 were produced by isolates from Hacettepe University Hospital in Ankara, Turkey. Total-DNA restriction profiles of these two isolates were identical, and both carried plasmids that gave identical restriction fragments on digestion with EcoRI (5). It is likely that the producer isolates differed only in the presence or absence of the second mutation in the β-lactamase gene (5).In the present study, we describe the discovery and characterization of a further ESBL mutant of OXA-10, also from P. aeruginosa isolates collected from Hacettepe University Hospital.     

Characterization of a P1-Like Bacteriophage Carrying an SHV-2 Extended-Spectrum β-Lactamase from an Escherichia coli Strain

P1 bacteriophages lysogenize bacteria as independent plasmid-like elements. We describe here a P1-like bacteriophage, RCS47, carrying a bla_SHV-2 gene, isolated from a clinical strain of Escherichia coli from phylogroup B1, and we report the prevalence of P1-like prophages in natural E. coli isolates. We found that 70% of the sequence of RCS47, a 115-kb circular molecule, was common to the reference P1 bacteriophage under GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF234172.1","term_id":"33338653","term_text":"AF234172.1"}}AF234172.1, with the shared sequences being 99% identical. RCS47 had acquired two main foreign DNA fragments: a 9,636-bp fragment mobilized by two IS26 elements containing a bla_SHV-2 gene, and an 8,544-bp fragment mobilized by two IS5 elements containing an operon encoding a dimethyl sulfoxide reductase. The reference P1 prophage plasmid replication gene belonged to the IncY incompatibility group, whereas that of RCS47 was from an unknown group. The lytic capacity of RCS47 and bla_SHV-2 gene transduction, through the lysogenization of RCS47 in the recipient E. coli strains, were not demonstrated. The prevalence of P1-like prophages in various animal and human E. coli strain collections, as determined by the PCR detection of repL, the lytic replication gene, was 12.6%. No differences in the prevalences of these prophages were found between extended-spectrum β-lactamase (ESBL)-producing and non-ESBL-producing strains (P = 0.69), but this prevalence was lower in phylogroup B2 than in the other phylogroups (P = 0.008), suggesting epistatic interactions between P1 family phages and the genetic background of E. coli strains. P1-like phages are part of the mobile elements that carry antibiotic resistance. The high prevalence of P1-like prophages suggests their role may be underestimated.     

Molecular and Biochemical Characterization of the Natural Chromosome-Encoded Class A β-Lactamase from Pseudomonas luteola

Pseudomonas luteola (formerly classified as CDC group Ve-1 and named Chryseomonas luteola) is an unusual pathogen implicated in rare but serious infections in humans. A novel β-lactamase gene, bla_LUT-1, was cloned from the whole-cell DNA of the P. luteola clinical isolate LAM, which had a weak narrow-spectrum β-lactam-resistant phenotype, and expressed in Escherichia coli. This gene encoded LUT-1, a 296-amino-acid Ambler class A β-lactamase with a pI of 6 and a theoretical molecular mass of 28.9 kDa. The catalytic efficiency of this enzyme was higher for cephalothin, cefuroxime, and cefotaxime than for penicillins. It was found to be 49% to 59% identical to other Ambler class A β-lactamases from Burkholderia sp. (PenA to PenL), Ralstonia eutropha (REUT), Citrobacter sedlakii (SED-1), Serratia fonticola (FONA and SFC-1), Klebsiella sp. (KPC and OXY), and CTX-M extended-spectrum β-lactamases. No gene homologous to the regulatory ampR genes of class A β-lactamases was found in the vicinity of the bla_LUT-1 gene. The entire bla_LUT-1 coding region was amplified by PCR and sequenced in five other genetically unrelated P. luteola strains (including the P. luteola type strain). A new variant of bla_LUT-1 was found for each strain. These genes (named bla_LUT-2 to bla_LUT-6) had nucleotide sequences 98.1 to 99.5% identical to that of bla_LUT-1 and differing from this gene by two to four nonsynonymous single nucleotide polymorphisms. The bla_LUT gene was located on a 700- to 800-kb chromosomal I-CeuI fragment, the precise size of this fragment depending on the P. luteola strain.Pseudomonas luteola (formely known as CDC group Ve-1 or Chryseomonas luteola) is a motile, strictly aerobic, gram-negative rod, producing a distinct yellow-orange pigment (4). This organism is nonfermentative, oxidase negative, and catalase positive. P. luteola has been isolated from many sources in nature (water, soil, and damp environments) and is considered to be a saprophyte or commensal organism only rarely pathogenic to humans (11, 19). Clinical infections due to this microorganism have rarely been reported (fewer than 25 cases) and have mostly presented as septicemia, meningitis, peritonitis, endocarditis, and ulcer infections, usually in association with surgical operations or the use of catheters or prostheses (11, 13-15, 17, 19, 20, 24, 36, 42). It has been suggested that this organism is likely to become more frequent as a nosocomial pathogen (19). The clinical isolates of P. luteola have generally been shown to be susceptible to extended-spectrum cephalosporins (ESC), aminoglycosides, and fluoroquinolones (11, 20, 36, 42). In most studies in which isolates were tested with a large panel of β-lactam antibiotics, resistance to original-spectrum and broad-spectrum cephalosporins was observed, whereas susceptibility to penicillins was variable (5, 13-15, 17, 20, 36). This β-lactam resistance phenotype suggests that this microbe may produce a natural β-lactamase.We report here the cloning and sequencing of the bla_LUT-1 gene, encoding the class A β-lactamase of the P. luteola clinical isolate LAM, which was isolated in January 2002 from a blood culture from a patient with an infected indwelling catheter. We investigated the biochemical characteristics of LUT-1. The presence, nucleotide diversity, and location of the bla_LUT gene were studied in five other genetically unrelated P. luteola strains.     

A Novel Extended-Spectrum β-Lactamase,SGM-1, from an Environmental Isolate of Sphingobium sp.

SGM-1 is a novel class A β-lactamase from an environmental isolate of Sphingobium sp. containing all of the distinct amino acid motifs of class A β-lactamases. It shares 77 to 80% amino acid sequence identity with putative β-lactamases that are present on the chromosome of all Sphingobium species whose genomes were sequenced and annotated. Thus, SGM-type β-lactamases are native to this genus. Antibiotic susceptibility testing classifies SGM-1 as an extended-spectrum β-lactamase, conferring the highest level of resistance to penicillins. Although SGM-1 contains the conserved cysteine residues characteristic of class A carbapenemases, it does not confer resistance to the carbapenem antibiotics imipenem, meropenem, or doripenem but does increase the MIC of ertapenem 8-fold. SGM-1 hydrolyzes penicillins and the monobactam aztreonam with similar catalytic efficiencies, ranging from 10⁵ to 10⁶ M⁻¹ s⁻¹. The catalytic efficiencies of SGM-1 for cefoxitin and ceftazidime were the lowest (10² to 10³ M⁻¹ s⁻¹) among the cephalosporins tested, while the catalytic efficiencies against all other cephalosporins varied from about 10⁵ to 10⁶ M⁻¹ s⁻¹. SGM-1 exhibited measurable but not significant activity toward the carbapenems tested. SGM-1 also showed high affinity for clavulanic acid, tazobactam, and sulbactam (K_i < 1 μM); however, only clavulanic acid significantly reduced the MICs of β-lactams.     

Effects of Klebsiella pneumoniae Carbapenemase Subtypes,Extended-Spectrum β-Lactamases,and Porin Mutations on the In Vitro Activity of Ceftazidime-Avibactam against Carbapenem-Resistant K. pneumoniae

Avibactam is a novel β-lactamase inhibitor with affinity for Klebsiella pneumoniae carbapenemases (KPCs). In combination with ceftazidime, the agent demonstrates activity against KPC-producing K. pneumoniae (KPC-Kp). KPC-Kp strains are genetically diverse and harbor multiple resistance determinants, including defects in outer membrane proteins and extended-spectrum β-lactamases (ESBLs). Mutations in porin gene ompK36 confer high-level carbapenem resistance to KPC-Kp strains. Whether specific mechanisms of antimicrobial resistance also influence the activity of ceftazidime-avibactam is unknown. We defined the effects of ceftazidime-avibactam against 72 KPC-Kp strains with diverse mechanisms of resistance, including various combinations of KPC subtypes and ESBL and ompK36 mutations. Ceftazidime MICs ranged from 64 to 4,096 μg/ml and were lowered by a median of 512-fold with the addition of avibactam. All strains exhibited ceftazidime-avibactam MICs at or below the CLSI breakpoint for ceftazidime (≤4 μg/ml; range, 0.25 to 4). However, the MICs were within two 2-fold dilutions of the CLSI breakpoint against 24% of the strains, and those strains would be classified as nonsusceptible to ceftazidime by EUCAST criteria (MIC > 1 μg/ml). Median ceftazidime-avibactam MICs were higher against KPC-3 than KPC-2 variants (P = 0.02). Among KPC-2-Kp strains, the presence of both ESBL and porin mutations was associated with higher drug MICs compared to those seen with either factor alone (P = 0.003 and P = 0.02, respectively). In conclusion, ceftazidime-avibactam displays activity against genetically diverse KPC-Kp strains. Strains with higher-level drug MICs provide a reason for caution. Judicious use of ceftazidime-avibactam alone or in combination with other agents will be important to prevent the emergence of resistance.     

OXA-253, a Variant of the Carbapenem-Hydrolyzing Class D β-Lactamase OXA-143 in Acinetobacter baumannii

The carbapenem-hydrolyzing class D β-lactamase OXA-253 was identified in an Acinetobacter baumannii clinical isolate belonging to sequence type 113 (ST113) in Brazil. OXA-253 shares 93.8% amino acid identity with OXA-143. The bla_OXA-253 gene is located on a ca. 20-kb plasmid. The genetic environment of the bla_OXA-253 gene shares the highest identity with ubiquitous GR2 group plasmids usually carrying bla_OXA-24/-40 genes.     

Biochemical and genetic characterization of carbapenem-hydrolyzing β-lactamase OXA-229 from Acinetobacter bereziniae

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 bla(OXA-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 bla(OXA-229) in A. bereziniae Nec as a source of carbapenem resistance was identified by quantitative real-time PCR.     

Characterization of OXA-181, a Carbapenem-Hydrolyzing Class D ��-Lactamase from Klebsiella pneumoniae

Klebsiella pneumoniae KP3 was isolated from a patient transferred from India to the Sultanate of Oman. K. pneumoniae KP3 was resistant to all β-lactams, including carbapenems, and expressed the carbapenem-hydrolyzing β-lactamase OXA-181, which differs from OXA-48 by four amino acid substitutions. Compared to OXA-48, OXA-181 possessed a very similar hydrolytic profile. The bla_OXA-181 gene was located on a 7.6-kb ColE-type plasmid and was linked to the insertion sequence ISEcp1. The ISEcp1-mediated one-ended transposition of bla_OXA-181 was also demonstrated.     

Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

PER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A (“A” indicates an insertion according to Ambler''s scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior.