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International audience; The emergence of multidrug-resistant Escherichia coli ST131 is a major worldwide public health problem in humans. According to the "one health" approach, this study investigated animal reservoirs of ST131, their relationships with human strains, and the genetic features associated with host colonization. High-quality genomes originating from human, avian, and canine hosts were classified on the basis of their accessory gene content using pangenomic. Pangenomic clusters and subclusters were specifi- cally and significantly associated with hosts. The functions of clustering accessory genes were mainly enriched in functions involved in DNA acquisition, interactions, and virulence (e.g., pathogenesis, response to biotic stimulus and interaction between organisms). Accordingly, networks of cooccurrent host interaction factors were significantly associated with the pangenomic clusters and the originating hosts. The avian strains exhibited a specific content in virulence factors. Rarely found in humans, they corresponded to pathovars responsible for severe human infections. An emerging subcluster significantly associated with both human and canine hosts was evidenced. This ability to significantly colonize canine hosts in addition to humans was associated with a specific content in virulence factors (VFs) and metabolic functions encoded by a new pathogenicity island in ST131 and an improved fitness that is probably involved in its emergence. Overall, VF content, unlike the determinants of antimicrobial resistance, appeared as a key actor of bacterial host adaptation. The host dimension emerges as a major driver of genetic evolution that shapes ST131 genome, enhances its diversity, and favors its dissemination.IMPORTANCE Until now, there has been no indication that the evolutionary dynamics of Escherichia coli ST131 may reflect independent and host-specific adaptation of this lineage outside humans. In contrast, the limited number of ST131 reports in animals supported the common view that it rather reflects a spillover of the human sector. This study uncovered a link between host, ST131 population structure, and virulence factor content which appeared to reflect adaptation to hosts. This study helps to better understand the reservoir of ST131, the putative transmission flux, associated risks and the evolutionary dynamics of this bacterial population and highlights a paradigm in which host colonization stands as a key ecological force of the ST131 evolution.

Genome changes are central to the adaptation of bacteria, especially under antibiotic pressure. The aim of this study was to report phenotypic and genomic adaptations undergone by an Enterobacter hormaechei clinical strain that became highly resistant to key antimicrobials during a 4-month period in a patient hospitalized in an intensive care unit (ICU). All six clinical E. hormaechei strains isolated in one ICU-hospitalized patient have been studied. MICs regarding 17 antimicrobial molecules have been measured. Single nucleotide polymorphisms (SNPs) were determined on the sequenced genomes. The expression of genes involved in antibiotic resistance among Enterobacter cloacae complex strains were determined by reverse transcription-quantitative PCR (qRT-PCR). All the strains belonged to sequence type 66 and were distant by a maximum of nine SNPs. After 3 months of hospitalization, three strains presented a significant increase in MICs for ceftazidime, cefepime, temocillin, ertapenem, tigecycline, ciprofloxacin, and chloramphenicol. Those resistant strains did not acquire additional antibiotic resistance genes but harbored a 16-bp deletion in the ramR gene. This deletion led to upregulated expression of RamA, AcrA, AcrB, and TolC and downregulated expression of OmpF. The ΔramR mutant harbored the same phenotype as the resistant clinical strains regarding tigecycline, chloramphenicol, and ciprofloxacin. The increased expression of RamA due to partial deletion in the ramR gene led to a cross-resistance phenotype by an increase of antibiotic efflux through the AcrAB-TolC pump and a decrease of antibiotic permeability by porin OmpF. ramR appears to be an important adaptative trait for E. hormaechei strains.

Major facilitator superfamily (MFS) efflux pumps have been shown to be important for bacterial cells to cope with biocides such as chlorhexidine (CHX), a widely used molecule in hospital settings. In this work, we evaluated the role of two genes, smvA and smvR, in CHX resistance in Enterobacter cloacae complex (ECC). smvA encodes an MFS pump whereas smvR, located upstream of smvA, codes for a TetR-type transcriptional repressor. To this aim, we constructed corresponding deletion mutants from the ATCC 13047 strain (CHX MIC, 2 mg/liter) as well as strains overexpressing smvA or smvR in both ATCC 13047 and three clinical isolates exhibiting elevated CHX MICs (16 to 32 mg/liter). Determination of MICs revealed that smvA played a modest role in CHX resistance, in contrast to smvR that modulated the ability of ECC to survive in the presence of CHX. In clinical isolates, the overexpression of smvR significantly reduced MICs of CHX (2 to 8 mg/liter). Sequence analyses of smvR and promoter regions pointed out substitutions in conserved regions. Moreover, transcriptional studies revealed that SmvR acted as a repressor of smvA expression even if no quantitative correlation between the level of smvA mRNA and MICs of CHX could be observed. On the other hand, overproduction of smvA was able to complement the lack of the major resistance-nodulation-cell division (RND) superfamily efflux pump AcrB and restored resistance to ethidium bromide and acriflavine. Although SmvA could expel biocides such as CHX, other actors, whose expression is under SmvR control, should play a critical role in ECC.

The pks genomic island present in Escherichia coli encodes polyketides (PK) and nonribosomal peptide (NRP) synthases. As discussed in an elegant study published recently ([1][1]), this leads to the synthesis of numerous and still not fully purified and characterized PK-NRP compounds that confer to

The clinical Klebsiella pneumoniae INSRA6884 strain exhibited nonsusceptibility to all penicillins tested (MICs of 64 to >2,048 μg/ml). The MICs of penicillins were weakly reduced by clavulanate (from 2,048 to 512 μg/ml), and tazobactam restored piperacillin susceptibility. Molecular characterization identified the genes bla GES-7 and a new β-lactamase gene, bla SHV-107 , which encoded an enzyme that differed from SHV-1 by the amino acid substitutions Leu35Gln and Thr235Ala. The SHV-107-producing Escherichia coli strain exhibited only a β-lactam resistance phenotype with respect to amoxicillin, ticarcillin, and amoxicillin-clavulanate combination. The kinetic parameters of the purified SHV-107 enzyme revealed a high affinity for penicillins. However, catalytic efficiency for these antibiotics was lower for SHV-107 than for SHV-1. No hydrolysis was detected against oxyimino-β-lactams. The 50% inhibitory concentration (IC 50 ) for clavulanic acid was 9-fold higher for SHV-107 than for SHV-1, but the inhibitory effects of tazobactam were unchanged. Molecular dynamics simulation suggested that the Thr235Ala substitution affects the accommodation of clavulanate in the binding site and therefore its inhibitory activity.

TEM-154, identified in Portugal in 2004, associated the substitutions observed in the extended-spectrum β-lactamase (ESBL) TEM-12 and in the inhibitor-resistant penicillinase (IRT) TEM-33. This enzyme exhibited hydrolytic activity against ceftazidime and a low level of resistance to clavulanic acid. Surprisingly, the substitution Met69Leu enhanced the catalytic efficiency of oxyimino β-lactams conferred by the substitution Arg164Ser. Its discovery confirms the dissemination of the complex mutant group of TEM enzymes in European countries.

Five different strains of bacteria belonging to the family Enterobacteriaceae were isolated from two patients hospitalized in the intensive care unit of the Central Military Hospital of Algiers, Algeria. All five strains, one Providencia stuartii strain, two Escherichia coli strains, and two Klebsiella pneumoniae strains, were intermediate or resistant to all β-lactams, including carbapenems. Synergy between imipenem and EDTA was observed for all five strains. The results of the PCR experiment confirmed the presence of a bla VIM gene in all five strains. The bla VIM genes were located as part of a class 1 integron on a 180-kb conjugative plasmid. They encoded a novel metallo-β-lactamase designated VIM-19, which differed from the parental enzyme VIM-1 by only two substitutions: Ser228Arg, previously observed in the closely related enzyme VIM-4, and Asn215Lys, not previously observed in other VIM-type carbapenemases. VIM-19 was further characterized after purification through determination of its kinetic constants. This enzyme was inhibited by EDTA and hydrolyzed penicillins, cephalosporins, and carbapenems, as observed for other VIM-type carbapenemases but with greater catalytic efficiency against penicillins than VIM-1. VIM-19 is the first carbapenemase enzyme identified from an isolate from Algeria. These results confirm the emergence of VIM-4-like enzymes in members of the family Enterobacteriaceae from Mediterranean countries.

β-Lactamases represent the major resistance mechanism of gram-negative bacteria against β-lactam antibiotics. The amino acid sequences of these proteins vary widely, but all are located in the periplasm of bacteria. In this study, we investigated the translocation mechanism of representative β-lactamases in an Escherichia coli model. N-terminal signal sequence analyses, antibiotic activity assay, and direct measurement of translocation of a green fluorescent protein (GFP) reporter fused to β-lactamases revealed that most were exported via the Sec pathway. However, the Stenotrophomonas maltophilia L2 β-lactamase was exported via the E. coli Tat translocase, while the S. maltophilia L1 β-lactamase was Sec dependent. These results show the possible Tat-dependent translocation of β-lactamases in the E. coli model system. In addition, the mutation of the cytoskeleton-encoding gene mreB , which may be involved in the spatial organization of penicillin-binding proteins, decreased the MIC of β-lactams for β-lactamase-producing E. coli . These findings provide new knowledge about β-lactamase translocation, a putative new target for addressing β-lactamase-mediated resistance.

The new β-lactamase SHV-72 was isolated from clinical Klebsiella pneumoniae INSRA1229, which exhibited the unusual association of resistance to the amoxicillin-clavulanic acid combination (MIC, 64 μg/ml) and susceptibility to cephalosporins, aztreonam, and imipenem. SHV-72 (pI 7.6) harbored the three amino acid substitutions Ile8Phe, Ala146Val, and Lys234Arg. SHV-72 had high catalytic efficiency against penicillins ( k cat / K m , 35 to 287 μM −1 ·s −1 ) and no activity against oxyimino β-lactams. The concentration of clavulanic acid necessary to inhibit the enzyme activity by 50% was 10-fold higher for SHV-72 than for SHV-1. Molecular-dynamics simulation suggested that the Lys234Arg substitution in SHV-72 stabilized an atypical conformation of the Ser130 side chain, which moved the Oγ atom of Ser130 around 3.5 Å away from the key Oγ atom of the reactive serine (Ser70). This movement may therefore decrease the susceptibility to clavulanic acid by preventing cross-linking between Ser130 and Ser70.

Escherichia coli CF349 exhibited a complex β-lactam resistance phenotype, including resistance to amoxicillin and ticarcillin alone and in combination with clavulanate and to some extended-spectrum cephalosporins. The double-disk synergy test was positive. CF349 harbored an 85-kb conjugative plasmid which encoded a β-lactamase of pI 5.9. The corresponding bla gene was identified by PCR and sequencing as a bla TEM gene. The deduced protein sequence revealed a new complex mutant of TEM-1 β-lactamase designated TEM-109 (CMT-5). TEM-109 contained both the substitutions Glu104Lys and Arg164His of the expanded-spectrum β-lactamase (ESBL) TEM-6 and Met69Leu of the inhibitor-resistant TEM-33 (IRT-5). TEM-109 exhibited hydrolytic activity against ceftazidime similar to that of TEM-6 ( k cat , 56 s −1 and 105 s −1 , respectively; K m values, 226 and 247 μM, respectively). The 50% inhibitory concentrations of clavulanate and tazobactam (0.13 μM and 0.27 μM, respectively) were 5- to 10-fold higher for TEM-109 than for TEM-6 (0.01 and 0.06 μM, respectively) but were almost 10-fold lower than those for TEM-33. The characterization of this novel CMT, which exhibits a low level of resistance to inhibitors, highlights the emergence of this new ESBL type.

A Proteus mirabilis clinical strain (7001324) was isolated from urine sample of a patient hospitalized in a long-term-care facility. PCR and cloning experiments performed with this strain identified a novel TEM-type β-lactamase (TEM-187) differing by four amino acid substitutions (Leu21Phe, Arg164His, Ala184Val, and Thr265Met) from TEM-1. This characterization provides further evidence for the diversity of extended-spectrum β-lactamases (ESBL) produced by P. mirabilis and for their potential spread to other Enterobacteriaceae due to a lack of sensitive detection methods used in daily practice.

TEM-24 (CAZ-6) extended-spectrum β-lactamase (ESBL) was detected in 1988 in Clermont-Ferrand, France, in Klebsiella pneumoniae ( bla TEM-24 ) and Enterobacter aerogenes ( bla TEM-24b ), and since 1994, a TEM-24-producing E. aerogenes clonal strain has been observed elsewhere in the country. To determine if the spread of this clonal strain was restricted to TEM-24-producing E. aerogenes strains, 84 E. aerogenes strains (non-TEM/SHV-producing strains, TEM-1- or -2-producing strains, and different ESBL-producing strains), isolated from 1988 to 1999 in Clermont-Ferrand ( n = 59) and in 11 other French hospitals in 1998 ( n = 25), were studied. A clonal strain was found for TEM-24- but also for TEM-3- and TEM-1- or 2-producing isolates. This study shows that there is a clonal strain dependent on acquisition of the TEM-type enzyme (TEM-24 and other TEM types).

Six clinical CTX-M-producing isolates of the family Enterobacteriaceae were detected between 1999 and 2000 in different French hospitals. Two strains produced CTX-M-1 and CTX-M-3 and four strains produced CTX-M-14, a mutant Ala-2313Val of CTX-M-9. A putative transposable element, ISEcp-1, was located 43 bp upstream of all the blaCTX-M genes. Two CTX-M-14-encoding plasmids exhibited similar restriction patterns. The CTX-M-1- and CTX-M-3-encoding plasmids were related to the CTX-M-1- and CTX-M-3-encoding plasmids previously reported in 1990 in France and in 1998 in Poland, respectively. The early extended-spectrum -lactamases (ESBLs) arose as the result of a few amino acid substitutions from the common plasmid-mediated TEM and SHV-1 -lactamases. At the beginning of the 1990s, a new class A type of ESBL was characterized in the first reports of the CTX-M-1 (MEN-1) enzyme (2, 3). These CTX-M-type enzymes are typical ESBLs, characterized by much greater hydrolytic activity against cefotaxime than against ceftazidime. Thus, they confer a high level of resistance to cefotaxime and have only marginal effects on the MIC of ceftazidime. The family of CTX-M-type ESBLs comprises at least 12 members belonging to four major phylogenetic branches on the basis of their amino acid sequence similarities (8): the CTX-M-1 (MEN-1) branch (CTX-M-1, CTX-M-3, and CTXM-10), the CTX-M-2 branch (CTX-M-2, Toho-1, and CTXM-4 to CTX-M-6), the CTX-M-9 branch (CTX-M-9 and Toho2), and the CTX-M-8 branch. These enzymes have been reported in several species of the family Enterobacteriaceae and in Vibrio cholerae serovar El Tor and have been isolated in three main geographical areas: South America (7, 8; M. Galas, F. Pasteran, R. Melano, A. Petroni, G. Lopez, A. Corso, A. Rossi, and Whonet Collaborative Group, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. E-109, p. 201, 1998), Eastern Europe (11, 12, 13, 20), and Japan (14, 19). We describe six clinical strains of the family Enterobacteriaceae (Table 1) which were collected in different French hospitals on the basis of their positive synergy test results (16) and their higher levels of resistance to cefotaxime than ceftazidime (MICs, 16 to 128 and 2 to 8 g/ml, respectively). Escherichia coli CF-1 and Enterobacter cloacae CF-2 were isolated at the teaching hospital of Clermont-Ferrand, France, from the urine of a patient hospitalized in 1999 and from a pulmonary sample of a patient admitted in 2000, respectively. E. coli Mnt-1 and Klebsiella pneumoniae Mnt-2 were isolated from blood and stool samples, respectively, of a Vietnamese child admitted to Montpellier hospital, Montpellier, France, in 1999. E. coli Roa-1 was isolated in Roannes hospital, Roannes, France, in 1999 from blood, and E. cloacae Ver-1 was isolated in Versailles hospital, Versailles, France, in 1999 from a urine sample (10). CTX-M-1-producing strain MEN (2) and CTX-M-3-encoding plasmid A1 (20) were used as references. Analytical isoelectric focusing was performed as described previously (7). The following -lactamases with known pIs were used as standards: TEM-1 (pI 5.4), SHV-1 (pI 7.6), and CTX-M-1 (pI 8.4). All strains tested produced an enzyme of pI 5.4, associated with a second -lactamase of alkaline pI: pI 8.4 for strains Ver-1 and CF-2 and pI 7.9 for strains CF-1, Mnt-1, Mnt-2, and Roa-1. PCR TEM and direct sequencing of the PCR product (23) identified the -lactamase of pI 5.4 as being the TEM-1 penicillinase. No PCR products were obtained with primers specific for blaSHV genes. Positive amplifications were obtained with primers CTX-MA (5-CGCTTTGCGATGTGCAG-3)

The OXA-48 carbapenemase is mainly encoded by ∼62-kb IncL/M plasmids. However, chromosome-mediated genes have been observed in Escherichia coli isolates. In this work, we investigated the genetic environment of OXA-48 in members of the family Enterobacteriaceae ( n = 22) to understand how the OXA-48-encoding gene is transferred into the E. coli chromosome. The OXA-48-encoding gene was located within intact Tn 1999.2 transposons in the ∼62-kb plasmids or within a truncated variant of Tn 1999.2 for the OXA-48-encoding genes located in the chromosomes of E. coli bacteria. The analysis of the Tn 1999.2 genetic environment revealed an inverted orientation of the transposon in five ∼62-kb plasmids (5/14 [35%]) and in all chromosome inserts ( n = 8). The sequencing of pRA35 plasmid showed that this orientation of Tn 1999.2 and the acquisition of an IS 1R insertion sequence generated a 21.9-kb IS 1R -based composite transposon encoding OXA-48 and designated Tn 6237 . The sequencing of a chromosomal insert encoding OXA-48 also revealed this new transposon in the E. coli chromosome. PCR mapping showed the presence of this element in all strains harboring an OXA-48-encoding chromosomal insert. However, different insertion sites of this transposon were observed in the E. coli chromosome. Overall, these findings indicate a plasticity of the OXA-48 genetic environment mediated by IS 1R insertion sequences. The insertion sequences can induce the transfer of the OXA-encoding gene into E. coli chromosomes and thereby promote its persistence and expression at low levels.

Serratia marcescens Rio-5, one of 18 extended-spectrum β-lactamase (ESBL)-producing strains isolated in several hospitals in Rio de Janeiro (Brazil) in 1996 and 1997, exhibited a high level of resistance to aztreonam (MIC, 512 μg/ml) and a distinctly higher level of resistance to cefotaxime (MIC, 64 μg/ml) than to ceftazidime (MIC, 8 μg/ml). The strain produced a plasmid-encoded ESBL with a pI of 7.5 whose bla gene was not related to those of other plasmid-mediated Ambler class A ESBLs. Cloning and sequencing revealed a bla gene encoding a novel class A β-lactamase in functional group 2be, designated BES-1 (Brazil extended-spectrum β-lactamase). This enzyme had 51% identity with chromosomal class A penicillinase of Yersinia enterocolitica Y56, which was the most closely related enzyme and 47 to 48% identity with CTX-M-type β-lactamases, which were the most closely related ESBLs. In common with CTX-M enzymes, BES-1 exhibited high cefotaxime-hydrolyzing activity ( k cat , 425 s −1 ). However, BES-1 differed from CTX-M enzymes by its significant ceftazidime-hydrolyzing activity ( k cat , 25 s −1 ), high affinity for aztreonam ( K i , 1 μM), and lower susceptibility to tazobactam (50% inhibitory concentration [IC 50 ], 0.820 μM) than to clavulanate (IC 50 , 0.045 μM). Likewise, certain characteristic structural features of CTX-M enzymes, such as Phe-160, Ser-237, and Arg-276, were observed for BES-1, which, in addition, harbored different residues (Ala-104, Ser-171, Arg-220, Gly-240) and six additional residues at the end of the sequence. BES-1, therefore, may be an interesting model for further investigations of the structure-function relationships of class A ESBLs.

β-Lactam resistance was studied in 1,072 consecutive P. mirabilis clinical strains isolated at the Clermont-Ferrand teaching hospital between April 1996 and March 1998. The frequency of amoxicillin resistance was 48.5%. Among the 520 amoxicillin-resistant isolates, three resistance phenotypes were detected: penicillinase (407 strains [78.3%]), extended-spectrum β-lactamase (74 strains [14.2%]), and inhibitor resistance (39 strains [7.5%]). The penicillinase phenotype isolates were divided into three groups according to the level of resistance to β-lactams, which was shown to be related to the strength of the promoter. The characterization of the different β-lactamases showed that amoxicillin resistance in P. mirabilis was almost always (97%) associated with TEM or TEM-derived β-lactamases, most of which evolved via TEM-2.

To estimate the diversity of extended-spectrum β-lactamases in Brazil, 18 strains from different species of the family Enterobacteriaceae exhibiting a positive double-disk synergy test were collected by a clinical laboratory from several hospitals in Rio de Janeiro, Brazil, in 1996 and 1997. Four strains ( Proteus mirabilis , Enterobacter cloacae , Enterobacter aerogenes , and Citrobacter amalonaticus ) hybridized with a 550-bp CTX-M probe. The P. mirabilis strain produced a CTX-M-2 enzyme. The E. cloacae , E. aerogenes , and C. amalonaticus isolates harbored a bla gene which was identified by cloning and sequencing as a bla CTX-M gene. E. coli HB101 transconjugants and the E. coli DH5α transformant harboring a recombinant plasmid produced a CTX-M β-lactamase with an isoelectric point of 7.6 conferring a resistance phenotype characterized by a higher level of resistance to cefotaxime than to ceftazidime, as observed with the other CTX-M enzymes. The deduced protein sequence showed a novel Ambler class A CTX-M enzyme, named CTX-M-8, which had 83 to 88% identity with the previously described CTX-M enzymes. The phylogenic study of the CTX-M family including CTX-M-8 revealed four CTX-M types, CTX-M-8 being the first member of a new phylum of CTX-M enzymes. The evolutionary distances between the four types of CTX-M were large, suggesting that the four clusters branched off early from a distant unknown enzyme and that intermediate enzymes probably existed.

From August 1996 to May 1997, six verotoxin-producing Escherichia coli (VTEC) strains were isolated from stool specimens of adults suffering from hemolytic-uremic syndrome (HUS). All the isolates were stx 2 positive and belonged to different serotypes: O6:H4, O91:H10, O91:H21, O rough:H16, OX3:H−, and O nontypeable:H−. The enterohemolysin (Ehly)-encoding genes were detected in two isolates, and none of the isolates harbors the intimin (Eae)-encoding gene. These findings suggest that stx 2 -positive non-O157:H7 VTEC is a major cause of HUS in adults and that several sources of pathogens are responsible for local endemic infections.

In this work, we characterized a new, 160-kb, bla OXA-48 -harboring IncL/M-type plasmid isolated from a Klebsiella pneumoniae strain from France. Moreover, we report the transfer of a 60-kb OXA-48-encoding plasmid from Klebsiella pneumoniae to other Enterobacteriaceae in two patients.

Currently, CTX-M β-lactamases are among the most prevalent and most heterogeneous extended-spectrum β-lactamases (ESBLs). In general, CTX-M enzymes are susceptible to inhibition by β-lactamase inhibitors. However, it is unknown if the pathway to inhibition by β-lactamase inhibitors for CTX-M ESBLs is similar to TEM and SHV β-lactamases and why bacteria possessing only CTX-M ESBLs are so susceptible to carbapenems. Here, we have performed a kinetic analysis and timed electrospray ionization mass spectrometry (ESI-MS) studies to reveal the intermediates of inhibition of CTX-M-9, an ESBL representative of this family of enzymes. CTX-M-9 β-lactamase was inactivated by sulbactam, tazobactam, clavulanate, meropenem, doripenem, ertapenem, and a 6-methylidene penem, penem 1. K i values ranged from 1.6 ± 0.3 μM (mean ± standard error) for tazobactam to 0.02 ± 0.01 μM for penem 1. Before and after tryptic digestion of the CTX-M-9 β-lactamase apo-enzyme and CTX-M-9 inactivation by inhibitors (meropenem, clavulanate, sulbactam, tazobactam, and penem 1), ESI-MS and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identified different adducts attached to the peptide containing the active site Ser70 (+52, 70, 88, and 156 ± 3 atomic mass units). This study shows that a multistep inhibition pathway results from modification or fragmentation with clavulanate, sulbactam, and tazobactam, while a single acyl enzyme intermediate is detected when meropenem and penem 1 inactivate CTX-M-9 β-lactamase. More generally, we propose that Arg276 in CTX-M-9 plays an essential role in the recognition of the C 3 carboxylate of inhibitors and that the localization of this positive charge to a “region of the active site” rather than a specific residue represents an important evolutionary strategy used by β-lactamases.

Inhibitor-resistant TEM (IRT) β-lactamases derive from TEM-1 or TEM-2 enzymes by point mutations in the corresponding coding gene (http://www.lahey.org/studies). Inhibitor-resistant SHV (IRS) β-lactamases are less commonly identified than IRT enzymes, and mainly in Klebsiella pneumoniae strains (3, 4, 5, 8, 9). In this study, we performed a phenotypic, molecular, and biochemical characterization of a new IRS enzyme, SHV-84, produced by a clinical Escherichia coli strain. E. coli INSRA4590 was isolated from a patient admitted in 1999 to Centro Hospitalar de Coimbra, Portugal. Antimicrobial susceptibility, isoelectric focusing for isoelectric point determination, identification of β-lactamase-encoding genes, and extraction and purification of a new β-lactamase were performed as previously described (1, 7). Antimicrobial susceptibility results were interpreted by using French Society of Microbiology criteria (2). E. coli INSRA4590 exhibited high-level resistance to the penicillins tested (except piperacillin) and to the combination of amoxicillin and clavulanic acid but was susceptible to the combination of piperacillin and tazobactam (Table ​(Table1);1); it was susceptible to all cephalosporins tested and to aztreonam. TABLE 1. MICs for clinical, transformant, and recipient E. coli strains and kinetic parameters of SHV-84 and SHV-1 β-lactamases The clinical strain expressed SHV-1 with a pI of 7.6 and the new SHV-84 β-lactamase with a pI of 7.4, which differed from SHV-1 by the amino acid substitution Lys234Arg. This mutation has been described in other IRS enzymes, such as SHV-56 (5) and SHV-72 (also encountered in Portugal) (8), in which it was associated with the substitution Leu35Gln and the substitutions Ile8Phe and Ala146Val, respectively. The recombinant SHV-encoding plasmid (pBK-SHV-84) and the corresponding transformant E. coli DH5α were obtained as previously described (8). The SHV-84-producing transformant DH5α-SHV-84 exhibited a β-lactam resistance phenotype similar to that of the clinical strain (Table ​(Table1),1), and the corresponding β-lactamase had a pI of 7.4. The kinetic constants of the purified enzyme (≥99% pure; data not shown) and the concentrations of inhibitors required to inhibit enzyme activity by 50% (IC50s) were determined as previously reported (6, 8) and compared with those of SHV-1. Unlike SHV-72, SHV-84 showed a lower affinity for penicillins (Km, 64 to 101 μM) and decreased catalytic activity against these antibiotics (kcat, 216 to 1,042 s−1) than SHV-1 (Km, 11 to 31 μM; kcat, 220 to 1,937 s−1) (Table ​(Table1).1). In SHV-72, mutations other than Lys234Arg might confer increased affinity of the enzyme for penicillins, as well as better catalytic activity against those antibiotics. SHV-84, like SHV-72, exhibited lower affinity for cephalothin (Km, 169 to 141 μM) than SHV-1. No hydrolysis of extended-spectrum cephalosporins was detected. However, SHV-84 was less susceptible to clavulanic acid (IC50, 2.21 μM) than SHV-1 (IC50, 0.17 μM), as observed for SHV-72 (IC50, 1.72 μM) (Table ​(Table1)1) (8). The IC50 of tazobactam for SHV-84 was 3.5-fold lower than that for SHV-1 (IC50s, 0.03 and 0.15 μM, respectively). In conclusion, this study underlines the importance of the Lys234Arg substitution in resistance to clavulanic acid in nature, since we demonstrate that this mutation alone is responsible for decreased susceptibility to β-lactamase inhibitors. These results corroborate those previously obtained by molecular dynamic simulation in a study using the model of the mutant SHV-72 enzyme, in which the authors suggested a change in the positioning of the Ser130 side chain induced by Arg234 (8).

In a 3-month period in 1998, 79 consecutive isolates of Enterobacteriaceae producing an extended-spectrum β-lactamase (ESBL) were collected. ESBLs were predominantly TEM derivatives (74 of 79): TEM-24-like (40 isolates), TEM-3-like (29 isolates), TEM-21 (3 isolates), and TEM-4 and TEM-52 (1 isolate each). Four isolates produced SHV derivatives SHV-4 (three isolates) and SHV-5 (one isolate), and one strain produced a CTX-M-3 enzyme. The high proportion of TEM-24-like-producing Enterobacter aerogenes isolates (36 of 79) suggests the occurrence of an epidemic strain in France.

In 2004, 65 CTX-M-producing Escherichia coli isolates were collected from infected patients in four French hospitals. The bla CTX-M-15 genes were predominant. Pulsed-field gel electrophoresis highlighted a clonal propagation of CTX-M-15-producing strains belonging to phylogenetic group B2, notably in the community. The main risk factors for acquiring these isolates were urinary tract infections or the presence of a urinary catheter in diabetic or renal failure patients.

By PCR, we screened for qnr genes 112 clinical isolates of extended-spectrum β-lactamase-producing Escherichia coli collected from hospitals in France during 2004. For the first time, 7.7% of CTX-M-producing E. coli isolates presented a plasmid-mediated resistance to quinolones. All strains harbored a qnrA gene located on a sul1 -type class 1 integron with similar structure to the In 36 integron.

The clinical strain Escherichia coli TO799 was resistant to penicillin-clavulanate combinations and ceftazidime and was not reproducibly detected as an extended-spectrum β-lactamase (ESBL) according to the standards of the Clinical Laboratory Standards Institute (CLSI; formerly NCCLS) and the national guidelines of the French Society for Microbiology (Comité de l'Antibiogramme de la Société Française de Microbiologie). A novel β-lactamase, designated TEM-125, was responsible for this phenotype. TEM-125 harbors a complex association of mutations previously described in the ESBL TEM-12 and in the inhibitor-resistant β-lactamase TEM-39. TEM-125 is the first complex mutant TEM to present hydrolytic activity against ceftazidime ( k cat , 3.7 s −1 ) together with a high level of resistance to clavulanate (50% inhibitory concentration, 13.6 μM). The discovery of such an ESBL, which is difficult to detect by the usual ESBL detection methods, confirms the emergence of a complex mutant TEM subgroup and highlights the need to evaluate detection methods so as to avoid possible therapeutic failures.

The TEM-24 extended-spectrum β-lactamase (ESBL), initially characterized in Klebsiella pneumoniae (3), is currently the predominant ESBL in France (4). This could be related to the spread of a TEM-24-producing Enterobacter aerogenes clone present in most French hospitals (1) but also found in Belgium (5) and Spain (2). TEM-24 enzyme has been recovered from an increasing number of species of the family Enterobacteriaceae (6, 8) and in Pseudomonas aeruginosa (7). In contrast, resistance to expanded-spectrum cephalosporins mediated by ESBLs has never been described in the genus Aeromonas, for which β-lactam resistance involves three chromosomally mediated enzymes: a cephalosporinase, a penicillinase, and a carbapenemase (9). We report the first characterization of a TEM-24-producing clinical Aeromonas strain. 16S ribosomal DNA (rDNA) and gyrB sequencing (10) showed that the strain was most closely related to Aeromonas caviae. This strain was recovered together with an ESBL-producing E. aerogenes isolate from the diarrheal feces of a 76-year-old man admitted to Montpellier Hospital for intestinal ischemia. Based on the resistance phenotype observed after the disk diffusion assay, both E. aerogenes and Aeromonas sp. isolates were suspected to produce ceftazidimase-type ESBL and AAC(6′)-I enzyme. Synergy was clearly observed between expanded-spectrum cephalosporins and clavulanate. The resistance phenotype was transferred in Escherichia coli C600 by mating experiments using Mueller-Hinton agar (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) containing ceftazidime (4 μg/ml). The MICs of β-lactams showed high levels of resistance with the E. aerogenes strain and the two transconjugants for penicillins (64 to >2,048 μg/ml), ceftazidime (512 to 1,048 μg/ml), and aztreonam (64 to 1,048 μg/ml). A low level of resistance or decrease in susceptibility was observed for cefotaxime (4 to 8 μg/ml). The Aeromonas sp. strain showed lower resistance levels (penicillins, 8 to 256 μg/ml; ceftazidime, 64 μg/ml; and aztreonam, 8 μg/ml). Clavulanate and tazobactam partially or completely restored the susceptibility to penicillins. ESBL characterization was performed for the two isolates by isoelectric focusing, ESBL-encoding gene amplification and sequencing, and plasmid content analysis (6, 7). The ESBLs were focused at a pI of 6.5. The sequence of ESBL-encoding genes shared 100% identity with the blaTEM-24 gene. Plasmids of approximately 180 kb, which displayed similar restriction patterns, were isolated from the two strains and their transconjugants. These results suggested an in vivo transfer of TEM-24-encoding plasmid from E. aerogenes to Aeromonas sp. in the intestinal tract. We have previously reported the transfer of 180-kb TEM-24-encoding plasmid from E. aerogenes to Providencia rettgeri CIP 107053 (6) and P. aeruginosa CIP 107051 (7). A comparative study of the restriction profiles obtained for the plasmids recovered from these organisms and from the strains analyzed here revealed similar restriction patterns (Fig. ​(Fig.1).1). Pulsed-field gel electrophoresis (PFGE) analysis of the E. aerogenes strains isolated in this study and previously (6, 7) revealed that they belong to the TEM-24-producing E. aerogenes clone prevalent in France. FIG. 1. EcoRI-SalI restriction patterns of 180-kb TEM-24-encoding plasmids obtained from transconjugants corresponding to the following clinical isolates: lane 2, P. rettgeri CIP 107053 (6); lane 3, P. aeruginosa CIP 107051 (7); lane 4, Aeromonas sp. (this study); ... This clone had disseminated the same TEM-24-encoding plasmid among various bacterial species for several years. The isolation of a TEM-24-producing Aeromonas sp. strain extends the list of TEM-24-harboring bacteria, and this wide host range is one of the factors responsible for the persistence and spread of the TEM-24-encoding plasmid.

In 2000, at the Université d'Auvergne teaching hospital in Clermont-Ferrand, France, 44 (6.2%) strains of Pseudomonas aeruginosa were found to be resistant to ceftazidime. After genotyping, 34 strains were selected. Nine had an additional β-lactamase: OXA-21 ( n = 6), PSE-1 (CARB-2) ( n = 2), or PER-1 ( n = 1). Ceftazidime resistance was related solely to the overproduction of the cephalosporinase in 30 strains. Sequencing of five bla AmpC genes encoding cephalosporinases with different pIs showed 99% identity with the ampC gene of P. aeruginosa PAO1.

The sequences of the bla TEM genes encoding TEM-92 in Proteus mirabilis and Providencia stuartii isolates were determined and were found to be identical. Except for positions 218 (Lys-6) and 512 (Lys-104), the nucleotide sequence of bla TEM-92 was identical to that of bla TEM-20 , including the sequence of the promoter region harboring a 135-bp deletion combined with a G-162→T substitution. The deduced amino acid sequence of TEM-92 differed from that of TEM-52 by the presence of a substitution (Gln-6→Lys) in the peptide signal.

We biochemically characterized the Klebsiella pneumoniae extended-spectrum SHV-55 enzyme carrying the amino acid substitutions Tyr7→Phe (as in SHV-28) and Gly238→Ser and Glu240→Lys (both found in SHV-5) identified in a previous study (5). The SHV-55 extended-spectrum β-lactamase differed from SHV-5 only in the signal peptide region (1). The blaSHV-55 gene was obtained as described by Mendonca et al. (6), and transformants were selected on Luria broth agar supplemented with 30 μg of kanamycin/ml and 16 μg of amoxicillin/ml. SHV-55 was extracted and purified according to the previously described protocol (6). The Michaelis constant (Km) and catalytic activity (kcat) of purified extracts of SHV-55 were obtained by using a computerized microacidimetric method and a 702 SM Titrino pH-stat apparatus (Metrohm, Herisau, Switzerland) (3). The complete hydrolysis time courses were analyzed, and the kinetic progress curves were fitted by nonlinear least-squares regression. These kinetic parameters were determined and compared to those of the SHV-1 enzyme for 10 β-lactams (Table ​(Table11). TABLE 1. Kinetic constants of SHV-55 and SHV-1 β-lactamasesa SHV-55 has a high affinity (Km, 5 to 10 μM) for penicillins, similar to that of SHV-5 (1) and higher than that of SHV-1 (Km, 11 to 31 μM). SHV-55 presented higher affinity values (Km, 9 to 58 μM) than SHV-1 (Km, 40 to 257 μM) for narrow-, extended-, and broad-spectrum cephalosporins and monobactams. This finding may be a consequence of the Gly238→Ser substitution present in the active sites of both SHV-55 and SHV-5, which pushes the β-strand out and away from the reactive Ser70 (2). This effect results in a slightly expanded active site that may improve binding and accommodate cephalosporins with bulky side chains (4). SHV-55 presented a higher affinity for cefotaxime than for ceftazidime (Kms, 21 and 58 μM, respectively), as did SHV-5 (1, 7). This finding is surprising because both enzymes possess the Glu240→Lys substitution, which increases hydrolytic activity against ceftazidime (8) due to the change in the electrostatic charge of the exposed group at position 240 (2). The enzymatic activities (kcats) of SHV-55 for penicillin G and amoxicillin were 84- and 45-fold lower, respectively, than those of SHV-1, and the catalytic efficiency (kcat/Km ratio) against penicillins was more than 10-fold higher for SHV-1 (kcat/Km ratio, 20 to 84 μM−1·s−1) than for SHV-55 (kcat/Km ratio, 2 to 5 μM−1·s−1). However, the enzyme activity and catalytic efficiency against extended- and broad-spectrum cephalosporins were higher for SHV-55 (kcat, 7 to 24 s−1, and kcat/Km ratio, 0.2 to 1 μM−1·s−1) than for SHV-1 (note, however, that the values for monobactam were undeterminable), although the catalytic efficiencies of the two enzymes against cephalothin were similar (kcat/Km ratios, 3.2 and 4.4 μM−1·s−1). This result may be due to the amino acid substitutions in SHV-55 causing conformational modifications in the active site. Fifty percent inhibitory concentrations (IC50s) indicated that SHV-55 was ninefold more susceptible to the inhibitor activity of clavulanate than SHV-1 (IC50s of clavulanate, 0.02 versus 0.17 μM). In conclusion, these results confirmed the extended-spectrum activity of the SHV-55 enzyme, which is important due to the magnitude of extended- and broad-spectrum SHV β-lactamases described to date and not biochemically characterized, in spite of the ease of sequencing genes (http://www.lahey.org/studies).

We report a case of postsurgical wound infection of polymicrobial etiology caused by Serratia marcescens and Pseudomonas aeruginosa following the use of a radial forearm free flap for oncological tongue reconstruction. S. marcescens was a producer of SHV-12 extended-spectrum β-lactamase (ESBL). This is the first report from India of this ESBL. S. marcescens and P. aeruginosa were resistant to the empirical perioperative antibiotics administered. Delay in the recognition of the type of infection and in the institution of appropriate therapy resulted in total loss of the free flap.

At the beginning of the 1990s, a new class A extended-spectrum β-lactamase (ESBL), MEN-1 (CTX-M-1), was characterized in Escherichia coli strains isolated from Italian and German patients (1, 2). CTX-M-1 was the first member of the CTX-M β-lactamase family, which now comprises nine members: CTX-M-1 (MEN-1) (1, 2), CTX-M-2 (2), Toho-1 (7), CTX-M-3 (6), CTX-M-4 (6), CTX-M-5 (4), Toho-2 (8), CTX-M-6 (5), CTX-M-7 (5), and CTX-M-8 (3). These ESBLs conferred higher cefotaxime MICs than those of ceftazidime. E. coli (1, 7–10) and Salmonella typhimurium (2, 5, 6, 11) strains are the species most frequently reported to produce CTX-M enzymes. These enzymes are reported mainly in three geographic areas: South America (2, 3), East Europe (4–7, 10, 11), and Japan (8, 9). During a multicenter survey of ESBLs in France in 1998 (C. De Champs, D. Sirot, C. Chanal, J. Sirot, and the French Study Group, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 149.C2-1485, p. 169, 1999), an Enterobacter cloacae strain (Ver-1) was selected for its resistance to broad-spectrum cephalosporins and a positive double-disk synergy test. This strain was isolated from a 56-year-old man admitted to the Versailles Hospital (Le Chesnay, France) with unbalanced non-insulin-dependent diabetes and severe arteriopathy. His last hospitalization was in 1996 for noninfected urinary tract retention. He had osteitis and a foot ulcer infected by a Staphylococcus aureus strain. After 2 days of treatment with oxacillin, the E. cloacae strain, Ver-1, was isolated from a urine sample collected for dysuria. This colonization was not treated. Ver-1 was also resistant to tetracycline, co-trimoxazole, gentamicin, and tobramycin. The ESBL phenotype was transferred to E. coli HB101, resistant to rifampin, at 37°C during an overnight mating assay on solid Mueller-Hinton medium containing rifampin (300 μg/ml). The E. coli transconjugant, designated TrVer-1, did not exhibit cotransferred resistance markers. Table ​Table11 shows the MICs of β-lactams, determined by the agar dilution method, for the strain E. cloacae Ver-1 and its E. coli transconjugant TrVer-1. These two strains were resistant to penicillins. MICs of cefotaxime (128 to 32 μg/ml) were 16- to 64-fold higher than those of ceftazidime (8 to 0.5 μg/ml). The β-lactam inhibitors clavulanate and tazobactam restored partially or totally the susceptibilities to piperacillin and cephalosporins. TABLE 1 MICs of β-lactams for E. cloacae isolate Ver-1 and its E. coli transconjugant TrVer-1 in comparison with wild-type E. cloacae and TEM-1-producing E. colia Sonicates of the clinical strain and its E. coli transconjugant were subjected to analytical isoelectric focusing over the pH range of 3 to 10. Both E. cloacae and its E. coli transconjugant produced a β-lactamase of isoelectric point 8.4, associated with a β-lactamase of pI 5.4. PCR and direct DNA sequencing identified the β-lactamase of pI 5.4 as TEM-1 penicillinase. No PCR products were obtained with primers specific for blaSHV. In contrast, positive amplification was obtained with primers CTX-M-3A (5′-GGTTAAAAAATCGCG-3′) and CTX-M-3B (5′-TTACAAACCGTCGGTGA-3′), which amplified the complete sequence of open reading frame blaCTX-M-3. The obtained DNA sequence of the PCR products exhibited 100% identity to the sequence blaCTX-M-3 (8). While only CTX-M-1 and -2 were characterized in 1990 and 1992 (1, 2), seven new CTX-M enzymes were described in 1998 and 1999, showing that the CTX-M family of ESBLs was small but rapidly growing. CTX-M-3 was first characterized in 1998 for Citrobacter freundii and E. coli strains at Praski Hospital in Poland (8). This enzyme has spread in other species of the Enterobacteriaceae (Klebsiella pneumoniae, Klebsiella oxytoca, E. cloacae, and Morganella morganii) and was the more frequently observed ESBL (11). Here, we report the first characterization of a CTX-M-3-producing strain isolated from a French patient with no history of travel the year before. The spread of the CTX-M-3 enzyme from East European countries cannot be excluded. Tassios et al. (11) have shown the probable spread of a CTX-M-4-producing S. typhimurium clone in Russia, Greece, and Hungary. However, the emergence of CTX-M enzymes from widespread environmental bacteria could also explain their spread. During the study of ESBLs (De Champs et al., 39th ICAAC) which led to characterization of these CTX-M-3-producing E. cloacae strains, 79 ESBL-producing strains were isolated. Only one CTX-M-producing strain was observed. Thus, this CTX-M-3-producing strain seems to be a sporadic isolate. However, in view of the spread of CTX-M-producing strains in East European countries, emergence of CTX-M-producing strains could be observed in France. The characterization of this CTX-M-3-producing strain highlights the feasibility of this process and constitutes a forewarning of the probable existence of CTX-M-producing strains in France.