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•Fresh surface waters play a critical role in human health and prosperity.•Water contamination is a serious problem, especially for disenfranchised populations.•Fecal contamination of irrigation waters can lead to the dissemination of drug-resistant bacteria.•Identification of E. coliwith blaNDM-5and YRIN-inserted PBP3, exacerbating AMR challenges.•Detection of K. pneumoniaecarrying blaSHV-187and mutations in the ompK37porin gene.

ABSTRACTIntroductionMetallo-β-lactamases (MBLs) are enzymes produced by bacteria that confer resistance to most β-lactam antibiotics, including carbapenems, which have the broadest spectrum of activity. This resistance mechanism poses a significant threat to public health as it drastically reduces treatment options for severe bacterial infections. Developing effective inhibitors against MBLs is crucial to restore susceptibility to β-lactam antibiotics.Areas coveredThis review aims to provide an updated analysis of patents describing novel MBL inhibitors and their potential therapeutic applications that were filed between January 2020 and May 2023.Expert opinionSignificant advancements were made in the development of selective MBL inhibitors with zinc-binding and zinc-chelating mechanisms of action. Dual inhibitors, targeting simultaneously both serine-β-lactamases (SBLs) and MBLs, represent an interesting alternative approach that is increasingly pertinent for the treatment of infections involving multiple β-lactamases from different Ambler classes. Most examples of MBL-specific inhibitors were focused on the treatment of MBL-mediated infections in Enterobacterales, where IMP-1 was a more difficult target compared with VIM-1 or NDM-1, and much less on Pseudomonas aeruginosaor Acinetobacter baumannii, which are more challenging to address.

•Activity of apramycin against a collection (n = 164) of 16S-RMTase producers.•Most of the 16S-RMTase-producing isolates (95%) were susceptible to apramycin.•Resistance rates were higher in P. aeruginosathan in A. baumanniior Enterobacterales.•One ST156 E. colihad a minimum inhibitory concentration (MIC) >64 mg/L due to acquisition of the aac(3)-IV gene.

•High rates of neonatal sepsis due to CPEs are associated with a high mortality rate.•NDM- and OXA-48–producing K. pneumoniaeand Enterobactersp.common in neonatal ICU.•Clonal spread of CPEs over the study period suggests endemic situation.•Isolates were susceptible to only two available antibiotics: colistin and amikacin.•Additional susceptibility to tigecyclin, eravacyclin, cefiderocol, and apramycin.

Carbapenem-resistant Enterobacterales (CRE) remain a significant public health threat, and, despite recent approvals, new antibiotics are needed. Severe infections caused by CRE, such as nosocomial pneumonia and bloodstream infections, are associated with a relatively high risk of morbidity and mortality. The recent approval of ceftazidime–avibactam, imipenem–relebactam, meropenem–vaborbactam, plazomicin, eravacycline and cefiderocol has broadened the armamentarium for the treatment of patients with CRE infections. Cefiderocol is a siderophore cephalosporin with overall potent in vitro activity against CRE. It is taken up via iron transport channels through active transport, with some entry into bacteria through traditional porin channels. Cefiderocol is relatively stable against hydrolysis by most serine- and metallo-beta-lactamases, including KPC, NDM, VIM, IMP and OXA carbapenemases—the most frequent carbapenemases detected in CRE. The efficacy and safety of cefiderocol has been demonstrated in three randomised, prospective, parallel group or controlled clinical studies in patients at risk of being infected by multidrug-resistant or carbapenem-resistant Gram-negative bacteria. This paper reviews the in vitro activity, emergence of resistance, preclinical effectiveness, and clinical experience for cefiderocol, and its role in the management of patients with CRE infections.

We isolated a highly colistin-resistant Escherichia coli, strain 58, from fresh chicken wings in Lebanon. Here, we performed in-depth phenotypic and genomic analyses to identify the resistome of the isolate, focusing on the determinants that encoded colistin resistance.

Alternative methods to RT-PCR for SARS-CoV-2 detection are investigated to provide complementary data on viral proteins, increase the number of tests performed, or identify false positive/negative results. Here, we have developed a simple mass spectrometry assay for SARS-CoV-2 in nasopharyngeal swab samples using common laboratory reagents. The method employs high sensitivity and selectivity targeted mass spectrometry detection, monitoring nine constitutive peptides representative of the three main viral proteins and a straightforward pellet digestion protocol for convenient routine applications. Absolute quantification of N, M, and S proteins was achieved by addition of isotope-labeled versions of best peptides. Limit of detection, recovery, precision, and linearity were thoroughly evaluated in four representative viral transport media (VTM) containing distinct total protein content. The protocol was sensitive in all swab media with limit of detection determined at 2 × 103pfu/mL, corresponding to as low as 30 pfu injected into the LC-MS/MS system. When tested on VTM-stored nasopharyngeal swab samples from positive and control patients, sensitivity was similar to or better than rapid immunoassay dipsticks, revealing a corresponding RT-PCR detection threshold at Ct ∼ 24. The study represents the first thorough evaluation of sensitivity and robustness of targeted mass spectrometry in nasal swabs, constituting a promising SARS-CoV-2 antigen assay for the first-line diagnosis of COVID-19 and compatible with the constraints of clinical settings. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01646.

The MAST Carba PAcE test is a colorimetric test used to detect carbapenemase-producing Gram-negative bacilli from cultured colonies. The performances of this test were compared with those of β-CARBA, Carba NP, and RAPIDEC Carba NP tests using a collection of 280 characterized isolates.

A novel real-time multiplex PCR assay, BD MAX Check-Points CPO, was evaluated to detect carbapenemase-producing organisms in clinical settings on the BD MAX system. A total of 175 well-characterized isolates (including 123 carbapenemase producers) and 128 rectal swab specimens (including 83 positives) of patients considered at high risk for carriage of carbapenemase producers were included. Bacterial suspensions were used to spike true-negative rectal swabs to mimic a clinical sample. Sample (50 μL), containing either the spiked or the patient's sample, was processed. The BD MAX Check-Points CPO assay detected carbapenemases KPC, VIM/IMP, NDM, and OXA-48–like producers with a high sensitivity and specificity of 97.1% and 98.8%, respectively. Rare variants of the IMP type (IMP-11, IMP-13, and IMP-14) and one rare and distantly related OXA-48 variant (OXA-535) remained undetected. With patients' rectal swabs, sensitivity and specificity were 92.8% and 97.8%, respectively. Failure of detection was due to weak inoculum. The time to result was short: approximately. 2.5 hours for 12 samples (including extraction and PCR). The automated sample-in results-out platform is an efficient, quick, and easy-to-use tool for the detection of the main five carbapenemases. The lack of distinction between producers of VIM and IMP may be limiting in countries where these enzymes are widespread, as in Asia, but not in France, where IMP producers are extremely rare.

ABSTRACTThe global spread of carbapenemase-producing Enterobacteriaceae(CPE) that are often resistant to most, if not all, classes of antibiotics is a major public health concern. The NDM-1 carbapenemase is among the most worrisome carbapenemases given its rapid worldwide spread. We have developed and evaluated a lateral flow immunoassay (LFIA) (called the NDM LFIA) for the rapid and reliable detection of NDM-like carbapenemase-producing Enterobacteriaceaefrom culture colonies. We evaluated the NDM LFIA using 175 reference enterobacterial isolates with characterized ß-lactamase gene content and 74 nonduplicate consecutive carbapenem-resistant clinical isolates referred for expertise to the French National Reference Center (NRC) for Antibiotic Resistance during a 1-week period (in June 2016). The reference collection included 55 non-carbapenemase producers and 120 carbapenemase producers, including 27 NDM producers. All 27 NDM-like carbapenemase producers of the reference collection were correctly detected in less than 15 min by the NDM LFIA, including 22 strains producing NDM-1, 2 producing NDM-4, 1 producing NDM-5, 1 producing NDM-7, and 1 producing NDM-9. All non-NDM-1 producers gave a negative result with the NDM LFIA. No cross-reaction was observed with carbapenemases (VIM, IMP, NDM, KPC, and OXA-48-like), extended-spectrum ß-lactamases (ESBLs) (TEM, SHV, and CTX-M), AmpCs (CMY-2, DHA-2, and ACC-1), and oxacillinases (OXA-1, -2, -9, and -10). Similarly, among the 74 referred nonduplicate consecutive clinical isolates, all 7 NDM-like producers were identified. Overall, the sensitivity and specificity of the assay were 100% for NDM-like carbapenemase detection with strains cultured on agar. The NDM LFIA was efficient, rapid, and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of NDM-like carbapenemase-producing Enterobacteriaceae.

In depth phenotypic and genomic analyses of a carbapenem-resistant Escherichia coliisolate, recovered from the feces of a farm dog in Lebanon, focusing on its antimicrobial resistance (AMR) patterns and the underlying resistome.

ABSTRACTHere, we characterized the first OXA-72-producing Acinetobacter baumanniiisolate (designated MAL) recovered from a urine sample from a Serbian patient. Antimicrobial susceptibility testing, plasmid analysis, and whole-genome sequencing (WGS) were performed to fully characterize the resistome of the A. baumanniiMAL clinical isolate. The isolate was multidrug resistant and remained susceptible only to colistin and tigecycline. PCR analysis revealed the presence of the carbapenemase OXA-72, an OXA-40 variant. Extraction by the Kieser method revealed the presence of two plasmids, and one of these, a ca. 10-kb plasmid, harbored the blaOXA-72gene. WGS revealed 206 contigs corresponding to a genome of 3.9 Mbp in size with a G+C content of 38.8%. The isolate belonged to sequence type 492 and to worldwide clone II (WWCII). Naturally occurring β-lactamase-encoding genes (blaADC-25and blaOXA-66) were also identified. Aminoglycoside resistance genes encoding one aminoglycoside adenyltransferase (aadA2), three aminoglycoside phosphatases (strA, strB, aphA6), and one 16S RNA methylase (armA) conferring resistance to all aminoglycosides were identified. Resistance to fluoroquinolones was likely due to mutations in gyrA, parC, and parE. Of note, the resistome matched perfectly with the antibiotic susceptibility testing results.

The fight against infectious diseases is probably one of the greatest public health challenges faced by our society, especially with the emergence of carbapenem-resistant gram-negatives that are in some cases pan-drug resistant. Currently, β-lactamase-mediated resistance does not spare even the newest and most powerful β-lactams (carbapenems), whose activity is challenged by carbapenemases. The worldwide dissemination of carbapenemases in gram-negative organisms threatens to take medicine back into the pre-antibiotic era since the mortality associated with infections caused by these “superbugs” is very high, due to limited treatment options. Clinically-relevant carbapenemases belong either to metallo-β- lactamases (MBLs) of Ambler class B or to serine-β-lactamases (SBLs) of Ambler class A and D enzymes. Class A carbapenemases may be chromosomally-encoded (SME, NmcA, SFC-1, BIC-1, PenA, FPH-1, SHV-38), plasmid-encoded (KPC, GES, FRI-1) or both (IMI). The plasmid-encoded enzymes are often associated with mobile elements responsible for their mobilization. These enzymes, even though weakly related in terms of sequence identities, share structural features and a common mechanism of action. They variably hydrolyse penicillins, cephalosporins, monobactams, carbapenems, and are inhibited by clavulanate and tazobactam. Three-dimensional structures of class A carbapenemases, in the apo form or in complex with substrates/inhibitors, together with site-directed mutagenesis studies, provide essential input for identifying the structural factors and subtle conformational changes that influence the hydrolytic profile and inhibition of these enzymes. Overall, these data represent the building blocks for understanding the structure-function relationships that define the phenotypes of class A carbapenemases and can guide the design of new molecules of therapeutic interest.

ABSTRACTFive GES-producing Enterobacteriaceaeisolates that displayed an extended-spectrum β-lactamase (ESBL) phenotype harbored two GES variants: GES-7 ESBL and GES-6 carbapenemase. In all isolates, the two GES alleles were located on the same integron that was inserted into an 80-kb IncM1 self-conjugative plasmid. Whole-genome sequencing suggested in vivohorizontal gene transfer of the plasmid along with clonal diffusion of Enterobacter cloacae. To our knowledge, this is the first description in Europe of clustered Enterobacteriaceaeisolates carrying two GES β-lactamases, of which one has extended activity toward carbapenems.

ABSTRACTThe Xpert Carba-R kit, version 2 (v2), which has been improved for the efficient detection of blaOXA-181and blaOXA-232genes, was tested on a collection of 150 well-characterized enterobacterial isolates that had a reduced susceptibility to carbapenems. The performance of the Xpert Carba-R v2 was high, as it was able to detect the five major carbapenemases (NDM, VIM, IMP, KPC, and OXA-48). Thus, it is now well adapted to the carbapenemase-producing Enterobacteriaceaeepidemiology of many countries worldwide.

ABSTRACTFifteen carbapenemase-producing Enterobacteriaceaeisolates and 12 carbapenemase-producing Pseudomonas aeruginosaisolates were recovered from patients hospitalized between August 2011 and March 2013 at the Hospital of Infectious Disease, Cluj-Napoca, Romania. One KPC-, nine NDM-1-, four OXA-48-, and one VIM-4-producing Enterobacteriaceaeisolates along with 11 VIM-2-producing and one IMP-13-producing P. aeruginosaisolates were recovered from clinical samples. All carbapenemase genes were located on self-conjugative plasmids and were associated with other resistance determinants, including extended-spectrum β-lactamases and RmtC methylases.

ABSTRACTThe epidemiology of carbapenemases worldwide is showing that OXA-48 variants are becoming the predominant carbapenemase type in Enterobacteriaceaein many countries. However, not all OXA-48 variants possess significant activity toward carbapenems (e.g., OXA-163). Two Serratia marcescensisolates with resistance either to carbapenems or to extended-spectrum cephalosporins were successively recovered from the same patient. A genomic comparison using pulsed-field gel electrophoresis and automated Rep-PCR typing identified a 97.8% similarity between the two isolates. Both strains were resistant to penicillins and first-generation cephalosporins. The first isolate was susceptible to expanded-spectrum cephalosporins, was resistant to carbapenems, and had a significant carbapenemase activity (positive Carba NP test) related to the expression of OXA-48. The second isolate was resistant to expanded-spectrum cephalosporins, was susceptible to carbapenems, and did not express a significant imipenemase activity, (negative for the Carba NP test) despite possessing a blaOXA-48-type gene. Sequencing identified a novel OXA-48-type β-lactamase, OXA-405, with a four-amino-acid deletion compared to OXA-48. The blaOXA-405gene was located on a ca. 46-kb plasmid identical to the prototype IncL/M blaOXA-48-carrying plasmid except for a ca. 16.4-kb deletion in the traoperon, leading to the suppression of self-conjugation properties. Biochemical analysis showed that OXA-405 has clavulanic acid-inhibited activity toward expanded-spectrum activity without significant imipenemase activity. This is the first identification of a successive switch of catalytic activity in OXA-48-like β-lactamases, suggesting their plasticity. Therefore, this report suggests that the first-line screening of carbapenemase producers in Enterobacteriaceaemay be based on the biochemical detection of carbapenemase activity in clinical settings.

ABSTRACTThe recognition of carbapenemase-producing Enterobacteriaceae(CPE) isolates is a major laboratory challenge, and their inappropriate or delayed detection may have negative impacts on patient management and on the implementation of infection control measures. We describe here a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-based method to detect carbapenemase activity in Enterobacteriaceae. After a 20-min incubation of the isolate with 0.5 mg/ml imipenem at 37°C, supernatants were analyzed by MALDI-TOF in order to identify peaks corresponding to imipenem (300 Da) and an imipenem metabolite (254 Da). A total of 223 strains, 77 CPE (OXA-48 variants, KPC, NDM, VIM, IMI, IMP, and NMC-A) and 146 non-CPE (cephalosporinases, extended-spectrum ß-lactamases [ESBLs], and porin defects), were tested and used to calculate a ratio of imipenem hydrolysis: mass spectrometry [MS] ratio = metabolite/(imipenem + metabolite). An MS ratio cutoff was statistically determined to classify strains as carbapenemase producers (MS ratio of =0.82). We validated this method first by testing 30 of our 223 isolates (15 CPE and 15 non-CPE) 10 times to calculate an intraclass correlation coefficient (ICC of 0.98), showing the excellent repeatability of the method. Second, 43 strains (25 CPE and 18 non-CPE) different from the 223 strains used to calculate the ratio cutoff were used as external controls and blind tested. They yielded sensitivity and specificity of 100%. The total cost per test is <0.10 U.S. dollars (USD). This easy-to-perform assay is time-saving, cost-efficient, and highly reliable and might be used in any routine laboratory, given the availability of mass spectrometry, to detect CPE.

ABSTRACTThe carbapenemase-encoding blaKPCgene, which is rapidly spreading in Gram-negative rods, is located on a Tn3-based transposon, Tn4401, which carries a polymorphic region giving rise to five isoforms (a, b, c, d, and e) that is located immediately upstream of the blaKPCgene and thus likely involved in its expression. Using 5′ rapid amplification of cDNA ends (5′RACE), we identified three potential promoter sequences (P1, P2, and P3) upstream of the blaKPCgene, of which only P1 (absent from isoforms cand d) and P2 (present in all isoforms, with a −35 box located inside the right inverted repeat of ISKpn7) were shown to be true promoters involved in expression. One representative of each different promoter combination of Tn4401, i.e., P2 alone (isoform c), P1-P2 (isoform a), and P1-P2-P3 (isoform b), was cloned into an Escherichia coliplasmid vector. Using reverse transcription-PCR (RT-PCR), the highest level of expression was obtained with isoform a(P1 and P2), which is also the most commonly encountered form in enterobacterial clinical isolates, followed by isoforms b(P1, P2, and P3) and c(P2 only). These differences in expression led to slight differences in MIC values of carbapenems. In silicoanalysis of the DNA sequence of isoform brevealed a stem-loop structure that is likely responsible for strong stops observed in 5′RACE experiments and for decreased expression compared to that with isoform a(P1 and P2). In addition, such structures could also be at the origin for the deletions observed in isoforms aand c. Taken together, these results indicate that the P1 and P2 promoters both contribute to the expression of the blaKPCgene and that the construct with the highest level of expression is that possessing isoform a, which is also the most commonly encountered form in clinical isolates.

ABSTRACTTen blaKPC-2-harboring Pseudomonas aeruginosaisolates from hospitals located in five different Colombian cities have been characterized. Isolates were multidrug resistant, belonged to five different pulsotypes, and possessed naturally chromosome-encoded blaAmpCand blaOXA-50genes and the acquired blaKPC-2gene. In most cases, the blaKPC-2genes were carried by plasmids of different sizes and were associated with Tn4401bor a new structure containing only part of the Tn4401sequence. This study revealed that several clones of P. aeruginosaproducing blaKPC-2are disseminating in Colombia.

ABSTRACTThe carbapenemase gene blaKPC, which is rapidly spreading worldwide, is located on a Tn3-based transposon, Tn4401. In a transposition-conjugation assay, Tn4401was able to mobilize blaKPC-2gene at a frequency of 4.4 × 10−6/recipient cell. A 5-bp target site duplication was evidenced upon each insertion without target site specificity. This study demonstrated that Tn4401is an active transposon capable of mobilizing blaKPCgenes at high frequency.

ABSTRACTAn in-house quantitative real-time PCR (qPCR) assay using TaqMan chemistry has been developed to detect NDM-1 carbapenemase genes from bacterial isolates and directly from stool samples. The qPCR amplification of blaNDM-1DNA was linear over 10 log dilutions (r2= 0.99), and the amplification efficiency was 1.03. The qPCR detection limit was reproducibly 1 CFU, or 10 plasmid molecules, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria harboring other β-lactam resistance genes. Feces spiked with decreasing amounts of enterobacterial isolates producing NDM-1 were spread on ChromID ESBL and on CHROMagar KPC media and were subjected to the qPCR. The limits of carbapenem-resistant bacterial detection from stools was reproducibly 1 × 101to 3 × 101CFU/100 mg feces with ChromID ESBL medium. The CHROMagar KPC culture medium had higher limits of detection (1 × 101to 4 × 103CFU/ml), especially with bacterial isolates having low carbapenem MICs. The limits of detection with the qPCR assay were reproducibly below 1 × 101CFU/100 mg of feces by qPCR assay. Samples spiked with NDM-1-negative bacteria were negative by qPCR. The sensitivity and specificity of the blaNDM-1qPCR assay on spiked samples were 100% in both cases. Using an automated DNA extraction system (QIAcube system), the qPCR assay was reproducible. The use of qPCR is likely to shorten the time for blaNDM-1detection from 48 h to 4 h and will be a valuable tool for outbreak follow-up in order to rapidly isolate colonized patients and assign them to cohorts.

ABSTRACTA new commercial low-density microarray which identifies common extended-spectrum β-lactamase plasmid-mediated cephalosporinase genes, as well as carbapenemase (blaKPCand blaNDM) genes, was evaluated. We tested 207 clinical and reference/collection isolates of the Enterobacteriaceaepossessing different blagenes. Overall, the sensitivity and specificity of the microarray were 100% for the detection of the plasmid-mediated blaAmpC, blaKPC, and blaNDMgenes using blagene sequencing as the reference method.

ABSTRACTThe Check-MDR CT102 microarray, aimed at identifying bacteria producing extended-spectrum ß-lactamase (ESBL) (SHV, TEM, and CTX-M) and carbapenemase (KPC, OXA-48, VIM, IMP, and NDM-1), was evaluated on a total of 144 Gram-negative strains expressing various ß-lactamases. The sensitivity and specificity were 100% for most tested genes, suggesting that this assay allows accurate identification of common ESBL and carbapenemase producers from bacterial cultures.

ABSTRACTExtended-spectrum β-lactamases (ESBLs) of the CTX-M type are increasingly being reported worldwide, with more than 90 known variants. Clinical Escherichia coliisolate Bre-1 was isolated in 2009 and displayed an unusual ESBL phenotype, made of a synergy image between expanded cephalosporins and clavulanic acid discs and susceptibility to penicillins. E. coliBre-1 harbored a novel CTX-M-encoding gene, designated blaCTX-M-93. CTX-M-93 differed from CTX-M-27 by only a single L169Q substitution. Compared to CTX-M-27, CTX-M-93 conferred higher MICs of ceftazidime for E. coli(MIC of 8 versus 1.5 μg/ml) and decreased MICs of other expanded-cephalosporins (MIC of cefotaxime of 1 versus 32 μg/ml) and penicillins (MIC of ticarcillin of 0.5 versus >256 μg/ml). A comparison of enzymatic properties revealed that the L169Q substitution led to a decreased Kmfor ceftazidime (25.5 versus 330 μM) but decreased hydrolytic activity against good substrates, such as cefotaxime (kcatof 0.6 versus 113 s−1), probably owing to the alteration of the omega loop positioning during the catalytic process. The blaCTX-M-93gene was surrounded by the ISEcp1and IS903elements and inserted onto a 150-kb non-self-transferrable IncF-type plasmid. E. coliBre-1 belongs to phylogroup D and is of multilocus sequence type (MLST) 624, a sequence type found only in rare Spanish CTX-M-14-producing E. coliisolates. We have characterized a novel CTX-M variant, CTX-M-93, lacking significant penicillin hydrolysis but with increased ceftazidime hydrolysis.

ABSTRACTSeventeen Klebsiella pneumoniaeisolates producing the OXA-48 carbapenemase, obtained from 10 patients hospitalized from April to June 2010, mostly in the medical intensive care unit of the Villeneuve-Saint-Georges Hospital in a suburb of Paris, France, were analyzed. Seven patients were infected, of whom five were treated at least with a carbapenem, and five patients died. Molecular analysis showed that the isolates belonged to a single clone that harbored a 70-kb plasmid carrying the blaOXA-48gene and coproduced CTX-M-15 and TEM-1 β-lactamases. This is the first reported outbreak of OXA-48-producing K. pneumoniaeisolates in France.

ABSTRACTDuring a PCR-based surveillance study of β-lactam resistance, 125 multidrug-resistant (MDR) Acinetobacter baumanniiisolates were obtained from 18 hospitals in Belgium from January 2008 to December 2009. Nine GES-positive A. baumanniiisolates were detected at 6 Belgian hospitals. DNA sequencing of the blaGESgenes identified GES-11, GES-12, and a novel variant GES-14, which differs from GES-11 by a single amino acid substitution (Gly170Ser). All index isolates were travel associated and originated from patients transferred from Turkey (n= 2), Egypt (n= 2), and Palestinian territories (Gaza) (n= 2). A nosocomial outbreak involving three additional patients occurred in a burn unit at a single hospital. No clonal relatedness could be established between the 6 index isolates by pulsed-field gel electrophoresis (PFGE) analysis. Three different alleles (the plasmid-located blaGES-11and blaGES-12and a likely chromosomally located novel variant blaGES-14) were detected as part of a class 1 integron, also including the aac6′Iband dfrA7genes. Restriction analysis of plasmids suggests a common origin for the plasmids bearing blaGES-11and blaGES-12. Cloning of the blaGESgenes in Escherichiacoliidentified GES-14 as hydrolyzing imipenem, while GES-12 showed the highest specific activity against ceftazidime. This report highlights the emergence of various blaGES-likegenes, especially those conferring carbapenem resistance in A. baumanniiand its importation in Western Europe from Middle Eastern countries.

ABSTRACTClass D β-lactamase-mediated resistance to β-lactams has been increasingly reported during the last decade. Those enzymes also known as oxacillinases or OXAs are widely distributed among Gram negatives. Genes encoding class D β-lactamases are known to be intrinsic in many Gram-negative rods, including Acinetobacter baumanniiand Pseudomonas aeruginosa, but play a minor role in natural resistance phenotypes. The OXAs (ca. 150 variants reported so far) are characterized by an important genetic diversity and a great heterogeneity in terms of β-lactam hydrolysis spectrum. The acquired OXAs possess either a narrow spectrum or an expanded spectrum of hydrolysis, including carbapenems in several instances. Acquired class D β-lactamase genes are mostly associated to class 1 integron or to insertion sequences.

ABSTRACTThe genetic environment of the blaOXA-18gene encoding a peculiar clavulanic acid-inhibitable Ambler class D extended-spectrum β-lactamase was determined from the prototype OXA-18-producing Pseudomonas aeruginosaMUS clinical isolate. An 8.2-kb genomic DNA fragment containing blaOXA-18was cloned from P. aeruginosaMUS. Although most oxacillinases are located in integrons, blaOXA-18lacked gene cassette-specific features. It was bracketed by two duplicated sequences containing ISCR19, a novel insertion sequence of the ISCRfamily of mobile elements; ΔintI1, a truncated integrase gene; and a truncated Δaac6′-Ibgene cassette. It is likely that ISCR19was at the origin of the blaOXA-18gene mobilization by a rolling-circle transposition event followed by homologous recombination. Furthermore, analysis of the cloned genomic DNA fragment revealed the presence of the integron-containing blaOXA-20gene. Concomitantly, three P. aeruginosaclinical isolates, displaying a synergy image as determined by double-disk diffusion tests on cloxacillin-containing plates, were isolated from three patients hospitalized in different wards over a 9-month period at the Saint-Luc University hospital (Brussels, Belgium). These isolates were positive by PCR for blaOXA-18and blaOXA-20genes, genetically related to P. aeruginosaMUS as determined by pulsed-field gel electrophoresis, and carried the same blaOXA-18/blaOXA-20-associated genetic structures. This report characterized the genetic elements likely at the origin of blaOXA-18gene mobilization in P. aeruginosaand suggests the spread of oxacillin-type extended-spectrum β-lactamases in P. aeruginosaat the Saint-Luc University hospital of Brussels, Belgium.

ABSTRACTGenetic structures surrounding the carbapenem-hydrolyzing Ambler class A blaKPCgene were characterized in several KPC-positive Klebsiella pneumoniaeand Pseudomonas aeruginosastrains isolated from the United States, Colombia, and Greece. The blaKPCgenes were associated in all cases with transposon-related structures. In the K. pneumoniaeYC isolate from the United States, the β-lactamase blaKPC-2gene was located on a novel Tn3-based transposon, Tn4401. Tn4401was 10 kb in size, was delimited by two 39-bp imperfect inverted repeat sequences, and harbored, in addition to the β-lactamase blaKPC-2gene, a transposase gene, a resolvase gene, and two novel insertion sequences, ISKpn6and ISKpn7. Tn4401has been identified in all isolates. However, two isoforms of this transposon were found: Tn4401a was found in K. pneumoniaeYC and K. pneumoniaeGR from the United States and Greece, respectively, and differed by a 100-bp deletion, located just upstream of the blaKPC-2gene, compared to the sequence of Tn4401b, which was found in the Colombian isolates. In all isolates tested, Tn4401was flanked by a 5-bp target site duplication, the signature of a recent transposition event, and was inserted in different open reading frames located on plasmids that varied in size and nature. Tn4401is likely at the origin of carbapenem-hydrolyzing β-lactamase KPC mobilization to plasmids and its further insertion into various-sized plasmids identified in nonclonally related K. pneumoniaeand P. aeruginosaisolates.

ABSTRACTA chromosomal β-lactamase gene from Bacillus clausiiNR, which is used as a probiotic, was cloned and expressed in Escherichia coli. It encodes a clavulanic acid-susceptible Ambler class A β-lactamase, BCL-1, with a pI of 5.5 and a molecular mass of ca. 32 kDa. It shares 91% and 62% amino acid identity with the chromosomally encoded PenP penicillinases from B. clausiiKSM-K16 and Bacillus licheniformis, respectively. The hydrolytic profile of this β-lactamase includes penicillins, narrow-spectrum cephalosporins, and cefpirome. This chromosome-encoded enzyme was inducible in B. clausii, and its gene is likely related to upstream-located regulatory genes that share significant identity with those reported to be upstream of the penicillinase gene of B. licheniformis. The blaBCL-1gene was located next to the known chromosomal aadD2gene and the erm34gene, which encode resistance to aminoglycosides and macrolides, respectively. Similar genes were found in a collection of B. clausiireference strains.

ABSTRACTA novel β-lactamase gene was cloned from the whole-cell DNA of an enterobacterial Citrobacter gilleniireference strain that displayed a weak narrow-spectrum β-lactam-resistant phenotype and was expressed in Escherichia coli. It encoded a clavulanic acid-inhibited Ambler class A β-lactamase, GIL-1, with a pI value of 7.5 and a molecular mass of ca. 29 kDa. GIL-1 had the highest percent amino acid sequence identity with TEM-1 and SHV-1, 77%, and 67%, respectively, and only 46%, 31%, and 32% amino acid sequence identity with CKO-1 (C. koseri), CdiA1 (C. diversus), and SED-1 (C. sedlaki), respectively. The substrate profile of the purified GIL-1 was similar to that of β-lactamases TEM-1 and SHV-1. The blaGIL-1gene was chromosomally located, as revealed by I-CeuI experiments, and was constitutively expressed at a low level in C. gillenii. No gene homologous to the regulatory ampRgenes of chromosomal class C β-lactamases was found upstream of the blaGIL-1gene, which fits the noninducibility of β-lactamase expression in C. gillenii. Rapid amplification of DNA 5′ ends analysis of the promoter region revealed putative promoter sequences that diverge from what has been identified as the consensus sequence in E. coli. The blaGIL-1gene was part of a 5.5-kb DNA fragment bracketed by a 9-bp duplication and inserted between the d-lactate dehydrogenase gene and the ydbHgenes; this DNA fragment was absent in other Citrobacterspecies. This work further illustrates the heterogeneity of β-lactamases in Citrobacterspp., which may indicate that the variability of Citrobacterspecies is greater than expected.

ABSTRACTThe genetic structures surrounding the plasmid-carried blaOXA-23oxacillinase gene, encoding resistance to carbapenems, were studied in Acinetobacter baumannii. ISAba1and the novel element ISAba4were detected upstream of the blaOXA-23gene, providing promoter sequences for its expression. These insertion elements were likely involved in transposition processes at the origin of acquisition of this β-lactamase gene.

ABSTRACTCTX-M extended-spectrum β-lactamases (ESBLs) are increasingly prevalent worldwide among Escherichia colibacteria, mostly in community-acquired urinary tract infections. Finding a fast and reliable technique for identification of CTX-M enzymes is becoming a challenge for the microbiology laboratory. A fast real-time PCR amplification technique, using degenerated primers specific for all the blaCTX-Malleles, coupled to real-time pyrosequencing was developed. The five CTX-M groups were unambiguously identified by pyrosequencing a 13-bp DNA region. Further sequencing of an additional 16-bp region allowed further division into subgroups. Phylogenetic trees constructed with the entire blaCTX-Mgenes and with both pyrosequenced regions (29 bp) gave similar results, suggesting that this technique, termed the real-time detection and sequencing method, has a powerful discriminatory ability. This high-throughput technique has been evaluated by screening 48 ESBL-producing E. coliisolates recovered from the Bicêtre hospital (France) in 2004. Forty-four of these strains were CTX-M positive by real-time PCR detection and direct pyrosequencing of the PCR products, which identified CTX-M-15 as the main CTX-M-type β-lactamase. Pulsed-field gel electrophoresis analysis of these strains revealed that several clones, of which one CTX-M-15-positive clone was predominant (60%), were identified both in nosocomial and in community-acquired isolates. The combination of real-time PCR with pyrosequencing represents a powerful tool for epidemiological studies of CTX-M producers. This assay has the potential to be used in a diagnostic laboratory since up to 96 bacterial isolates may be screened in less than 3 h.

ABSTRACTCarbapenem-resistant Acinetobacter baumanniiisolates were obtained from 17 patients between September 2004 and August 2005 at the Academisch Ziekenhuis Vrije Universiteit Brussel, Brussels, Belgium. These multidrug-resistant isolates, which belonged to a single clone, remained susceptible to colistin and tigecycline only and produced the carbapenem-hydrolyzing oxacillinase OXA-58. This study highlights the importance of the intercountry spread of this ß-lactamase-mediated resistance mechanism and its epidemic evolution.

ABSTRACTA pyrosequencing technique was used for identification of extended-spectrum ß-lactamases (ESBLs) of GES type. These ß-lactamases are isolated increasingly emerging in gram-negative bacteria worldwide. This rapid and reliable identification method is interesting, since GES variants, including not only expanded-spectrum cephalosporins but also carbapenems, cephamycins, and monobactams, are the only ESBLs that possess different hydrolysis profiles.

Objectives: Strains of Acinetobacter baumannii producing the extended-spectrum β-lactamase (ESBL) PER-1 are widespread in Turkey and have also been reported from Korea and France. In contrast, A. baumannii producing the ESBL VEB-1 have only been reported from France, where one strain was responsible for a nationwide outbreak in 2003–2004. Here we describe the emergence of strains of A. baumannii producing VEB-1 and PER- 1 in Belgium.Methods: Belgian hospitals were alerted in December 2003 to the emergence in France of VEB-1-producing A. baumannii susceptible only to meropenem and colistin. Isolates with a compatible susceptibility profile were sent to a single central laboratory for VEB-1 confirmation, molecular characterization and typing.Results: From December 2003 to March 2005, three hospitals located close to the French border and one in the Brussels area reported isolation of eight A. baumannii isolates compatible with the French epidemic clone. Using PCR, six were identified as VEB-1-positive and two as PER-1-positive. All the VEB-1-positive isolates were clonally related by PFGE and by integron analysis to the French epidemic strain. The PER-1-positive strains were indistinguishable by PFGE but not related to the known French isolate or to several Turkish isolates. Both genes were chromosomally encoded.Conclusions: This work illustrates the inter-country spread of VEB-1-producing A. baumannii isolates as well as the emergence of PER-1-producing A. baumannii strains in Belgium.

ABSTRACTTwo clinical isolates of Pseudomonas aeruginosa, TL-1 and TL-2, were isolated from a patient transferred from Bangladesh and hospitalized for osteomyelitis in Paris, France. P. aeruginosaTL-1 expressed the extended-spectrum β-lactamase VEB-1a and was susceptible only to imipenem and colistin, while P. aeruginosaTL-2 expressed only the naturally occurring blaAmpCgene at a basal level and exhibited a wild-type β-lactam resistance phenotype. In TL-1, the typical 5′-end conserved sequence (5′-CS) region of class 1 integrons usually present upstream of the blaVEB-1agene was replaced by a truncated 3′-CS and a 135-bp repeated element (Re). Downstream of the blaVEB-1agene, an insertion sequence, ISPa31disrupted by ISPa30, and an orf513sequence, belonging to a common region (conserved region 1 [CR1]) immediately upstream of the aphA-6gene, were present. Further downstream, a second truncated 3′-CS region in direct repeat belonged to In51, an integron containing two gene cassettes (aadA6and the OrfD cassette). Thus, the overall structure corresponded to a sul-type class 1 integron termed In121. Genetic analyses revealed that both isolates were clonally related and differed by a ca. 100-kb fragment that contained In121. Both isolates contained another integron, In122, that carried three gene cassettes: aadB, dfrA1, and the OrfX cassette. This work identifies for the first time the spread of Re-associated blaVEBgenes located on a sul-type integron. It also reports for the first time a CR1 element in P. aeruginosathat is associated with an aminoglycoside resistance aphA-6gene that is expressed from a composite promoter.

ABSTRACTCarbapenem-resistant Acinetobacter baumanniiisolates were obtained from 24 patients between March and May 2004 at the Centre Hospitalier de Polyne´sie Franc¸aise, Tahiti, French Polynesia. The isolates were multidrug resistant, produced the carbapenemase OXA-23, and belonged to a single clone presenting several subtypes, suggesting an endemic situation. This study further illustrates the global spread of this kind of ß-lactamase-mediated resistance.

ABSTRACTThe spread of the plasmid-mediated carbapenem-hydrolyzing oxacillinase OXA-58 was detected in Acinetobactersp. clinical isolates from southern Europe, the Balkans, and central Turkey. It may contribute significantly to the emergence of carbapenem resistance in Acinetobacterspp., at least in this part of the world.

ABSTRACTAlthough enterococci expressing acquired vancomycin resistance phenotype have been reported increasingly worldwide, they have been rarely reported in France. From August to December 2004 we faced an outbreak of vancomycin-resistant Enterococcus faecium(VRE) isolates in the nephrology department at Bice^tre Hospital (K.-Bice^tre, France). The expression of the glycopeptide resistance varied among the 26 VRE isolates, with vancomycin MICs ranging from 12 to >256 µg/ml, whereas teicoplanin MICs ranged from 4 to 48 µg/ml. However, several strains appeared to be susceptible to glycopeptides according to disk diffusion testing and expressed resistance only after subculture with glycopeptides. In addition, a heterogeneous expression of glycopeptide resistance was also observed. This so-called VanD-like phenotype of resistance (low-level resistance to vancomycin and mostly susceptibility to teicoplanin) was surprisingly associated with a vanAgene. Plasmid extraction and mating-out experiments indicated that the vanAgene was located on a 200-kb self-transferable plasmid. Pulsed-field gel electrophoresis identified mostly dissemination of a single clone, whereas diffusion of the VanA-positive plasmid in different genomic backgrounds had also occurred. The vanAgene was part of a vanA-type operon for expression of resistance located on a Tn1546-like transposon. Sequencing of this transposon identified insertion of insertion sequence IS16in the vanYgene that encodes a d,d-carboxypeptidase that might explain in part the peculiar VanD-type phenotype of resistance. This report is the first description of a VRE outbreak in France and underlines the difficulty in detecting this organism due to variability on the expression of the glycopeptide resistance trait, if any.

ABSTRACTA chromosomally encoded oxacillinase, OXA-22, had been characterized from Ralstonia pickettiiPIC-1 that did not explain by itself the resistance profile of this strain to β-lactams. Thus, further analysis of the genetic background of this species led to the identification of another oxacillinase, OXA-60, that was expressed only after β-lactam induction. This chromosomally encoded oxacillinase shared 19% amino acid identity with OXA-22. It has a narrow-spectrum hydrolysis profile that includes imipenem. OXA-60-like enzymes were identified in several R. pickettiistrains. Gene inactivation and induction studies of the blaOXA-60and blaOXA-22genes in R. pickettiiidentified the relative contribution of each oxacillinase to the resistance profile of R. pickettiito β-lactams.

ABSTRACTCloning, sequencing, and biochemical analysis identified a novel AmpC-type β-lactamase conferring resistance to extended-spectrum cephalosporins in an Escherichia coliclinical isolate. This enzyme, exhibiting 14 amino acid substitutions compared to a reference AmpC cephalosporinase of E. coli, hydrolyzed ceftazidime and cefepime significantly.

ABSTRACTA Pseudomonas aeruginosaclinical strain isolated from a patient hospitalized in a New Delhi, India, hospital was resistant to expanded-spectrum cephalosporins, imipenem, and aztreonam. A blaVEB-1-like gene named blaVEB-1a, which codes for the extended-spectrum β-lactamase VEB-1a, was identified. The genetic environment of blaVEB-1awas peculiar: (i) no 5′ conserved sequence (5′-CS) region was present upstream of the β-lactamase gene, whereas blaVEB-1-like genes are usually associated with class 1 integrons; (ii) blaVEB-1awas inserted between two truncated 3′-CS regions in a direct repeat; and (iii) four 135-bp repeated DNA sequences (repeated elements) were located on each side of the blaVEB-1agene. Expression of the blaVEB-1agene was driven by a strong promoter located in one of these repeated sequences. In addition, cloning of the β-lactamase content of this P. aeruginosaisolate followed by expression in Escherichia coliidentified the naturally occurring AmpC β-lactamase and a gene encoding an OXA-2-like β-lactamase located in a class 1 integron, In78, in which an insertion sequence, ISpa7, was inserted within its 5′-CS region.