Search

In Europe, evidence for the spread of extended-spectrum [beta]-lactamase (ESBL)-producing Escherichia coli from animals to humans via food is unclear (1). Few studies have been conducted in low- and middle-income [...]

Background: Avibactam, relebactam and vaborbactam are β-lactamase inhibitors that proved their efficiency against KPC-producing Enterobacterales. Regarding their inhibitor activity towards Ambler's class A extended spectrum β-lactamases (ESBL) and Ambler's class C cephalosporinase (AmpC), they should be active on most of the carbapenem-resistant non-carbapenemase-producing Enterobacterales (CR non-CPE). Objectives: Determine the in vitro activity of ceftazidime-avibactam, imipenem-relebactam and meropenem-vaborbactam and comparators against CR non-CPE. Methods: MICs to ceftazidime/avibactam, imipenem/relebactam, meropenem/vaborbactam, but also temocillin, ceftolozane/tazobactam, ertapenem, colistin, eravacycline and tigecycline were determined by broth microdilution (ThermoFisher) on a collection of 284 CR non-CPE (inhibition zone diameter < 22 mm to meropenem). Whole genome sequencing was performed on 90 isolates to assess the genetic diversity as well as resistome. Results: According to EUCAST breakpoints, susceptibility rates of ceftazidime, imipenem, meropenem and ertapenem used at standard dose were 0.7%, 45.1%, 14.8% and 2.5%, respectively. Increased exposure of ceftazidime, imipenem and meropenem led to reach 3.5%, 68.3% and 67.7% susceptibility, respectively. Using the EUCAST clinical breakpoints, susceptibility rates of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam were 88.4%, 81.0% and 80.6%, respectively. Susceptibility rates of temocillin, ceftolozane/tazobactam, tigecycline, eravacycline, and colistin were 0%, 4.6%, 27.8%, 54.9% and 90.1%. MICs distributions with and without the presence of the inhibitor demonstrated a better ability of avibactam and relebactam compared to vaborbactam to restore susceptibility to the associated β-lactam. Conclusions: This study demonstrated the in vitro efficacy of ceftazidime/avibactam, imipenem/relebactam and to a lesser extent meropenem/vaborbactam against CR non-CPE. Moreover, to test all β-lactams/β-lactamases inhibitors combinations without a priori for CRE, non-CPE is crucial since resistance to one of the β-lactam/β-lactamase inhibitor combinations does not predict resistance to another molecule, depending on the resistance mechanisms involved. [ABSTRACT FROM AUTHOR]

Early detection of multidrug resistant bacteria is of paramount importance for implementing appropriate infection control strategies and proper antibacterial therapies. We have evaluated a novel real-time PCR assay using fluorescent probes and 3base® technology, the EasyScreenTM ESBL/CPO Detection Kit (Genetic Signatures, Newtown, Australia), for the detection of 15 β-lactamase genes (blaVIM, blaNDM, blaIMP, blaOXA-48, blaKPC, blaOXA-23, blaOXA-51, blaSME,blaIMI, blaGES,blaTEM,blaSHV, blaCTX-M,blaCMY, blaDHA) and colistin resistance mcr-1 gene from 341 bacterial isolates (219 Enterobacterales, 66 P. aeruginosa and 56 A. baumannii) that were grown on Mueller–Hinton (MH) agar plates. One colony was suspended in provided extraction buffer, which lyses and converts the nucleic acids into a 3base®-DNA form (cytosines are converted into uracil, and subsequently thymine during PCR). The converted bacterial DNA is then added to the 6 PCR mixes, with primers for three targets plus one internal control. The EasyScreenTM ESBL/CPO Detection Kit was able to detect the 5-major (NDM, VIM, IMP, KPC, OXA-48) and 2-minor (IMI, Sme) carbapenemases and their variants irrespective of the species expressing them with nearly 100% sensitivity and specificity. With cephalosporinases CMY (82% of sensitivity) and DHA (87% of sensitivity) detection of chromosomally encoded variants was less efficient. Similarly, the chromosomally encoded OXA-51 variants were not consistently detected in A. baumannii. Despite being capable of efficiently detecting blaCTX-M-, blaTEM-, blaSHV- and blaGES-like genes, the EasyScreen™ ESBL/CPO Detection Kit was not able to distinguish between penicillinases and ESBL-variants of TEM and SHV and between GES-ESBLs and GES-carbapenemases. As GES enzymes are still rare, their detection as an ESBL or a carbapenemase remains important. Detection of mcr-1 was efficient, but none of the other mcr-alleles were detected in the 341 bacterial isolates tested. The EasyScreenTM ESBL/CPO Detection Kit is adapted for the detection of the most prevalent carbapenemases encountered in Gram-negatives isolated worldwide. [ABSTRACT FROM AUTHOR]

Enterobacteriaceae are inhabitants of the intestinal flora J-J and are among the most common human pathogens, causing infections such as cystitis and pyelonephritis with fever, septicemia, pneumonia, peritonitis, meningitis, and [...]

While there are numerous variants of CIM that have been proposed [[1]], the "rCIM-A" name has already been published almost a year ago to define a rapid CIM (rCIM) protocol that optimizes results for chromosomal cephalosporinase (AmpC) hyper-producing Enterobacterales [[3]]. The Carbapenem Inactivation Method became "rapid" with the work by Muntean et al. [[6]] with final test results in less than 3 h, a time-to-result similar to those of other rapid CPE confirmation methods (colorimetric, spectrometry, etc.), thus changing the paradigm of CIM testing. As stated by Gauthier et al. interpretation of growth of I E. coli i ATCC 25922 on Mueller-Hinton agar in relation to the antibiotic is not always interpretable at 6 h [[5]]. References 1 Mitteregger D., Wessely J., Barisi'c I., Bedeni'c B., Kosak D., Kundi M. A Variant Carbapenem Inactivation Method (CIM) for Acinetobacter Baumannii Group with Shortened Time-to-Result: RCIM-A. Pathogens. [Extracted from the article]

Background: OXA-244, a single amino acid variant of OXA-48, demonstrates weaker hydrolytic activity towards carbapenems and temocillin compared with OXA-48. Of note, these antimicrobials are present in high concentrations in several carbapenemase-producing Enterobacterales (CPE) screening media. As a result, some screening media fail to grow OXA-244-producing isolates, while the prevalence of OXA-244 producers is constantly increasing in France.Methods: Here, we evaluate the performance of three commercially available CPE screening media [ChromID® CARBA SMART (bioMérieux), Brilliance™ CRE (Thermo Fisher) and mSuperCARBA™ (MAST Diagnostic)] for their ability to detect OXA-244 producers (n = 101). As OXA-244 producers may also express an ESBL, two additional ESBL screening media were tested (Brilliance™ ESBL and ChromID® BLSE). MICs of temocillin and imipenem were determined by broth microdilution. The clonality of OXA-244-producing Escherichia coli isolates (n = 97) was assessed by MLST.Results: Overall, the sensitivity of the ChromID® CARBA SMART, Brilliance™ CRE and mSuperCARBA™ media were 14% (95% CI = 8.1%-22.5%), 54% (95% CI = 43.3%-63.4%) and 99% (95% CI = 93.8%-100%), respectively, for the detection of OXA-244 producers. Among the 101 OXA-244-producing isolates, 96% were E. coli and 77%-78% grew on ESBL screening media. MLST analysis identified five main STs among OXA-244-producing E. coli isolates: ST38 (n = 37), ST361 (n = 17), ST69 (n = 12), ST167 (n = 11) and ST10 (n = 8).Conclusions: Our results demonstrated that the mSuperCARBA™ medium is very efficient in the detection of OXA-244 producers, unlike the ChromID® CARBA SMART medium. The high prevalence of ESBLs among OXA-244 producers allowed detection of 77%-78% of them using ESBL-specific screening media. [ABSTRACT FROM AUTHOR]

Objectives: Escherichia coli is characterized by three resistance patterns to β-lactams/β-lactamase inhibitors (BLs/BLIs): (i) resistance to ampicillin/sulbactam and susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (RSS); (ii) resistance to ampicillin/sulbactam and amoxicillin/clavulanic acid, and susceptibility to piperacillin/tazobactam (RRS); and (iii) resistance to ampicillin/sulbactam, amoxicillin/clavulanic acid and piperacillin/tazobactam (RRR). These resistance patterns are acquired consecutively, indicating a potential risk of developing resistance to piperacillin/tazobactam, but the precise mechanism of this process is not completely understood.Methods: Clinical isolates incrementally pressured by piperacillin/tazobactam selection in vitro and in vivo were used. We determined the MIC of piperacillin/tazobactam in the presence and absence of piperacillin/tazobactam pressure. We deciphered the role of the blaTEM genes in the new concept of extended-spectrum resistance to BLs/BLIs (ESRI) using genomic analysis. The activity of β-lactamase was quantified in these isolates.Results: We show that piperacillin/tazobactam resistance is induced in E. coli carrying blaTEM genes. This resistance is due to the increase in copy numbers and transcription levels of the blaTEM gene, thus increasing β-lactamase activity and consequently increasing piperacillin/tazobactam MICs. Genome sequencing of two blaTEM-carrying representative isolates showed that piperacillin/tazobactam treatment produced two types of duplications of blaTEM (8 and 60 copies, respectively). In the clinical setting, piperacillin/tazobactam treatment of patients infected by E. coli carrying blaTEM is associated with a risk of therapeutic failure.Conclusions: This study describes for the first time the ESRI in E. coli. This new concept is very important in the understanding of the mechanism involved in the acquisition of resistance to BLs/BLIs. [ABSTRACT FROM AUTHOR]

Background: KPC-like carbapenemases have spread worldwide with more than 30 variants identified that differ by single or double amino-acid substitutions.Objectives: To describe the steady-state kinetic parameters of KPC-28, which differs from KPC-2 by a H274Y substitution and the deletion of two amino acids (Δ242-GT-243).Methods: The blaKPC-2, blaKPC-3, blaKPC-14 and blaKPC-28 genes were cloned into a pTOPO vector for susceptibility testing or into pET41b for overexpression, purification and subsequent kinetic parameter (Km, kcat) determination. Molecular docking experiments were performed to explore the role of the amino-acid changes in the carbapenemase activity.Results: Susceptibility testing revealed that Escherichia coli producing KPC-28 displayed MICs that were lower for carbapenems and higher for ceftazidime and ceftazidime/avibactam as compared with KPC-2. The catalytic efficiencies of KPC-28 and KPC-14 for imipenem were 700-fold and 200-fold lower, respectively, than those of KPC-2, suggesting that Δ242-GT-243 in KPC-28 and KPC-14 is responsible for reduced carbapenem hydrolysis. Similarly, the H274Y substitution resulted in KPC-28 in a 50-fold increase in ceftazidime hydrolysis that was strongly reversed by clavulanate.Conclusions: We have shown that KPC-28 lacks carbapenemase activity, has increased ceftazidime hydrolytic activity and is strongly inhibited by clavulanate. KPC-28-producing E. coli isolates display an avibactam-resistant ESBL profile, which may be wrongly identified by molecular and immunochromatographic assays as the presence of a carbapenemase. Accordingly, confirmation of carbapenem hydrolysis will be mandatory with assays based solely on blaKPC gene or gene product detection. [ABSTRACT FROM AUTHOR]

We compared extended-spectrum β-lactamase-producing Escherichia coli isolates from meat and fish, gut-colonized women, and infected patients in Cambodia. Nearly half of isolates from women were phylogenetically related to food-origin isolates; a subset had identical multilocus sequence types, extended-spectrum β-lactamase types, and antimicrobial resistance patterns. Eating sun-dried poultry may be an exposure route. [ABSTRACT FROM AUTHOR]

Highlights • Genomic analysis of NDM-producing Acinetobacter baumannii from Tunisia. • The clonal relationship revealed that these isolates were highly related. • This clone corresponds to a clone recovered from France 5 years ago. • Conjugation and electroporation experiments suggested that the bla NDM-1 gene is likely to be chromosomally located. ABSTRACT Background New Delhi metallo-β-lactamase (NDM)-producing Acinetobacter baumannii have been described in several countries worldwide, and studies have suggested that Acinetobacter spp. could play the role of intermediate progenitor of the bla NDM-1 gene between environmental progenitor and Enterobacteriaceae. Materials and methods In total, 246 carbapenem-resistant A. baumannii isolates from a teaching hospital in Sousse, Tunisia were investigated between 1st June 2013 and 31st December 2015 to detect metallo-ß-lactamase (MBL) production. Polymerase chain reaction (PCR), antibiotic susceptibility testing, and genetic and whole-genome sequencing tools were used to study the underlying carbapenem resistance mechanisms. Results PCR screening of the 246 carbapenem-resistant A. baumannii isolates revealed that 242 of 246 isolates harboured carbapenemase genes (seven of 246 positive for bla NDM-1 , four of 246 positive for bla NDM-1 and bla OXA-23 , 231 positive for bla OXA-23). Conjugation and electroporation experiments suggested that the bla NDM-1 gene is likely to be chromosomally located. All the NDM-1-producing A. baumannii isolates were clonally related, and belonged to ST85 according to the Pasteur Institute's multi-locus sequence typing scheme. Analysis of the immediate genetic environment of the bla NDM-1 gene revealed that the gene was located within a truncated isoform of Tn 125 transposon (ΔTn 125). The bla OXA-23 gene was located within transposon Tn 2008. Conclusion This study showed the dissemination of a single clone of NDM-1-producing A. baumannii in a Tunisian hospital. Countries in north Africa may constitute a significant reservoir for NDM-1-producing A. baumannii. The spread of the bla NDM-1 gene in A. baumannii was linked to clonal spread in this study. [ABSTRACT FROM AUTHOR]

Objectives: To describe a novel plasmid-borne class D carbapenemase (CHDL) named OXA-427 identified in several Enterobacteriaceae clinical isolates from nine patients in one Belgian hospital.Methods: OXA-427-producing isolates were analysed by an electrochemical imipenem hydrolysis method (BYG Carba test), Carba NP test, conventional phenotypic assays and by molecular methods (PCR, whole sequencing of the OXA-427-encoding plasmid and cloning). The antimicrobial resistance profile of OXA-427 was analysed by expression of the cloned gene in Escherichia coli DH10B and J53.Results: Eleven OXA-427-producing Enterobacteriaceae isolates of various species were identified from clinical specimens of nine patients between March 2012 and June 2014. OXA-427 shares only 22%-29% amino acid identity with OXA-48-like enzymes and other acquired CHDL (e.g. OXA-23, -24/40 and -58 of Acinetobacter spp.). Conversely, it appeared closely related to the chromosomal class D β-lactamase of Aeromonas media, Aeromonas hydrophila and Aeromonas sobria (99%, 89% and 77% of identity, respectively). When expressed in E. coli, OXA-427 hydrolysed imipenem and conferred resistance to extended-spectrum cephalosporins (mostly ceftazidime), penicillins including temocillin, and reduced susceptibility to carbapenems. The blaOXA-427 gene was located in a 45 kb resistance island on a 177 kb IncA/C plasmid.Conclusions: OXA-427 is a novel CHDL most closely related to chromosomal class D β-lactamase of A. media WS. It confers resistance to penicillins, ceftazidime and aztreonam and in some instances to carbapenems. OXA-427, which is not detectable by classical molecular tests, caused a protracted outbreak in one university hospital over a 2 year period. [ABSTRACT FROM AUTHOR]

Objectives: There is an urgent need for accurate and fast diagnostic tests capable of identifying carbapenemase producers. Here, we assessed the performance of a new multiplex lateral flow assay (OKN K -SeT) for the rapid detection of OXA-48-like, KPC and NDM carbapenemase-producing Enterobacteriaceae from culture colonies.Methods: Two hundred collection isolates with characterized β-lactamase content and 183 non-duplicate consecutive isolates referred to two National Reference Centres over a 2 month period in 2016 were used to evaluate the OKN K -SeT assay.Results: The assay correctly detected all 42 OXA-48-like-, 27 KPC- and 30 NDM-producing isolates from the collection panel, including 7 isolates that co-produced NDM and OXA-181 carbapenemases. No cross-reactivity was observed with non-targeted carbapenemases ( n  =   41) or with non-carbapenemase producers ( n  =   60). Prospectively, all OXA-48-like ( n  =   69), KPC ( n  =   9) and NDM ( n  =   19) carbapenemase-producing Enterobacteriaceae isolates were correctly detected, while 11 carbapenemase producers not targeted by the assay went undetected [VIM ( n  =   8) and OXA-23/OXA-58-like ( n  =   3)]. Overall, the sensitivity and specificity of the assay were 100%.Conclusions: The OKN assay is efficient, rapid and easy to implement in the workflow of a clinical microbiology laboratory for the confirmation of OXA-48, NDM and KPC carbapenemases. This test represents a powerful diagnostic tool as it enables the rapid detection of the most clinically important carbapenemases without the need for more costly and less frequently available molecular assays. [ABSTRACT FROM AUTHOR]

Objectives: There is an urgent need for accurate and fast diagnostic tests to identify carbapenemase-producing bacteria. Here, we have evaluated a novel colorimetric test (the β-CARBA™ test; Bio-Rad) to detect carbapenemase-producing Gram-negative bacilli from cultured colonies.Methods: The performance of the β-CARBA™ test was compared with that of the Carba NP test (or the CarbAcineto NP test) and RAPIDEC ® CARBA NP (bioMérieux) using a collection of 290 isolates with characterized β-lactamase content. This collection included 199 carbapenemase producers (121 Enterobacteriaceae, 36 Pseudomonas and 42 Acinetobacter baumannii ) and 91 non-carbapenemase producers (55 Enterobacteriaceae, 20 Pseudomonas and 16 A. baumannii ).Results: The β-CARBA™ test correctly detected 84.9% of the carbapenemase producers, including all KPC and IMP, 96.4% of VIM, 85.3% of NDM, 80.5% of OXA-48-like and 91.2% of A. baumannii -related OXA carbapenemases (OXA-23, OXA-40, OXA-58, OXA-143 and overexpressed OXA-51). All rare metallo-β-lactamases (SPM, AIM, GIM, DIM and SIM) were detected. Importantly, all non-KPC Ambler class A carbapenemases were not detected, including GES variants with carbapenemase activity ( n  = 6), IMI ( n  = 3), NMC-A ( n  = 1), SME ( n  = 2), FRI-1 ( n  = 1) and BIC-1 ( n  = 1). All non-carbapenemase producers gave a negative result except with OXA-163-, OXA-405- and one TEM-3-producing Citrobacter freundii . The overall sensitivity and specificity of the β-CARBA™ test were 84.9% and 95.6%, respectively. This test is easy to perform and to interpret by non-specialized staff members.Conclusions: Despite lack of specificity towards non-KPC Ambler class A and OXA-48-like carbapenemases, the β-CARBA™ test could complete the existing panel of tests available for the confirmation of carbapenemases in Gram-negatives. [ABSTRACT FROM AUTHOR]

Beta-Lactamase Database (BLDB) is a comprehensive, manually curated public resource providing up-to-date structural and functional information focused on this superfamily of enzymes with a great impact on antibiotic resistance. All the enzymes reported and characterised in the literature are presented according to the class (A, B, C and D), family and subfamily to which they belong. All three-dimensional structures of β-lactamases present in the Protein Data Bank are also shown. The characterisation of representative mutants and hydrolytic profiles (kinetics) completes the picture and altogether these four elements constitute the essential foundation for a better understanding of the structure-function relationship within this enzymes family. BLDB can be queried using different protein- and nucleotide-based BLAST searches, which represents a key feature of particular importance in the context of the surveillance of the evolution of the antibiotic resistance. BLDB is available online athttp://bldb.euwithout any registration and supports all modern browsers. [ABSTRACT FROM PUBLISHER]

Objectives KPC-producing Pseudomonas aeruginosa are increasingly isolated in the Americas and in the Caribbean islands. Here, we determined the whole-plasmid sequence of two plasmids carrying the blaKPC-2 gene from multidrug-resistant P. aeruginosa clinical isolates from Colombia. Methods The two plasmids, pCOL-1 and pPA-2, were transferred to Escherichia coli recipient strain TOP10 and completely sequenced using high-throughput pyrosequencing for pCOL-1 and classical Sanger sequencing for pPA-2. Results Both plasmids could be transferred to E. coli by transformation and displayed no other resistance marker besides KPC. Plasmid pCOL-1 was 31 529 bp in size, contained 31 open reading frames (ORFs) and belonged to the IncP-6 replicon group. It exhibited genes involved in replication, mobilization and partitioning, but none involved in conjugation. Plasmid pPA-2 was 7995 bp in size and contained seven ORFs. It exhibited a replicase gene of IncU, but was lacking genes involved in mobilization, partitioning and conjugation. Only 2072 bp matched Tn4401, including the blaKPC-2 gene, part of ISKpn6 and a 73 bp segment located upstream of the blaKPC-2 gene, containing the P1 promoter. Sequence identity was interrupted by a Tn3 transposon, itself interrupted by an IS26 element inserted within the β-lactamase blaTEM-1 gene. Conclusions Here we present the genetic features of the very first plasmids carrying the blaKPC-2 gene from P. aeruginosa. The emergence of the blaKPC-2 gene on unrelated plasmids, differing in size and in incompatibility group, and harbouring different genetic structures containing the blaKPC-2 genes in P. aeruginosa isolates suggests that this resistance trait may follow a dissemination scheme in P. aeruginosa similar to that seen in Enterobacteriaceae. [ABSTRACT FROM PUBLISHER]

Abstract: Klebsiella pneumoniae carbapenemase (KPC)-type enzymes have largely disseminated worldwide among K. pneumoniae isolates. In this study, 11 non-K. pneumoniae KPC-producing enterobacterial isolates from four hospitals located in different Colombian cities were genetically investigated. All isolates were multidrug-resistant and harboured the bla KPC-2 gene along with several other acquired β-lactamase genes. The bla KPC-2 gene was associated with transposon Tn4401b inserted in different loci of plasmids varying in size and replicon type. The presence of KPC-2 in different enterobacterial species from different cities within Colombia underlines the spread of KPC beyond K. pneumoniae. [Copyright &y& Elsevier]

The veb1 gene cassette encodes the extended spectrum b-lactamase, VEB-1 that is increasingly isolated from worldwide Gram-negative rods. Veb1 is commonly inserted into the variable region of different class 1 integrons in which it is always associated with a downstream-located aadB gene cassette encoding an aminoglycoside adenylyltransferase. In Pseudomonas aeruginosa, the majority of veb1-containing integrons also carry an insertion sequence, IS1999 that is inserted upstream of the veb1 gene cassette and disrupts the integron specific recombination site, attI1. Investigation of the recombination properties of the sites surrounding veb1 revealed that insertion of IS1999 reduces significantly the recombination frequency of attI1 and that veb1 attC is not efficient for recombination in contrast to aadB attC. Subsequent sequence optimisation of veb1 attC by mutagenesis, into a more consensual attC site resembling aadB attC, successfully improved recombination efficiency. Overall, this work gives some insights into the organisation of veb1-containing integrons. We propose that IS1999 and the nature of veb1 attC stabilize the veb1 gene cassette environment likely by impairing recombination events upstream or downstream of veb1, respectively. [ABSTRACT FROM AUTHOR]

Objectives Carbapenem-resistant Gram-negative bacilli are reported increasingly and represent an emerging public health concern. Laboratory detection of extended-spectrum β-lactamase (ESBL), plasmid-mediated cephalosporinase (pAmpC) and carbapenemase producers remains a challenge for microbiology laboratories and is important to avoid clinical failure due to inappropriate antimicrobial therapy and to prevent nosocomial outbreaks. We evaluated a novel microarray, the ‘Check-MDR CT103 array’ test (Check-Points, Wageningen, The Netherlands), that employs highly specific DNA markers to identify the β-lactamase genes of ESBLs (TEM, SHV and CTX-M, and discriminates between ESBL and non-ESBL TEM and SHV variants), of pAmpC (CMY-2-like, DHA, FOX, ACC-1, ACT/MIR and CMY-1-like/MOX) and of carbapenemases (KPC, OXA-48, VIM, IMP and NDM). Methods One-hundred-and-eighty-seven well-characterized Gram-negative bacilli isolates possessing different bla genes were tested. Total DNAs were extracted using a Qiagen DNA mini kit. The ‘Check-MDR CT103 array’ was used as recommended by the manufacturer. Results The system correctly identified representatives of the three ESBL gene families tested, including differentiation between non-ESBL and ESBL TEM and SHV variants. All blaCTX-M genes were classified into the appropriate family group (i.e. CTX-M-1 group, CTX-M-2 group, CTX-M-9 group and CTX-M-8/25/26 group). In addition, the clinically relevant plasmid-encoded cephalosporinase and carbapenemase genes were also reliably detected. Specificities and sensitivities of 100% were recorded for most bla genes. Conclusions The ‘Check-MDR CT103 array’ is a powerful high-throughput tool for rapid identification of ESBL, pAmpC and carbapenemase producers in culture. Because of its rapid performance, this platform is a valuable tool for epidemiological or infection control studies. [ABSTRACT FROM PUBLISHER]

Abstract: Plasmid-mediated 16S rRNA methylases such as ArmA, which confer high levels of resistance to aminoglycosides, are increasingly reported in Enterobacteriaceae. This study investigated the molecular mechanism of β-lactam and aminoglycoside resistance in extended-spectrum β-lactamase (ESBL)-producing Salmonella enterica serotype Infantis isolated at the 53-bed neonatology ward of University Hospital Benabib in Constantine, Algeria. From September 2008 to January 2009, 200 S. enterica isolates were obtained from 138 patients (age range 8–80 months) hospitalised in the neonatology ward. Most isolates were from stool cultures, but also from two blood cultures and one gastric fluid. The isolates were multidrug-resistant and produced TEM-1 and CTX-M-15 enzymes as well as the 16S RNA methylase ArmA. The armA, bla CTX-M-15 and bla TEM-1 genes were located on the same 140-kb self-transferable plasmid belonging to the IncL/M incompatibility group. All of the S. Infantis isolates belonged to a single clone. Increased infection control measures and thorough biodecontamination of the rooms led to control of the outbreak but did not eradicate the epidemic strain. This study further illustrates the global emergence of ArmA methylase and its frequent association with bla CTX-M genes. Spread of 16S RNA methylase determinants at the same level as bla CTX-M genes in Enterobacteriaceae may seriously compromise the efficacy of aminoglycosides for treating Gram-negative infections. [Copyright &y& Elsevier]

Abstract: Four Klebsiella pneumoniae isolates (KP1–4) were recovered sequentially from an infected patient. Whilst KP1–3 were resistant to all β-lactams except carbapenems, KP4 recovered after 24 days of imipenem-containing treatment showed additional resistance to carbapenems. No carbapenem hydrolysis could be identified in KP4. Molecular characterisation revealed that KP1–4 were indistinguishable by pulsed-field gel electrophoresis, contained a 95-kb self-transferable plasmid harbouring bla CTXM-15 and bla TEM-1 genes and a 65-kb plasmid that was not transferred by conjugation into Escherichia coli, and harboured the plasmid-mediated bla DHA-1 AmpC β-lactamase gene. In addition, KP4 failed to express OmpK36 owing to a point mutation leading to a premature stop of the protein. This study demonstrates development of carbapenem resistance related to loss of OmpK36 expression in a K. pneumoniae isolate harbouring extended-spectrum β-lactamase and plasmid-mediated cephalosporinase genes following prolonged imipenem exposure. [Copyright &y& Elsevier]

Objectives: CTX-M extended-spectrum β-lactamases (ESBLs) are emerging worldwide. Fast and reliable detection techniques may become mandatory for implementing proper treatment and infection control measures. Here, a blaCTX-M-specific LightCycler real-time PCR (LC-PCR) assay based on hybridization probes was developed. [ABSTRACT FROM PUBLISHER]

The article focuses on class D beta-lactamase gene expression in Ralstonia pickettii which is an environmental bacterium that may also be responsible for human infections. R. pickettii produces two unrelated, inducible and chromosomally encoded oxacillinases, OXA-22 and OXA-60. After one hour induction, the beta-lactamase production reached a maximal level.

VEB-1 extended-spectrum β-lactamase-producing Acinetobacter baumannii was responsible for an outbreak in hospitals in France. A national alert was triggered in September 2003 when 4 hospitals reported clusters of A. baumannii infection with similar susceptibility profiles. Case definitions and laboratory guidelines were disseminated, and prospective surveillance was implemented; strains were sent to a single laboratory for characterization and typing. From April 2003 through June 2004, 53 hospitals reported 290 cases of A. baumannii infection or colonization; 275 isolates were blaVEB-1-positive and clonally related. Cases were first reported in 5 districts of northern France, then in 10 other districts in 4 regions. Within a region, interhospital spread was associated with patient transfer. In northern France, investigation and control measures led to a reduction of reported cases after January 2004. The national alert enabled early control of new clusters, demonstrating the usefulness of early warning about antimicrobial drug resistance. [ABSTRACT FROM AUTHOR]

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal β-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These β-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C β-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC β-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC β-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C β-lactamases that explain inducibility of β-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C β-lactamases. [Copyright &y& Elsevier]

Investigates the Β-lactamase gene content and epidemiology of ceftazidime-resistant Pseudomonas aeruginosa isolates at the University Hospital in Thailand during a four-month period in 1999. Contribution of class A Β-lactamase genes to resistance to cephalosporins in P. aeruginosa clinical isolates; Clinical features of P. aeruginosa isolates and source patients; Antibiotic susceptability of P. aeruginosa clinical isolates.

The structure of the β-lactamase SME-1 from Serratia marcescens, a class A enzyme characterized by its significant activity against imipenem, has been determined to 2.13 Å resolution. The overall structure of SME-1 is similar to that of other class A β-lactamases. In the active-site cavity, most of the residues found in SME-1 are conserved among class A β-lactamases, except at positions 104, 105 and 237, where a tyrosine, a histidine and a serine are found, respectively, and at position 238, which is occupied by a cysteine forming a disulfide bridge with the other cysteine residue located at position 69. The crucial role played by this disulfide bridge in SME-1 was confirmed by site-directed mutagenesis of Cys69 to Ala, which resulted in a mutant unable to confer resistance to imipenem and all other β-lactam antibiotics tested. Another striking structural feature found in SME-1 was the short distance separating the side chains of the active serine residue at position 70 and the strictly conserved glutamate at position 166, which is up to 1.4 A shorter in SME4 compared with other class A β-lactamases. Consequently, the SME-1 structure cannot accommodate the essential catalytic water molecule found between Ser70 and Glu166 in the other class A β-lactamases described so far, suggesting that a significant conformational change may be necessary in SME-1 to properly position the hydrolytic water molecule involved in the hydrolysis of the acyl-enzyme intermediate. [ABSTRACT FROM AUTHOR]

Discusses the characterization of the genetic environment of the gene encoding the Escherichia coli extended-spectrum beta-lactamase that revealed the presence of a plasmid-located class 1 integron, In53. Condition showing that In53 is a truncated integron located on a composite transposon named Tn2000.

The article presents a study that deals with the prevalence of 16S rRNA methylase genes among representative extended-spectrum ß-lactamase (ESBL)-producing Salmonella enterica Senftenberg isolates from Algeria. The isolates were resistant to aminoglycosides recovered in the neonatology ward from January 1982 to December 2005. Included in the study were 12 non-duplicate ESBL-producing S. enterica serotype Senftenberg clinical isolates with variable levels of resistance to aminoglycosides. Based on their findings, the authors highlighted the role of Salmonella as a reservoir for ESBL genes and for 16S rRNA methylase genes in the community.

OXA-48-producing Enterobacterales have now widely disseminated globally. A sign of their extensive spread is the identification of an increasing number of OXA-48 variants. Among them, three are particularly interesting, OXA-163, OXA-247 and OXA-405, since they have lost carbapenem activities and gained expanded-spectrum cephalosporin hydrolytic activity subsequent to a four amino-acid (AA) deletion in the β5–β6 loop. We investigated the mechanisms responsible for substrate specificity of OXA-405. Kinetic parameters confirmed that OXA-405 has a hydrolytic profile compatible with an ESBL (hydrolysis of expanded spectrum cephalosporins and susceptibility to class A inhibitors). Molecular modeling techniques and 3D structure determination show that the overall dimeric structure of OXA-405 is very similar to that of OXA-48, except for the β5–β6 loop, which is shorter for OXA-405, suggesting that the length of the β5–β6 loop is critical for substrate specificity. Covalent docking with selected substrates and molecular dynamics simulations evidenced the structural changes induced by substrate binding, as well as the distribution of water molecules in the active site and their role in substrate hydrolysis. All this data may represent the structural basis for the design of new and efficient class D inhibitors. [ABSTRACT FROM AUTHOR]

The article discusses the endoscopy-linked transmission of carbapenem-resistant Klebsiella pneumoniae producing KPC-2 beta-lactamase. Researchers found that all Klebsiella pneumoniae carbapenemase (KPC) isolates were identical to a previously determined Klebsiella pneumoniae ST258-type clone that became an epidemic in Greece. The article details the case study of an 85-year-old individual suffering from bladder cancer who incurred an extended-spectrum beta lactamase (ESBL).

IS1999 and a point mutant derivative, IS1999.2, have been described inserted upstream of emerging antibiotic resistance genes blaVEB-1 and blaOXA-48. 5′ Rapid amplification of cDNA ends experiments revealed that expression of these β-lactamase genes was driven by the outward-directed promoter, Pout, located in the IS1999 elements. These findings led us to study IS1999-mediated gene mobilization. Thus, the transposition properties of IS1999 and of IS1999-based composite transposons, made of two copies of IS1999 in different orientations, were investigated. IS1999 or IS1999-based composite transposons were capable of transposing onto the conjugative plasmid pOX38-Gen. Sequence analysis of the insertion sites revealed that IS1999 inserted preferentially into DNA targets containing the consensus sequence NGCNNNGCN. Transposition was more efficient when at least one left inverted repeat end was located at an outside end of the transposon. The transposition frequency of IS1999.2 was 10-fold lower than that of IS1999, and transposition frequencies of the putative natural transposon, Tn1999, were below detection limits of our transposition assay. This reduced transposition frequency of IS1999.2-based elements may result from a lower transcription of the transposase gene, as revealed by reverse transcription-PCR analyses. [ABSTRACT FROM AUTHOR]

• Eleven bla PER-2 -harboring plasmids from Enterobacterales were fully sequenced. • The bla PER-2 gene was encoded on a variety of plasmid types (IncA, C, FIB, HI1B, N2). • IS Pa12 -composite transposons are suggested to be involved in bla PER-2 origin recruitment and dissemination. • A novel IS Pa12 -composite transposon, Tn 7390 , carrying the bla PER-2 gene was identified. • IS Kox2 -like elements seem to play a role in bla PER-2 gene dissemination. The production of PER-like extended-spectrum β-lactamases has recently been associated with reduced susceptibility to the last resort drugs aztreonam/avibactam and cefiderocol. PER-2 has been mainly confined to Argentina and neighboring countries. Until now, only three plasmids harboring bla PER-2 genes have been characterized but very little is known about the involvement of different plasmid groups in its dissemination. The diversity of genetic platforms associated with bla PER-2 genes from a collection of PER-producing Enterobacterales was analysed by describing the close environment and the plasmid backbones. Full sequences of 11 plasmids were obtained by short read (Illumina) and long read (Oxford Nanopore or PacBio) sequencing technologies. De novo assemblies, annotation and sequence analysis were performed by Unicycler, Prokka and BLAST. Plasmid analysis revealed that the bla PER-2 gene is encoded on plasmids of different incompatibility groups (A, C, FIB, HI1B, N2), indicating that this gene may have been disseminated through a variety of plasmids. Comparison with the few publicly available nucleotide sequences describing the bla PER-2 genetic environment, including those from the environmental species Pararheinheimera spp. (considered as the progenitor of bla PER genes), indicates a role of IS Pa12 in bla PER-2 gene mobilization from the chromosome of Pararheinheimera spp. Also, the bla PER-2 gene was carried by a novel IS Pa12 -composite transposon, Tn 7390. In addition, its association with IS Kox2 -like elements in the close genetic environment in all plasmids analysed suggests a role of these insertion sequence elements in further dissemination of bla PER-2 genes. [ABSTRACT FROM AUTHOR]

As carbapenemase-producing Enterobacteriaceae (CPE) and extended-spectrum β-lactamase–producing Enterobacteriaceae (ESBL-E) are becoming a major public health issue, there is an urgent need for accurate and fast diagnostic tests. The ELITe InGenius is a fully automated sample-to-result system designed for the extraction and detection by multiplex real-time polymerase chain reaction of carbapenemases KPC, NDM, VIM, IMP, and OXA-48–like variants and CTX-M group 1 and 9-producers from diverse sample matrices such as colonies, positive blood cultures, and rectal swabs. CRE and ESBL ELITe MGB® kits were evaluated on 153 cultured colonies of enterobacterial isolates with characterized β-lactamase content, on 30 spiked blood cultures, and the CRE kit was also evaluated on 53 clinical rectal swabs collected prospectively during a 3-month period and 10 spiked rectal swabs. CRE ELITe MGB® kit’s performances reached 100% sensitivity and 100% specificity, while for the ESBL ELITe kit, 100% sensitivity and 96.6% specificity were observed, with a sample to result of less than 3 h and a total percentage of agreement with expected results of 99.6% (255/256). [ABSTRACT FROM AUTHOR]

The dissemination of Gram-negative bacteria (GNB) producing extended-spectrum β-lactamases (ESBLs), plasmid-encoded cephalosporinases (pAmpCs) and carbapenemases is a matter of great clinical concern. In this study, we evaluated a new low-density DNA array ‘Check-MDR CT103 XL’ (Check-Points, Wageningen, The Netherlands) that identifies the most clinically relevant β-lactamase genes of ESBLs ( bla TEM , bla SHV , bla CTX-M , bla BEL , bla PER , bla GES and bla VEB ), pAmpCs ( bla CMY-2-like , bla DHA , bla FOX , bla ACC-1 , bla ACT/MIR and bla CMY-1-like/MOX ) and carbapenemases ( bla KPC , bla OXA-48 , bla VIM , bla IMP , bla NDM , bla GIM , bla SPM and bla OXA-23 , -24 and -58 ) in cultured bacteria. In total, 223 GNB isolates with well-characterised resistance mechanisms to β-lactams were analysed. A specificity and sensitivity of 100% were recorded for most bla genes, with a slightly lower signal observed for bla IMP . The Check-MDR CT103 XL array proved highly accurate for the identification of epidemiologically relevant ESBL, pAmpC and carbapenemase genes harboured in Enterobacteriaceae, Pseudomonas and Acinetobacter spp. The Check-MDR CT103 XL assay is a significant improvement compared with Check-MDR CT103 and it highlights the ability of this array to evolve rapidly to adjust to the current needs for the detection of resistance mechanisms to β-lactam agents. [ABSTRACT FROM AUTHOR]

Eleven carbapenem-resistant Klebsiella pneumoniae isolates were recovered from the military hospital of Tunis, Tunisia. Three of these isolates carried the carbapenemase bla OXA-232 gene recently identified in France. These isolates were clonally related and co-expressed the extended-spectrum ß-lactamase, CTX-M-15. This work identified the occurrence of OXA-232 producers in North-African countries. [ABSTRACT FROM AUTHOR]

The occurrence of resistances in Gram negative bacteria is steadily increasing to reach extremely worrying levels and one of the main causes of resistance is the massive spread of very efficient β-lactamases which render most β-lactam antibiotics useless. Herein, we report the development of a series of imino-analogues of β-lactams (namely azetidinimines) as efficient non-covalent inhibitors of β-lactamases. Despite the structural and mechanistic differences between serine-β-lactamases KPC-2 and OXA-48 and metallo-β-lactamase NDM-1, all three enzymes can be inhibited at a submicromolar level by compound 7dfm , which can also repotentiate imipenem against a resistant strain of Escherichia coli expressing NDM-1. We show that 7dfm can efficiently inhibit not only the three main clinically-relevant carbapenemases of Ambler classes A (KPC-2), B (NDM-1) and D (OXA-48) with Ki' s below 0.3 μM, but also the cephalosporinase CMY-2 (class C, 86% inhibition at 10 μM). Our results pave the way for the development of a new structurally original family of non-covalent broad-spectrum inhibitors of β-lactamases. [Display omitted] • Forty azetidinimines synthesized and evaluated for their activity against carbapenemases. • Several compounds are submicromolar inhibitors of both MBLs (NDM-1) and SBLs of class A (KPC-2) and D (OXA-48). • One compound is also active against 4 other MBLs (NDM-4, NDM-7, NDM-9 and VIM-1), CTX-M-15 (class A) and CMY-2 (class C). • The same compound can repotentiate imipenem against a NDM-1-producing clinical isolate of E. coli. [ABSTRACT FROM AUTHOR]

• Second description worldwide of LMB-1, a novel subclass B3 MBL in Citrobacter freundii 164 from Argentina. • LMB-1 confers reduced susceptibility to carbapenems and gives inconsistent results with carbapenemase confirmatory tests. • The bla LMB-1 gene was located on a 176-kb IncA/C2 plasmid. • The genetic environment of bla LMB-1 indicates an IS CR -like mobilization from the chromosome of Rheinheimera pacifica. • C. freundii 164 expressed CMY-150, a novel CMY-2 variant that increases ceftazidime and aztreonam MICs when expressed in E. coli. Carbapenemase-producing Enterobacterales expressing OXA-48, KPC, NDM, VIM or IMP enzymes are increasingly reported worldwide. We have characterized LMB-1, a novel metallo-β-lactamase (MBL) of Ambler class B3 from Citrobacter freundii 164 (Cf 164) clinical isolate from Buenos Aires, Argentina. Cf 164 displayed reduced susceptibility to carbapenems but gave inconsistent results with carbapenemase confirmatory tests, indicating the presence of a weak carbapenemase. Analysis of whole-genome sequencing (WGS) of Cf 164 using Resfinder revealed four β-lactamase genes coding for CTX-M-8, PER-2, TEM-1 and CMY-150, a novel chromosomally-encoded CMY variant. Kinetic parameters of purified CMY-150 did not reveal any carbapenemase activity. However, CMY-150 conferred higher minimum inhibitory concentrations (MICs) to E. coli for ceftazidime and aztreonam compared with CMY-2. The in-house-developed β-lactamase search software (ResMiner) in WGS data revealed a novel subclass B3 MBL named LMB-1. LMB-1 conferred resistance to penicillins and expanded-spectrum cephalosporins and reduced susceptibility to carbapenems in E. coli. The bla LMB-1 gene was located on a 176-kb IncA/C2 plasmid. LMB-1 shared 99% amino acid sequence identity with the MBL encoded in the chromosome of Rheinheimera pacifica, it's likely progenitor. Despite repeated attempts, LMB-1 could not be purified, thus only specific activities could indicate hydrolysis of carbapenems. Here we report on CMY-150, a novel CMY-2 variant that confers increased ceftazidime and aztreonam MICs to E. coli and the first description of LMB-1 in Argentina. This work underlines the need for several carbapenemase-producing Enterobacteriaceae (CPE) confirmatory tests, as this novel enzyme might have been missed using only one. Image, graphical abstract [ABSTRACT FROM AUTHOR]