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This article discusses the discovery of the OXA-198 carbapenemase gene cassette in the genus Candidatus Accumulibacter. The Candidatus Accumulibacter genus is known for its role in enhanced biological phosphate removal in wastewater treatment. The OXA-198 gene was first identified in clinical Pseudomonas aeruginosa isolates and later in Enterobacterales. The study found that the blaOXA-198-like gene was present in three different regions of Denmark, but unfortunately, the isolates were not cultivated. The genetic contexts of the blaOXA-198-like genes were analyzed, and it was determined that they were not acquired from other sources. The study suggests that Candidatus Accumulibacter may be the progenitor of OXA-198-like carbapenemase-encoding genes. This finding is significant as it provides insight into the origins of carbapenemase genes and may aid in future research and prevention efforts. [Extracted from the article]

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. [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]

Acinetobacter baumannii is an opportunistic pathogen in healthcare facilities responsible for nosocomial infections mostly in immunocompromised patients. Colistin resistance is increasingly reported worldwide in A. baumannii . Here we describe the in vivo selection of colistin and rifampicin resistance in carbapenem-resistant A. baumannii . Antimicrobial susceptibility testing, plasmid analysis and whole-genome sequencing (WGS) were performed to fully characterise the resistome of two clinical isolates ( Ab S1 and Ab S2) selected during treatment. Clinical isolate Ab S1 remained susceptible to colistin, rifampicin and tigecycline, whilst Ab S2 was susceptible only to tigecycline. PCR analysis revealed the presence of a bla OXA-23-like carbapenemase gene. Kieser extraction revealed an ca. 74 kb plasmid harbouring bla OXA-23 . WGS revealed genomes of 3.8 Mbp in size with a G + C content of 38.9%, and both belonged to ST281 according to the Oxford MLST scheme and ST641 according to the Institut Pasteur scheme. The resistome was also composed of naturally occurring β-lactamases, i.e. ADC-25 cephalosporinase and OXA-82 oxacillinase, aminoglycoside resistance genes [ aac(3)-Ia , aadA1 and aph(3')-VIa ( aphA6 )], and mutations in DNA gyrases explaining fluoroquinolone resistance. Single nucleotide polymorphism analysis revealed that both isolates were identical except for a 30-nucleotide duplication within the pmrB gene and a point mutation in the rpoB gene resulting in colistin and rifampicin resistance, respectively. This study highlights the genomic plasticity of A. baumannii under antibiotic pressure. The 10-amino acid duplication in PmrB affects colistin susceptibility by regulating lipopolysaccharide modification through the PmrAB two-component system. These findings provide further information on the molecular mechanisms leading to colistin resistance in A. baumannii . [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]

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]

Objectives Outbreaks of OXA-48-like carbapenemase producers are increasingly reported in many European countries and are often the result of difficulties in detection, especially for isolates with MICs of carbapenems that remain in the susceptibility range. Methods An in-house real-time quantitative PCR (qPCR) assay using TaqMan chemistry to detect blaOXA-48-like genes was compared with bacterial culturing on ChromID ESBL and SUPERCARBA media of spiked stool samples with several species producing OXA-48 variants. Results qPCR amplification using plasmid DNA was linear over 10 log dilutions (r2 = 0.998 and slope = −3.14), with an amplification efficiency of 1.10, and the detection limit of the assay was reproducibly estimated at 10 plasmid molecules/PCR. No cross-reaction was detected with DNA extracted from several multidrug-resistant bacteria harbouring other β-lactam resistance genes. The blaOXA-48 qPCR assay was capable of detecting 10–50 cfu of OXA-48 producers/100 mg of faeces. ChromID ESBL was capable of detecting OXA-48 producers (1 × 101 to 3 × 102 cfu/100 mg of faeces), as long as the isolates exhibited a high level of resistance to cephalosporins due to an associated extended-spectrum β-lactamase. The SUPERCARBA screening medium was capable of detecting all the OXA-48-like producers (1–3 × 101 cfu/100 mg of faeces), except those producing OXA-163, a variant lacking carbapenem-hydrolysing activity. Conclusions The qPCR is likely to shorten the time for blaOXA-48 detection from 48 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. [ABSTRACT FROM PUBLISHER]

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]

Carbapenemases increasingly have been reported in Enterobacteriaceae in the past 10 years. Klebsiella pneumoniae carbapenemases have been reported in the United States and then worldwide, with a marked endemicity at least in the United States and Greece. Metalloenzymes (Verona integron-encoded metallo-β-lactamase, IMP) also have been reported worldwide, with a higher prevalence in southern Europe and Asia. Carbapenemases of the oxacillinase-48 type have been identified mostly in Mediterranean and European countries and in India. Recent identification of New Delhi metallo-β-lactamase-1 producers, originally in the United Kingdom, India, and Pakistan and now worldwide, is worrisome. Detection of infected patients and carriers with carbapenemase producers is necessary for prevention of their spread. Identification of the carbapenemase genes relies mostly on molecular techniques, whereas detection of carriers is possible by using screening culture media. This strategy may help prevent development of nosocomial outbreaks caused by carbapenemase producers, particularly K. pneumoniae. [ABSTRACT FROM AUTHOR]

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]

New resistance problems have emerged recently among hospital and community-acquired pathogens such as in Staphylococcus aureus, Enterococcus faecium and Pseudomonas aeruginosa. Hospital-acquired and now community-acquired methicillin-resistant S. aureus are emerging worldwide whereas vancomycin-resistant S. aureus remain extremely rare. Hospital-acquired outbreaks of vancomycin-resistant enterococci and multidrug resistant Pseudomonas aeruginosa infections are increasingly reported worldwide. Whereas novel molecules are being developed for treating Gram-positive infections, difficult to non possible-to-treat pandrug-resistant P. aeruginosa infections may become a therapeutic challenge soon. [Copyright &y& Elsevier]

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 beta-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 bla(VEB-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 resist. [ABSTRACT FROM AUTHOR]

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]

The integron-borne bla[sub VEB-1] gene encodes an extended-spectrum β-lactamase. This gene was associated mostly with IS1999 and rarely with an additional IS2000 element in Pseudomonas aeruginosa isolates from Thailand, whereas IS1999 was only very rarely associated with bla[sub VEB-1] in Enterobacteriaceae. Expression experiments and promoter study identified promoter sequences in IS1999 that increased the expression of VEB-1 in P. aeruginosa. [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.

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.

Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a last resort therapy with extremely drug resistant Gram-negative bacteria. Here, we developed a rapid, easy and miniaturized colistin susceptibility assay based on microfluidics, which allows for culture and high-throughput analysis of bacterial samples. Specifically, a simple microfluidic platform that can easily be operated was designed to encapsulate bacteria in nanoliter droplets and perform a fast and automated bacterial growth detection in 2 h, using standardized samples. Direct bright-field imaging of compartmentalized samples proved to be a faster and more accurate detection method as compared to fluorescence-based analysis. A deep learning powered approach was implemented for the sensitive detection of the growth of several strains in droplets. The DropDeepL AST method (Droplet and Deep learning-based method for AST) developed here allowed the determination of the colistin susceptibility profiles of 21 fast-growing Enterobacterales (E. coli and K. pneumoniae), including clinical isolates with different resistance mechanisms, showing 100 % categorical agreement with the reference broth microdilution (BMD) method performed simultaneously. Direct AST of bacteria in urine samples on chip also provided accurate results in 2 h, without the need of complex sample preparation procedures. This method can easily be implemented in clinical microbiology laboratories, and has the potential to be adapted to a variety of antibiotics, especially for last-line antibiotics to optimize treatment of patients infected with multi-drug resistant strains. [Display omitted] • Simplified workflow for bacteria encapsulation and detection in nanoliter droplets. • Automated and deep learning powered approach for droplet classification. • Digital susceptibility testing for colistin using clinical isolates in 2 hours. • Categorization of bacteria strains in complete agreement with the reference method. • Direct urine analysis on chip for rapid detection of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Background Carbapenem-resistant Pseudomonas aeruginosa are being increasingly described worldwide. Here, we investigated the molecular mechanisms underlying carbapenem resistance in an extremely drug-resistant P. aeruginosa isolate from a neonatal intensive care unit in Morocco. Materials and methods P. aeruginosa strain O82J1 was identified using MALDI-TOF-MS. Carba NP, immunochromatographic assay NG Carba5 and antimicrobial susceptibility testing using disc diffusion and microbroth were performed. Whole-genome sequencing using the Illumina and MinION technologies and different software packages available at the Center of Genomic Epidemiology were used to predict the resistome, sequence type and plasmid types. Results P. aeruginosa O82J1 co-expressed two metallo-β-lactamases, bla NDM-1 and bla VIM-2, and was susceptible to colistin and apramycin only. It belonged to ST773 that is frequently reported worldwide as a high-risk P. aeruginosa clone. The bla VIM-2 gene was integron-borne on a IncP-2 465-kb plasmid, whereas the bla NDM-1 gene was chromosomally encoded and embedded in an integrative conjugative element, probably at the origin of its acquisition. A total of 23 antimicrobial resistance genes were detected including a bla PER-1 ESBL gene, and an 16S-rRNA methyltransferase gene rmtB. Conclusions The isolation of XDR P. aeruginosa isolates expressing several carbapenemases in a neonatal intensive care unit is of great concern due to the reduced treatment options, relying only on colistin, but not recommended in neonates, and apramycin, not yet approved for human therapy. Concerns were further elevated due to the resistance to cefiderocol and ATM/AVI, two novel and last-resort antibiotics recommended to treat infections caused by Gram-negative bacteria, particularly XDR P. aeruginosa in adults. [ABSTRACT FROM AUTHOR]

Retrotransposition of LINEs and other retroelements increases repetition in mammalian genomes and can cause deleterious mutations. Recent insertions of two full-length L 1s, L1spa and L1Orl, caused the disease phenotypes of the spastic and Orleans reeler mice respectively. Here we show that these two recently retrotransposed L1s are nearly identical in sequence, have two open reading frames and belong to a novel subfamily related to the ancient F subfamily. We have named this new subfamily TF (for transposable) and show that many full-length members of this family are present in the mouse genome. The TF 5′ untranslated region has promoter activity, and TF-type RNA is abundant in cytoplasmic ribonucleoprotein particles, which are likely intermediates in retrotransposition. Both L1spa and L 1Orl have reverse transcriptase activity in a yeast-based assay and retrotranspose at high frequency in cultured cells. Together, our data indicate that the TF subfamily of L1s contains a major class of mobile elements that is expanding in the mouse genome. [ABSTRACT FROM AUTHOR]

Studies the variations in DNA sequence insertion and compares the restriction fragment length polymorphism (RFLP) of subclones made from individual viable cells recovered from a stab culture of the resting Escherichia coli K-12 strain W3110. Nature of polymorphism; Pedigree of the old stab culture; Physiological studies with genetic variants; Nature of DNA rearrangements.

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.

A letter to the editor is presented about the prevalence of nosocomial infections caused by multidrug-resistant Acinetobacter baumannii in Russia.

We characterized a collection of IMI-like--producing Enterobacter spp. isolates (n = 112) in France. The main clone corresponded to IMI-1--producing sequence type 820 E. cloacae subspecies cloacae that was involved in an outbreak. Clinicians should be aware of potential antimicrobial resistance among these bacteria. [ABSTRACT FROM AUTHOR]

Background VRE are increasingly described worldwide. Screening of hospitalized patients at risk for VRE carriage is mandatory to control their dissemination. Here, we have developed the Bfast [VRE Panel] PCR kit, a rapid and reliable quantitative PCR assay for detection of vanA , vanB , vanD and vanM genes, from solid and liquid cultures adaptable to classical and ultrafast real-time PCR platforms. Methods Validation was carried out on 133 well characterized bacterial strains, including 108 enterococci of which 64 were VRE. Analytical performances were determined on the CFX96 Touch (Bio-Rad) and Chronos Dx (BforCure), an ultrafast qPCR machine. Widely used culture plates and broths for enterococci selection/growth were tested. Results All targeted van alleles (A, B, D and M) were correctly detected without cross-reactivity with other van genes (C, E, G, L and N) and no interference with the different routinely used culture media. A specificity and sensitivity of 100% and 99.7%, respectively, were determined, with limits of detection ranging from 21 to 238 cfu/reaction depending on the targets. The Bfast [VRE Panel] PCR kit worked equally well on the CFX and Chronos Dx platforms, with differences in multiplexing capacities (five and four optical channels, respectively) and in turnaround time (45 and 16 minutes, respectively). Conclusions The Bfast [VRE Panel] PCR kit is robust, easy to use, rapid and easily implementable in clinical microbiology laboratories for ultra-rapid confirmation of the four main acquired van genes. Its features, especially on Chronos Dx, seem to be unmatched compared to other tools for screening of VRE. [ABSTRACT FROM AUTHOR]

Ventilator-associated pneumonia (VAP) is associated with increased hospital stay and high morbidity and mortality in critically ill patients. The aims of this study were to (i) determine the incidence of multidrug-resistant (MDR) pathogens in the first episodes of VAP and to assess potential differences in bacterial profiles of subjects with early- versus late-onset VAP. This was a retrospective cohort study over a period of 18 months including all patients who had a first episode of VAP confirmed by positive bacterial culture. Subjects were distributed into two groups according to the number of intubation days: early-onset VAP (<5 days) or late-onset VAP (≥5 days). The primary endpoint was the nature of causative pathogens and their resistance profiles. Sixty patients were included, 29 men and 31 women, with an average age of 38 ± 16 years. The IGS 2 at admission was 40.5 [32–44] and APACHE was 19 [15–22]. Monomicrobial infections were diagnosed in 77% of patients (n = 46). The most frequently isolated bacteria were A. baumannii, 53% (n = 32); P. aeruginosa in 37% (n = 22); Enterobacterales in 28% (n = 17) and S. aureus in 5% (n = 3). Ninety-seven percent of the bacteria were MDR. The VAP group comprised 36 (60%) episodes of early-onset VAP and 24 (40%) episodes of late-onset VAP. There was no significant difference in the distribution of the bacterial isolates, nor in terms of antibacterial resistances between early- and late-onset VAPs. Our data support recent observations that there is no microbiological difference in the prevalence of potential MDR pathogens or in their resistance profiles associated with early- versus late-onset VAPs, especially in countries with high rates of MDR bacteria. [ABSTRACT FROM AUTHOR]

This article discusses a study on antimicrobial resistance (AMR) in Lebanon, specifically focusing on the presence of the blaNDM-5 gene in E. coli isolates. The study found that blaNDM-5-harbouring E. coli ST617 was present in Syrian refugees, their animals, and the camp environment, posing a health risk to both the refugees and the hosting community. The study emphasizes the importance of integrated surveillance programs and One Health studies to address AMR in vulnerable populations and highlights the role of war, mass migration, and displacement in the spread of AMR. Funding for the study was provided by various organizations, and ethical considerations were taken into account. [Extracted from the article]

This article discusses the emergence of a multidrug-resistant strain of Enterobacter xiangfangensis (Exn692) in a patient with COVID-19 and diabetes in Lebanon. The strain showed resistance to 37 antibiotics from different classes and carried genes that encoded resistance to these antibiotics. However, it was susceptible to a few antibiotics. The study emphasizes the challenges in treating infections caused by multidrug-resistant bacteria and the importance of effective antimicrobial stewardship. It also highlights the need for monitoring the evolution of Enterobacter xiangfangensis and developing accurate identification tools to understand its epidemiology, pathogenesis, and antibiotic resistance properties. [Extracted from the article]

The recently released CHROMagar™ ESBL/CHROMagar™ mSuperCARBA bi-plate medium was evaluated for the detection of ESBL- and carbapenemase-producing Enterobacteriaceae. Spiked stools were used to mimic in vivo stool colonization. Two-hundred enterobacterial isolates were tested. Respective sensitivities of 93.9% and 97.8% were obtained for the detection of ESBL and carbapenemase producers. [ABSTRACT FROM AUTHOR]

Background: The fecal carriage of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE) is a major driver of the global spread of these antibiotic resistance determinants. Here we determined the rate of fecal ESBL-PE carriage in pediatric hospitals and community-serving healthcare centers serving adults and children in the Gaza Strip, Palestine. Methods: A total of 373 fecal and rectal samples were collected from different hospitals and clinics in Gaza. The antibiotic susceptibility was determined using the disk diffusion method and interpreted according to CLSI guidelines. The bacterial isolates were tested for ESBL production using phenotypic methods (double disk synergy test and growth on selective chromogenic media). BlaCTX−M, blaSHV, and blaTEM genes were sought by PCR. Results: Out of the 373 isolates tested, 138 (37%) were considered ESBL positive as revealed by phenotypic tests. The prevalence of ESBLs among hospitalized patients was 39.1% (hospital setting) whereas, among outpatients attending community healthcare centers, it was 35.1% (community setting). ESBL production among Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Proteus mirabilis, and Klebsiella aerogenes isolates was 52.8%, 39.1%, 26.7%, 2.8%, and 2.1% respectively. Meropenem and amikacin were the most effective antibiotics against ESBL producers (68.9% and 73.6% susceptibility, respectively), while only 15.2%, 22.5%, and 24.6% remained susceptible to ceftazidime, cefotaxime, and ceftriaxone, respectively. Out of 138 phenotypically ESBL-positive isolates, 98 randomly chosen were screened for blaCTX−M, blaTEM, and blaSHV genes. The prevalence rate of blaCTX−M was 45.9%, while blaTEM and blaSHV genes were detected in 16.8% and 5.2% of CTX-M-negative isolates (corresponding mostly for K. pneumoniae isolates in the case of SHV-PCR), respectively. Conclusions: The study revealed an alarmingly high prevalence of fecal carriage of ESBL-producing Enterobacterales among hospitalized children but also in the community of the Gaza Strip. In addition, 30% of ESBL-producers were already resistant to carbapenems, the treatment of choice of infections with ESBL-producers. [ABSTRACT FROM AUTHOR]

This article discusses the emergence of carbapenem resistance in Enterobacterales, specifically through the dissemination of carbapenemase-producing bacteria. The study characterizes two new variants of the VIM-1 enzyme, called VIM-29 and VIM-86, which were isolated in multidrug-resistant Enterobacterales in France. The isolates were resistant to all β-lactam antibiotics except aztreonam/avibactam. The study also analyzes the genetic environment of the VIM-29 and VIM-86 genes and identifies common resistance determinants. The plasmids carrying these genes were found to be self-conjugative. The hydrolytic abilities of VIM-29 and VIM-86 were compared to other prevalent VIM variants, and it was found that they displayed similar resistance profiles. The study concludes that these new variants contribute to the growing problem of carbapenem resistance. [Extracted from the article]

A letter about multidrug-resistant (MDR) Pseudomonas aeruginosa isolates from hospitalized patients in Brussels, Belgium is presented. The susceptibility of the bacteria to carbapenems based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and genetic sequencing is tested. It is suggested that Metallo Beta Lactamase (MBL) producers that are susceptible should be reported to help in epidemioloical analysis.

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).

Cefiderocol resistance is increasingly reported in New Delhi metallo-β-lactamase–producing Enterobacterales. Genomic and phenotypic analysis of Escherichia coli sequence type 361, a primary clone causing carbapenemase spread in France, revealed mutations leading to cefiderocol resistance. Continued genomic surveillance of carbapenem-resistant Enterobacterales could clarify prevalence of cefiderocol-resistant E. coli in Europe. [ABSTRACT FROM AUTHOR]

Background Cefiderocol is a catechol-substituted cephalosporin with potent in vitro activity against carbapenem-resistant (CR) Gram-negative bacteria (GNB). Cefiderocol susceptibility testing is complex because iron concentrations need to be taken into consideration. Here, we assessed the clinical performance of Bruker's UMIC® Cefiderocol and corresponding iron-depleted CAMHB to determine MIC by broth microdilution (BMD) for clinically relevant GNB. Methods MICs of cefiderocol for 283 GN clinical isolates were determined by BMD using iron-depleted CAMHB. Frozen panels were used as a reference. The concentration range of cefiderocol was 0.03–32 mg/L. The isolates, with different degrees of susceptibility to cefiderocol, included Enterobacterales (n  = 180) , Pseudomonas aeruginosa (n  = 49), Acinetobacter baumannii (n  = 44) and Stenotrophomonas maltophilia (n  = 10). Results The rates of categorical agreement (CA), essential agreement (EA) and bias were calculated to evaluate the performance of the UMIC® Cefiderocol, as compared with the reference method. Overall, the UMIC® Cefiderocol showed 90.8% EA (95% CI: 86.9%–93.7%) with a bias of −14.5% and a CA of 90.1% (95% CI: 86.1%–93.1%). For Enterobacterales, the UMIC® Cefiderocol showed 91.7% EA (95% CI: 86.7%–94.9%) with a bias of −25.0% and a CA of 87.8% (95% CI: 82.2%–91.8%). For non-fermenters, the UMIC® Cefiderocol showed 89.3% EA (95% CI: 81.9%–93.9%) (not significantly different from 90.0%, Student t -test) with a bias of −3.9% and a CA of 94.2% (95% CI: 87.7%–97.3%). Conclusions UMIC® Cefiderocol is a valid method for the determination of cefiderocol MICs even if higher than expected discrepancies were observed with NDM-producing Enterobacterales, which presented in most cases MIC values close to the breakpoint. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to article "Endoscopy-Related Infection: Relic of the Past?" in the 2008 issue is presented.

Background As carbapenemase-producing Enterobacterales are increasingly reported worldwide, their rapid detection is crucial to reduce their spread and prevent infections and outbreaks. Lateral flow immunoassays (LFIAs) have become major tools for the detection of carbapenemases. However, as for most commercially available assays, only the five main carbapenemases are targeted. Objectives Here, we have developed and evaluated an LFIA prototype for the rapid and reliable detection of the increasingly identified GES-type β-lactamases. Methods The GES LFIA was validated on 103 well-characterized Gram-negative isolates expressing various β-lactamases grown on Mueller–Hinton (MH) agar, chromogenic, and chromogenic/selective media. Results The limit of detection of the assay was 106 cfu per test with bacteria grown on MH agar plates. GES LFIA accurately detected GES-type β-lactamases irrespective of the culture media and the bacterial host. The GES LFIA was not able to distinguish between GES-ESBLs and GES-carbapenemases. Because GES enzymes are still rare, their detection as an ESBL or a carbapenemase remains important, especially because extensive use of carbapenems to treat ESBL infections may select for GES variants capable of hydrolysing carbapenems. Conclusions The GES LFIA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of GES-type β-lactamases. Combining it with immunochromatographic assays targeting the five main carbapenemases (KPC, NDM, VIM, IMP and OXA-48) would improve the overall sensitivity for the most frequently encountered carbapenemases and ESBLs, especially in non-fermenters. [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]

Culture media is fundamental in clinical bacteriology for the detection and isolation of bacterial pathogens. However, in-house media preparation could be challenging in low-resource settings. InTray® cassettes (Biomed Diagnostics) could be a valid alternative as they are compact, ready-to-use media preparations. In this study, we evaluate the use of two InTray media as a subculture alternative for the diagnosis of bloodstream infections: the InTray® Müller-Hinton (MH) chocolate and the InTray® Colorex™ Screen. The InTray MH chocolate was evaluated in 2 steps: firstly, using simulated positive blood cultures (reference evaluation study), and secondly, using positive blood cultures from a routine clinical laboratory (clinical evaluation study). The Colorex Screen was tested using simulated poly-microbial blood cultures. The sensitivity and specificity of the InTray MH chocolate were respectively 99.2% and 90% in the reference evaluation study and 97.1% and 88.2% in the clinical evaluation study. The time to detection (TTD) was ≤20 h in most positive blood cultures (99.8% and 97% in the two studies, respectively). The InTray® MH Chocolate agar showed good performance when used directly from clinical blood cultures for single bacterial infections. However, mixed flora is more challenging to interpret on this media than on Colorex™ Screen, even for an experienced microbiologist. [ABSTRACT FROM AUTHOR]

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]

During the latent phase of herpes simplex virus type 1 (HSV-1) infection, the latency-associated transcripts (LATs) are the most abundant viral transcripts present in neurons, but some immediate-early viral transcripts, such as those encoding ICP0, have also been reported to be transcribed in latently infected mouse trigeminal ganglia (TG). A murine oro-ocular model of herpetic infection was used to study ICP0 gene expression in the major anatomical sites of HSV-1 latency, including the TG, superior cervical ganglion, spinal cord, and hypothalamus. An HSV-1 recombinant strain, SC16 110LacZ, revealed ICP0 promoter activity in several neurons in latently infected ganglia, and following infection with wild-type HSV-1 strain SC16, in situ hybridization analyses identified ICP0 transcripts in the nuclei of neurons at times consistent with the establishment of latency. Reverse transcription (RT)-PCR assays performed on RNA extracted from latently infected tissues indicated that ICP0 transcripts were detected in all anatomical sites of viral latency. Furthermore, quantitative real-time RT-PCR showed that neurons differentially expressed the LATs and ICP0 transcripts, with splicing of ICP0 transcripts being dependent on the anatomical location of latency. Finally, TG neurons were characterized by high-level expression of LATs and detection of abundant unspliced ICP0 transcripts, a pattern markedly different from those of other anatomical sites of HSV-1 latency. These results suggest that LATs might be involved in the maintenance of HSV-1 latency through the posttranscriptional regulation of ICP0 in order to inhibit expression of this potent activator of gene expression during latency. [ABSTRACT FROM AUTHOR]