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Background Polymyxins are currently considered a last-resort treatment for infections caused by MDR Gram-negative bacteria. Recently, the emergence of carbapenemase-producing Enterobacteriaceae has accelerated the use of polymyxins in the clinic, resulting in an increase in polymyxin-resistant bacteria. Polymyxin resistance arises through modification of lipid A, such as the addition of phosphoethanolamine (pETN). The underlying mechanisms involve numerous chromosome-encoded genes or, more worryingly, a plasmid-encoded pETN transferase named MCR. Currently, detection of polymyxin resistance is difficult and time consuming. Objectives To develop a rapid diagnostic test that can identify polymyxin resistance and at the same time differentiate between chromosome- and plasmid-encoded resistances. Methods We developed a MALDI-TOF MS-based method, named the MALDIxin test, which allows the detection of polymyxin resistance-related modifications to lipid A (i.e. pETN addition), on intact bacteria, in <15 min. Results Using a characterized collection of polymyxin-susceptible and -resistant Escherichia coli, we demonstrated that our method is able to identify polymyxin-resistant isolates in 15 min whilst simultaneously discriminating between chromosome- and plasmid-encoded resistance. We validated the MALDIxin test on different media, using fresh and aged colonies and show that it successfully detects all MCR-1 producers in a blindly analysed set of carbapenemase-producing E. coli strains. Conclusions The MALDIxin test is an accurate, rapid, cost-effective and scalable method that represents a major advance in the diagnosis of polymyxin resistance by directly assessing lipid A modifications in intact bacteria. [ABSTRACT FROM AUTHOR]

Background: There is an urgent need for accurate and rapid diagnostic tests to identify carbapenemase producing enterobacteria (CPE). Here, we have evaluated the Carbapenem Inactivation Method (CIM) test to detect CPEs from cultured colonies. Methods: A total of 256 enterobacterial isolates were used to evaluate the performance of the CIM in comparison to Carba NP test and molecular detection used a reference method. Ninety three well-characterized isolates (including 29 non-CPE and 63 CPEs of worldwide origin) with decreased susceptibility to at least one carbapenem were used to (i) evaluate the efficacy of CIM test and (ii) to compare it to the Carba NP test. We also tested different carbapenems to determine the best substrate for this test. Finally, the CIM test was then evaluated prospectively against 164 isolates referred to the French National Reference Center (NRC) for Antimicrobial Resistance from may 2016 to july 2016. Results: Based on the results of this retrospective study, sensitivity and specificity of the CIM and the Carba NP test were 92.1% and 100%, respectively. We demonstrated that the meropenem was the best substrate to perform the CIM test since sensitivity and specificity were 81.1% and 100% using ertapenem disk, and 100% and 65,6% using imipenem disk, and respectively. Taking in account the results of retrospective and prospective studies, CIM and Carba NP tests have similar sensitivity, specificity, positive predictive value and negative predictive values being 96.3%, 98.9%, 99.0% and 98.4% for the CIM test versus 96.9%, 100%, 100% and 100% for the Carba NP test. Conclusions: Our results confirm that the CIM test may be a useful tool for the reliable confirmation of carbapenemase-activity in enterobacterial isolates, especially in clinical microbiological laboratories with limited resources, no trained personnel, and no specialized equipment. [ABSTRACT FROM AUTHOR]

Background: Polymyxins are currently considered a last-resort treatment for infections caused by MDR Gram-negative bacteria. Recently, the emergence of carbapenemase-producing Enterobacteriaceae has accelerated the use of polymyxins in the clinic, resulting in an increase in polymyxin-resistant bacteria. Polymyxin resistance arises through modification of lipid A, such as the addition of phosphoethanolamine (pETN). The underlying mechanisms involve numerous chromosome-encoded genes or, more worryingly, a plasmid-encoded pETN transferase named MCR. Currently, detection of polymyxin resistance is difficult and time consuming.Objectives: To develop a rapid diagnostic test that can identify polymyxin resistance and at the same time differentiate between chromosome- and plasmid-encoded resistances.Methods: We developed a MALDI-TOF MS-based method, named the MALDIxin test, which allows the detection of polymyxin resistance-related modifications to lipid A (i.e. pETN addition), on intact bacteria, in <15 min.Results: Using a characterized collection of polymyxin-susceptible and -resistant Escherichia coli, we demonstrated that our method is able to identify polymyxin-resistant isolates in 15 min whilst simultaneously discriminating between chromosome- and plasmid-encoded resistance. We validated the MALDIxin test on different media, using fresh and aged colonies and show that it successfully detects all MCR-1 producers in a blindly analysed set of carbapenemase-producing E. coli strains.Conclusions: The MALDIxin test is an accurate, rapid, cost-effective and scalable method that represents a major advance in the diagnosis of polymyxin resistance by directly assessing lipid A modifications in intact bacteria. [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]

Highlights • Development of a rapid and reliable multiplex polymerase chain reaction for mcr -1 to mcr- 5 gene detection. • Sensitivity and specificity of 100% using a collection of mcr -positive isolates. • Prospective evaluation revealed 100% positive and negative predictive values. ABSTRACT Plasmid-mediated colistin resistance is increasingly described worldwide in Enterobacteriaceae from animal and human isolates. Diffusion of these resistance traits among carbapenem-resistant enterobacterial isolates is of particular concern as colistin has become the last resort antibiotic for treating human infections with these organisms. Therefore, being able to monitor the presence of these transferable colistin resistance genes (mcr-1 to mcr-5 -variants) is crucial. This paper describes the development of a multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes in Enterobacteriaceae. Five primer pairs were designed to amplify mcr-1, mcr-2, mcr-3, mcr-4 and mcr -5 gene products in a multiplex PCR. This assay was validated retrospectively on colonies of 50 Escherichia coli , 44 Klebsiella pneumoniae and 12 Salmonella enterica isolates of animal and human origin, all well characterized, and validated prospectively on 450 carbapenem-resistant enterobacterial isolates received by the French National Reference Centre. In addition, 82 Aeromonas spp. and 10 Shewanella spp. known to be the progenitors of mcr-3 and mcr-4 alleles, respectively, were screened. Mcr-multiplex PCR assay displayed 100% specificity, sensitivity, negative predictive value and positive predictive value. The assay was able to detect all variants of the different mcr alleles, and was able to detect chromosomally encoded mcr-4- like variants present in two Shewanella bicestrii JAB-1 and Shewanella woodyi S539. In conclusion, a rapid and robust multiplex PCR assay able to detect all known mcr gene families described in Enterobacteriaceae was developed and validated. This type of test is critical for the epidemiological surveillance of plasmid-encoded resistance, especially in carbapenem-resistant bacteria. [ABSTRACT FROM AUTHOR]

Background Carbapenem-resistant Klebsiella pneumoniae (CRKP) strains are of particular concern, especially strains with mobilizable carbapenemase genes such as bla KPC, bla NDM or bla OXA-48, given that carbapenems are usually the last line drugs in the β-lactam class and, resistance to this sub-class is associated with increased mortality and frequently co-occurs with resistance to other antimicrobial classes. Objectives To characterize the genomic diversity and international dissemination of CRKP strains from tertiary care hospitals in Lisbon, Portugal. Methods Twenty CRKP isolates obtained from different patients were subjected to WGS for species confirmation, typing, drug resistance gene detection and phylogenetic reconstruction. Two additional genomic datasets were included for comparative purposes: 26 isolates (ST13, ST17 and ST231) from our collection and 64 internationally available genomic assemblies (ST13). Results By imposing a 21 SNP cut-off on pairwise comparisons we identified two genomic clusters (GCs): ST13/GC1 (n  = 11), all bearing bla KPC-3, and ST17/GC2 (n  = 4) harbouring bla OXA-181 and bla CTX-M-15 genes. The inclusion of the additional datasets allowed the expansion of GC1/ST13/KPC-3 to 23 isolates, all exclusively from Portugal, France and the Netherlands. The phylogenetic tree reinforced the importance of the GC1/KPC-3-producing clones along with their rapid emergence and expansion across these countries. The data obtained suggest that the ST13 branch emerged over a decade ago and only more recently did it underpin a stronger pulse of transmission in the studied population. Conclusions This study identifies an emerging OXA-181/ST17-producing strain in Portugal and highlights the ongoing international dissemination of a KPC-3/ST13-producing clone from Portugal. [ABSTRACT FROM AUTHOR]

Background: Carbapenem-resistant Enterobacteriaceae are considered by WHO as "critical" priority pathogens for which novel antibiotics are urgently needed. The dissemination of carbapenemase-producing Escherichia coli (CP-Ec) in the community is a major public health concern. However, the global molecular epidemiology of CP-Ec isolates remains largely unknown as well as factors contributing to the acquisition of carbapenemase genes. Methods: We first analyzed the whole-genome sequence and the evolution of the E. coli sequence type (ST) 410 and its disseminated clade expressing the carbapenemase OXA-181. We reconstructed the phylogeny of 19 E. coli ST enriched in CP-Ec and corresponding to a total of 2026 non-redundant isolates. Using the EpiCs software, we determined the significance of the association between specific mutations and the acquisition of a carbapenemase gene and the most probable order of events. The impact of the identified mutations was assessed experimentally by genetic manipulations and phenotypic testing. Results: In 13 of the studied STs, acquisition of carbapenemase genes occurred in multidrug-resistant lineages characterized by a combination of mutations in ftsI encoding the penicillin-binding protein 3 and in the porin genes ompC and ompF. Mutated ftsI genes and a specific ompC allele related to that from ST38 inducing reduced susceptibility to diverse β-lactams spread across the species by recombination. We showed that these mutations precede in most cases the acquisition of a carbapenemase gene. The ompC allele from ST38 might have contributed to the selection of CP-Ec disseminated lineages within this ST. On the other hand, in the pandemic ST131 lineage, CP-Ec were not associated with mutations in ompC or ftsI and show no signs of dissemination. Conclusions: Lineages of CP-Ec have started to disseminate globally. However, their selection is a multistep process involving mutations, recombination, acquisition of antibiotic resistance genes, and selection by β-lactams from diverse families. This process did not yet occur in the high-risk lineage ST131. [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]