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Introduction: Antibiotic resistance (AR) is a worldwide concern and the description of AR have been discovered mainly because of their implications in human medicine. Since the recent burden of whole-genome sequencing of microorganisms, the number of new AR genes (ARGs) have dramatically increased over the last decade. Areas covered: In this review, we will describe the different methods that could be used to characterize new ARGs using classic or innovative methods. First, we will focus on the biochemical methods, then we will develop on molecular methods, next-generation sequencing and bioinformatics approaches. The use of various methods, including cloning, mutagenesis, transposon mutagenesis, functional genomics, whole genome sequencing, metagenomic and functional metagenomics will be reviewed here, outlining the advantages and drawbacks of each method. Bioinformatics softwares used for resistome analysis and protein modeling will be also described. Expert opinion: Biological experiments and bioinformatics analysis are complementary. Nowadays, the ARGs described only account for the tip of the iceberg of all existing resistance mechanisms. The multiplication of the ecosystems studied allows us to find a large reservoir of AR mechanisms. Furthermore, the adaptation ability of bacteria facing new antibiotics promises a constant discovery of new AR mechanisms. [ABSTRACT FROM AUTHOR]

In this study, the draft genome sequences of two different carbapenem-resistant Acinetobacter baumannii clinical strains isolated from the same blood culture sample of an Iranian patient were determined. The strain A. baumannii 554S harbouring blaoxa72 gene belonged to ST 307 whereas A. baumannii 554L carrying blaoxa23 gene belonged to ST 2. We found that this sample contains two different isolates of A. baumannii, each phenotypically and genetically different. [ABSTRACT FROM AUTHOR]

Enterobacter cloacae complex belong to a group for which colistin resistance is not well documented, due to their frequent heteroresistance. Previous studies have examined the role of the two-component systems PhoP-PhoQ and its negative regulator mgrB or the arnBCADTEF operon. We investigated the molecular basis of colistin heteroresistance using genome analysis and random mutagenesis in a strain of Enterobacter cloacae and identified the arnA gene as responsible for colistin resistance., (Copyright © 2024. Published by Elsevier Ltd.)

Antimicrobial resistance in Enterobacteriaceae is a public health problem. Rodents, can be a potential vector for transmission of multidrug resistant bacteria between animals, humans, and environment. The aim of our study was to assess the level of Enterobacteriaceae present in the intestines of rats collected from different locations in Tunisia, then to determine their antimicrobial susceptibility profiles, to screen extended spectrum β-lactamases-producing strains and determine the molecular mechanism of β-lactams resistance. Between July 2017 and June 2018, 55 strains of Enterobacteriaceae were isolated from 71 rats captured in various locations in Tunisia. Antibiotic susceptibility testing was performed using the disc diffusion method. Genes encoding ESBL and mcr genes were investigated by RT-PCR, standard PCR and sequencing when these genes were found. Fifty-five strains of Enterobacteriaceae were identified. The overall prevalence of ESBL production found in our study was 12.7 % (7/55) of which two E. coli strains were DDST positive, one isolated from a house-caught rat and one from the veterinary clinic and harbored the bla TEM-128 gene. In addition, the other five strains were DDST negative and harbored the bla TEM gene, including three strains isolated from collective restaurant (n = 2: bla TEM-163 ; n = 1: bla TEM-1 ), one strain isolated from the veterinary clinic (bla TEM-82 ), and one strain isolated from a house (bla TEM-128 ). The results of our study suggest that rodents may play a role in the spread of antimicrobial resistant E. coli, highlighting the need to protect the environment and monitor antimicrobial resistant bacteria in rodents to prevent their spread to other wildlife and humans., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Background: Carbapenemase-producing Enterobacteriaceae (CPE) represent an increasing threat to public health, especially in hospitals., Objectives: To investigate an outbreak of CPE in a thoracic-oncology unit by using whole genome sequencing (WGS) and to describe the control measures taken to limit the epidemic, including fecal microbiota transplantation (FMT)., Methods: A retrospective study between December 2016 and October 2017 was performed to investigate an outbreak of CPE in a thoracic-oncology unit at the North Hospital in Marseille, France. The isolates were identified, and antimicrobial susceptibility tests were performed. All CPE were sequenced using MiSeq and/or MinIon technologies. Nucleotide variations between plasmids and similarity within the same species were investigated. The origin of this outbreak, its spread, and the decolonization of patients in the ward were also studied., Results: Four Citrobacter freundii , one Enterobacter cloacae and four E. hormaechei OXA-48 carbapenemase producers were isolated in eight patients hospitalized the same year in a thoracic-oncology ward. The bla OXA-48 gene was present in a Tn 1999.2 transposon located in IncL/M plasmids, with single nucleotide variants (SNV) ranging from 0 to 5. All C. freundii strains belonged to the same ST22 and had more than 99.6% similarity between them. Two strains of E. hormaechei ST1007 were almost identical at 99.98%, while the others belonged to a different ST (ST98, ST114, ST133). No single source was identified. FMT resulted in decolonization in 4/6 patients., Conclusions: WGS demonstrated the dissemination of the bla OXA-48 gene by both clonal ( C. freundii ST22 and E. hormaechei ST1007) and plasmid spread (pOXA-48 IncL/M). The origin of this outbreak appeared to be both external and internal to the ward. This evidence of cross-infection supports the urgent need for the implementation of infection control measures to prevent CPE dissemination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hadjadj, Cassir, Saïdani, Hoffman, Brouqui, Astoul, Rolain and Baron.)

Introduction: The role of wildlife in the transmission of antimicrobial resistant (AMR) is suspected but scarcely reported in current studies. Therefore, we studied the dynamics and prevalence of antibiotic-resistant Enterobacterales in antibiotic-limited areas of Senegal., Materials and Methods: We collected fecal samples from monkeys and apes (N = 226) and non-fecal environmental samples (N = 113) from Senegal in 2015 and 2019. We grew the samples on selective media, subsequently isolated AMR Enterobacterales , and then sequenced their genomes., Results: We isolated 72 different Enterobacterales among which we obtained a resistance rate of 65% for colistin (N = 47/72) and 29% for third generation-cephalosporin (C3G) (29%, N = 21/72). Interestingly, almost 46% of our isolates, among Enterobacter sp., Citrobacter cronae and Klebsiella aerogenes , belong to 34 new STs. Moreover, the genes bla CTX - M -15 , bla TEM 1 B , sul2 , dfrA14 , qnrs , aph ( 3'' ), aph ( 6 ), tetA , and tetR harbored within a transposon on the IncY plasmid of ST224 Escherichia coli were transferred and inserted into a ST10 E . coli phage coding region., Conclusion: Wildlife constitutes a rich, unexplored reservoir of natural microbial diversity, AMR genes and international resistant clones pathogenic in humans. The presence of a transposon that carries AMR genes is intriguing since no antibiotics are used in the non-human primates we studied., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Abdallah, Kuete Yimagou, Hadjadj, Mediannikov, Ibrahim, Davoust, Barciela, Hernandez-Aguilar, Diatta, Sokhna, Raoult, Rolain and Baron.)

Alternative strategies against multidrug-resistant (MDR) bacterial infections are suggested to clinicians, such as drug repurposing, which uses rapidly available and marketed drugs. We gathered a collection of MDR bacteria from our hospital and performed a phenotypic high-throughput screening with a 1280 FDA-approved drug library. We used two Gram positive (Enterococcus faecium P5014 and Staphylococcus aureus P1943) and six Gram negative (Acinetobacter baumannii P1887, Klebsiella pneumoniae P9495, Pseudomonas aeruginosa P6540, Burkholderia multivorans P6539, Pandoraea nosoerga P8103, and Escherichia coli DSM105182 as the reference and control strain). The selected MDR strain panel carried resistance genes or displayed phenotypic resistance to last-line therapies such as carbapenems, vancomycin, or colistin. A total of 107 compounds from nine therapeutic classes inhibited >90% of the growth of the selected Gram negative and Gram positive bacteria at a drug concentration set at 10 µmol/L, and 7.5% were anticancer drugs. The common hit was the antiseptic chlorhexidine. The activity of niclosamide, carmofur, and auranofin was found against the selected methicillin-resistant S. aureus. Zidovudine was effective against colistin-resistant E. coli and carbapenem-resistant K. pneumoniae. Trifluridine, an antiviral, was effective against E. faecium. Deferoxamine mesylate inhibited the growth of XDR P. nosoerga. Drug repurposing by an in vitro screening of a drug library is a promising approach to identify effective drugs for specific bacteria.

Here, we develop a robust and sensitive real-time PCR assay which allows the simultaneous detection of vanA and vanB genes using common primers. The system was designed using the Primer3 online software. The specificity of primers and probes was first checked by in silico PCR and by BlastN analysis. The genomic DNA of 255 bacterial isolates, including Enterococcus spp., Gram-negative, and Gram-positive strains, as well as a collection of 50 stool and 50 rectal swab samples, were tested to evaluate the specificity of the new real-time PCR (RT-PCR) system. The results of the designed RT-PCR were 100% specific and 100% positive on tested vancomycin resistant isolates harboring either the vanA or vanB gene. RT-PCR assays were negative for all other bacterial species tested including vancomycin-sensitive Enterococci and Enterococcus strains harboring vanC genes. The limit of detection of vanA and vanB genes by RT-PCR assay was 47 CFU/mL and 32 CFU/mL, respectively. The rapid and accurate detection of vancomycin-resistant Enterococci is the cornerstone for minimizing the risk of nosocomial transmissions and outbreaks. We believe that this assay will strengthen routine diagnostics and surveillance programs.

In this study, we aim to characterize the genetic environment of the plasmid-mediated colistin resistance gene mcr-1 in 25 Escherichia coli and seven Klebsiella pneumoniae strains from different countries and continents. Multilocus sequence typing, conjugation experiments, plasmid typing, and the presence and location of the insertion sequence IS Apl1 were investigated. Whole genome sequencing of four E. coli was performed to analyse the genetic environment of the mcr-1 gene. Colistin minimum inhibitory concentration of mcr-1 strains varied from 3 to 32 µg/mL. Six E. coli sequence types were detected: ST 4015, ST 3997, ST 10, ST 93, ST 48, and ST 648. IncHI2, IncI2, and IncP plasmid types were predominant and were unrelated to a specific country of origin. IS Apl1 was found in 69% of analysed plasmids that were mainly around the mcr-1 gene. Analysis of four closed mcr-1 plasmids revealed the integration of mcr-1 into hotspots. We found that the spread of mcr-1 gene was due to the diffusion of a composite transposon and not to the diffusion of a specific plasmid or a specific bacterial clone. The ease with which the mcr-1 gene integrates into various regions facilitates its dissemination among bacteria and explains its large diffusion all over the world, both in animals and in humans., Competing Interests: The authors declare no conflict of interest.