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Nadjette Bourafa,1–3 Wafaa Chaalal,1,4 Sofiane Bakour,1 Rym Lalaoui,1 Nafissa Boutefnouchet,2 Seydina M Diene,1 Jean-Marc Rolain1 1Aix Marseille Universite, MEPHI, IHU-Mediterranee Infection, Marseille, France; 2Laboratoire de Microbiologie et Biochimie Appliquée, Département de Biochimie, Faculté des Sciences, Université Badji Mokhtar Annaba, Algeria; 3Département de Biologie, Faculté des Sciences de la Nature et de la Vie, Université Mohamed Cherif Messaadia-Souk-ahras, Algeria; 4Laboratoire de Microbiologie Appliquée, Faculté des Sciences de la Nature et de la Vie, Université d’Oran, Es Senia, Oran, Algeria Objectives: The aims of this study were to investigate the occurrence of carbapenem-resistant Gram-negative bacilli (GNB) isolated from inpatients and outpatients in Algeria between July and September 2015, and to screen their resistance mechanisms and genetic relatedness.Materials and methods: A total of 68 non-redundant isolates were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) antibiotic susceptibility testing was performed using disk diffusion and Etest methods. Carbapenemase activity was carried out using modified Carba NP test, EDTA assay, and the modified Hodge test. Molecular characterization of carbapenemases and extended-spectrum β-lactamase (ESBL) genes were detected by standard PCR and sequencing. Genotyping of carbapenem-resistant isolates was performed by multilocus sequence typing (MLST) analysis.Results: Of the 68 GNB isolates, 13 (19%) showed reduced susceptibility to carbapenems, including, four Klebsiella pneumoniae, one Escherichia coli, six Acinetobacter baumannii, and two Pseudomonas aeruginosa. blaOXA-48 gene was detected in the five Enterobacteriaceae isolates, and blaOXA-23 was identified in all A. baumannii isolates. OprD mutations were revealed in the two P. aeruginosa isolates. A total of 11 out of the 13 carbapenem-resistant GNB were detected in inpatients, and the two remaining strains were isolated from outpatients. Molecular typing showed the presence of four sequence types (STs) among the OXA-48-producing K. pneumoniae isolates: ST101, ST147, ST163, and ST2017. ST533 was identified for the OXA-48 producing E. coli isolate. All of the A. baumannii and P. aeruginosa were assigned to the international clonal lineages ST2 and ST654, respectively.Conclusion: This study reports the first detection of the epidemic multidrug-resistant lineage, K. pneumoniae ST147 coproduced blaOXA-48 and ESBL genes in Algeria and represents the first description of OXA-48-producing E. coli ST533 and K. pneumoniae ST163 and ST2017. In addition, this study describes for the first time the emergence of OXA-48-producing E. coli and K. pneumoniae in the community in Algeria, leading to major problems for managing microbial infections. Keywords: Gram-negative bacilli, carbapenem resistance, epidemic lineages, Algeria

The current study aimed to determine the molecular mechanisms of carbapenem and colistin resistance among the clinical isolates of Klebsiella pneumoniae from hospitalized patients admitted to a university hospital in Eastern Algeria. In total, 124 non-duplicate isolates of K. pneumoniae were collected from September 2018 to April 2019. Bacterial identification was performed using MALDI-TOF MS. The presence of extended spectrum β-lactamase (ESBL) genes, carbapenemase genes, chromosomal mutation and mcr genes in colistin-resistant K. pneumoniae were evaluated by PCR. ESBLs represented a rate of 49.1% and harbored bla CTX-M , bla TEM and bla SHV genes. Concerning carbapenems, 12 strains (9.6%) were resistant to ertapenem (MIC: 1-32 μg/mL), of which one strain (0.8%) was also resistant to imipenem (MIC: 32 μg/mL). Among these strains, nine (75%) harbored bla OXA-48 gene. Seven strains (5.6%) expressed resistance to colistin (MIC: 2-32 μg/mL), of which two harbored mcr-8 and mgrB genes simultaneously. The existence of a double resistance to colistin in the same strain is new in Algeria, and this could raise concerns about the increase in levels of resistance to this antibiotic (MIC: 32 μg/mL). The mgrB gene alone was observed in five isolates (71.4%), including two strains harboring bla OXA-48 . This is the first report revealing the presence of K. pneumoniae strains carrying the bla OXA-48 gene as well as a mutation in the mgrB gene. Large-scale surveillance and effective infection control measures are also urgently needed to prevent the outbreak of various carbapenem- and colistin-resistant isolates.

Background: The Grand Magal of Touba (GMT) associates with risks of infection, but no study on the circulation of resistant bacteria has yet been conducted., Materials and Methods: qPCR was performed on rectal samples from GMT pilgrims between 2018 and 2021, before and after their participation in the gathering. Rectal samples from between 2018 and 2020 were also cultured on specific media, and antibiotic susceptibility testing was performed., Results: Forty-one of the 296 (13.8%) pilgrims had at least one gastrointestinal symptom and 91/290 (31.4%) acquired pathogenic bacteria, mostly Escherichia coli. A total of 54.7% of pilgrims reported washing their hands more frequently than usual and 89.2% used soap. One hundred and five (36.2%) acquired at least one resistance gene, notably CTX-M A (21.0%), SHV (16.5%) and TEM (8.2%). The strains isolated by culture were mostly E. coli. These bacteria were found to be sensitive to carbapenems and resistant to amoxicillin and amoxicillin-clavulanic acid. The acquisition of enteroaggregative E. coli was independently associated with CTX-M A and TEM acquisition., Conclusion: Pilgrims presented a risk for acquisition of CTX-M A after the GMT. Surveillance of the prevalence of resistant bacteria and the occurrence of associated clinical infections among pilgrims are necessary in the future., 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 © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: The colonization of patients by carbapenemase-producing Enterobacterales (CPE) has been associated with heightened mortality, especially in vulnerable individuals within intensive care units (ICUs). Our study aimed to comprehensively assess CPE prevalence among ICU patients across the Mediterranean region pre-COVID-19, conducting a multicenter prevalence study in the first quarter of 2019., Methods: We collected clinical data and rectal or fecal samples from 256 ICU patients for CPE testing. Additionally, we performed whole-genome sequencing on 40 representative CPE strains to document their molecular characteristics., Results: Among the 256 patients, CPE was detected in 73 samples (28.5%), with prevalence varying from 3.3 to 69.0% across participating centers. We observed 13 colistin-resistant CPE strains, affecting three ICUs. Genetic analysis revealed highly diverse E. coli and K. pneumoniae strains, predominantly from international high-risk clones. Notably, bla OXA-48 and bla NDM-1 were the most prevalent carbapenemase genes. Molecular typing uncovered potential patient clusters in six centers. Significantly, longer hospital stays were associated with increased CPE carriage ( p  < 0.001). Nine centers across Morocco, Tunisia, Egypt, and Lebanon voluntarily participated., Discussion: Our study provides CPE prevalence in Mediterranean ICUs and reaffirms established CPE presence in this setting but also provides updates on the molecular diversity of CPE strains. These findings highlight the imperative of reinforcing infection control measures in the participating ICUs to curtail escalated mortality rates, and of strictly applying isolation measures around patients originating from the Mediterranean region when transferred to other healthcare institutions., 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 © 2024 Dos Santos, Diene, Benouda, Zerouali, Ghaith, El-Mahdy, El Tayeb, Boutiba, Hammami, Chrabieh, Daoud, Mereghetti, Francois, Van Der Mee-Marquet and Mediterranean infection control group.)

Introduction: The rapid evolution of the antibacterial resistance problem worldwide, including the Mediterranean countries, constitutes a real threat to public health. This study aims to characterize carbapenemase encoding genes among Gram-negative bacteria collected from some Tunisian hospitals., Methodology: Twenty-two clinical carbapenem-resistant Gram-negative bacteria were recovered, and identified by the matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method. Antibiotic resistance was tested by disk diffusion method on Muller-Hinton Agar. The minimum inhibitory concentration (MIC) for imipenem was revealed by the E-test method. Carbapenemase encoding genes were screened by polymerase chain reaction (PCR). Genetic relatedness was performed by the pulsed field gel electrophoresis (PFGE) method., Results: Our isolates, identified as K. pneumoniae (n = 7), P. mirabilis (n = 1), A. baumannii (n = 13), and P. aeruginosa (n = 1), presented high MIC values for imipenem. Enterobacerales were resistant to carbapenems due to OXA-48 production. Only, four K. pneumoniae harbored the blaNDM-1 gene. VIM-2 production was detected in P. aeruginosa. However, OXA-23 production was observed in A. baumannii isolates, one of which co-produced the KPC-2 enzyme that was identified for the first time in Tunisia in this species. A high genetic diversity was demonstrated by pulsed-field gel electrophoresis in K. pneumoniae and A. baumannii after XbaI and ApaI digestion respectively., Conclusions: Our findings highlight the spread of various unrelated clones of carbapenemase-producers in some Tunisian hospitals as well as the spread of several carbapenemase types., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2023 Dhouha Miniaoui, Olfa Dziri, Yomna Ben Lamine, Allaaeddin A El Salabi, Elham O Omar, Khouloud Slimene, Raoudha Dziri, Sophia Bouhalila-Besbes, Linda Hadjadj, Aymen Mabrouk, Ahmed I Elbousify, Seydina M Diene, Jean-Marc Rolain, Chedly Chouchani.)

Background: To determine the prevalence of antimicrobial-resistant genes in carbapenem-resistant Escherichia coli (CRECO). Methods: A total of 290 carbapenem-resistant bacteria were collected from tertiary care hospitals in Lahore (Pakistan). These isolates were confirmed by VITEK 2 and matrix-assisted laser desorption/ionization time of flight. The minimum inhibitory concentration was performed by VITEK 2. Sequence typing, resistant gene identification, DNA hybridization and replicate typing were also performed. Results: 33 out of 290 (11.3%) were CRECO and carried bla NDM ; 69, 18 and 12% were NDM-1, NDM-5 and NDM-7, respectively, with 100% resistance to β-lactams and β-lactam inhibitors. ST405 and ST468 were mostly identified. NDM-ECO carried approximately 50-450 kb of plasmids and 16 (55%) were associated with IncA/C. Conclusion: NDM-1-producing E. coli are highly prevalent in clinical settings.

Antimicrobial resistance, particularly resistance to carbapenems, has become one of the major threats to public health. Seventy-two isolates were collected from patients and hospital environment of Ibn Sina Hospital, Sirte, Libya. Antibiotic susceptibility tests, using the disc diffusion method and E-Test strips, were performed to select carbapenem-resistant strains. The colistin (CT) resistance was also tested by determining the minimum inhibitory concentration (MIC). RT-PCR was conducted to identify the presence of carbapenemase encoding genes and plasmid-mediated mcr CT resistance genes. Standard PCR was performed for positive RT-PCR and the chromosome-mediated CT resistance genes ( mgrB, pmrA, pmrB, phoP, phoQ ). Gram-negative bacteria showed a low susceptibility to carbapenems. Molecular investigations indicated that the metallo-β-lactamase New Delhi metallo-beta-lactamases-1 was the most prevalent ( n  = 13), followed by Verona integron-encoded metallo-beta-lactamase (VIM) enzyme (VIM-2 [ n  = 6], VIM-1 [ n  = 1], and VIM-4 [ n  = 1]) that mainly detected among Pseudomonas spp . The oxacillinase enzyme OXA-23 was detected among six Acinetobacter baumannii , and OXA-48 was detected among one Citrobacter freundii and three Klebsiella pneumoniae, in which one coharbored the Klebsiella pneumoniae carbapenemase enzyme and showed resistance to CT (MIC = 64 μg/mL) by modification in pmrB genes. In this study, we report for the first time the emergence of Pseudomonas aeruginosa carrying the bla NDM-1 gene and belonging to sequence type773 in Libya. Our study reported also for the first time CT resistance by mutation in the pmrB gene among Enterobacteriaceae isolates in Libya.

Antibiotic resistance is a public health problem. The emergence of carbapenemase-producing Enterobacterales (CPE) infections is a concern, particularly in Senegal. (1) Methods: Between January 2019 and July 2022, 240 isolates of enterobacteria resistant to third-generation cephalosporins and imipenem from biological samples from Fann Hospital (Dakar) and Hôpital Paix (Ziguinchor) were selected. The isolates were identified by MALDI-TOF mass spectrometry, and susceptibility tests were performed by the disk diffusion method. Antibiotic-resistance genes for class A beta-lactamases, carbapenemases, and plasmid resistance to colistin resistance ( mcr-1-8 ) were screened by RT-PCR. (2) Results: The 240 enterobacteria were composed of: Escherichia coli (60.83%), Klebsiella pneumoniae (21.67%), Enterobacter cloacae (13.75%), Citrobacter freundii (2.08%), Serratia marcescens (0.83%), Klebsiella aerogenes (0.42%), and Proteus mirabilis (0.42%). Class A beta-lactamase genes were found in 229 isolates (70.41% bla TEM , 37.5% bla SHV , 83.75% bla CTX-A , and 0.42% bla CTX-B ). The carbapenemase genes bla OXA-48 and bla NDM were found in 25 isolates, including 14 isolates with bla OXA-48 , 13 isolates with bla NDM , and 2 isolates with both genes simultaneously. The mcr-8 gene was found in one isolate of E. cloacae . (3) Conclusions: The epidemiology of antibiotic-resistance genes in enterobacteria in Senegal shows the emergence of CPEs. This phenomenon is worrying, and rigorous surveillance is necessary to avoid further spread.

Introduction : The antimicrobial resistance (AMR) of bacteria is increasing rapidly against all classes of antibiotics, with the increasing detection of carbapenem-resistant isolates. However, while growing prevalence has been reported around the world, data on the prevalence of carbapenem resistance in developing countries are fairly limited. In this study, we investigated and determined the resistance rate to carbapenems among multidrug-resistant Gram-negative bacteria (MDR-GNB) isolated in Djibouti and characterized their resistance mechanisms. Results : Of the 256 isolates, 235 (91.8%) were identified as Gram-negative bacteria (GNB). Of these GNBs, 225 (95.7%) isolates exhibited a multidrug resistance phenotype, and 20 (8.5%) isolates were resistant to carbapenems, including 13 Escherichia coli, 4 Acinetobacter baumannii , 2 Klebsiella pneumoniae and 1 Proteus mirabilis . The most predominant GNB in this hospital setting were E. coli and K. pneumoniae species. Carbapenemase genes such as bla OXA-48 and bla NDM-5 were identified, respectively, in six and four E. coli isolates, whereas the carbapenemase bla NDM-1 was identified in three E. coli , two K. pneumoniae , one P. mirabilis and one A. baumannii . Moreover, three A. baumannii isolates co-hosted bla OXA-23 and bla NDM-1 . Materials and Methods : A total of 256 clinical strains collected between 2019 and 2020 were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). Antibiotic susceptibility testing was performed using disk diffusion and E-test methods. Real-time polymerase chain reaction (RT-PCR), standard PCR and sequencing were used to investigate genes encoding for extended-spectrum-β-lactamases, carbapenemases and colistin resistance genes. Conclusions : We report, for the first time, the presence of MDR-GNB clinical isolates and the emergence of carbapenem-resistant isolates in Djibouti. In addition to performing antimicrobial susceptibility testing, we recommend phenotypic and molecular screening to track the spread of carbapenemase genes among clinical GNB isolates.

β-lactamase enzymes have generated significant interest due to their ability to confer resistance to the most commonly used family of antibiotics in human medicine. Among these enzymes, the class B β-lactamases are members of a superfamily of metallo-β-lactamase (MβL) fold proteins which are characterised by conserved motifs (i.e., HxHxDH) and are not only limited to bacteria. Indeed, as the result of several barriers, including low sequence similarity, default protein annotation, or untested enzymatic activity, MβL fold proteins have long been unexplored in other organisms. However, thanks to search approaches which are more sensitive compared to classical Blast analysis, such as the use of common ancestors to identify distant homologous sequences, we are now able to highlight their presence in different organisms including Bacteria, Archaea, Nanoarchaeota, Asgard, Humans, Giant viruses, and Candidate Phyla Radiation (CPR). These MβL fold proteins are multifunctional enzymes with diverse enzymatic or non-enzymatic activities of which, at least thirteen activities have been reported such as β-lactamase, ribonuclease, nuclease, glyoxalase, lactonase, phytase, ascorbic acid degradation, anti-cancer drug degradation, or membrane transport. In this review, we (i) discuss the existence of MβL fold enzymes in the different domains of life, (ii) present more suitable approaches to better investigating their homologous sequences in unsuspected sources, and (iii) report described MβL fold enzymes with demonstrated enzymatic or non-enzymatic activities.

Heteroresistance to colistin can be defined as the presence of resistant subpopulations in an isolate that is susceptible to this antibiotic. Colistin resistance in Gram-negative bacteria is more frequently related to chromosomal mutations and insertions. This work aimed to study heteroresistance in nine clinical isolates of Klebsiella pneumoniae producing OXA-48 and to describe genomic changes in mutants with acquired resistance in vitro. Antimicrobial susceptibility was determined by broth microdilution (BMD) and heteroresistance by population analysis profiling (PAP). The proteins related to colistin resistance were analyzed for the presence of mutations. Additionally, PCR of the mgr B gene was performed to identify the presence of insertions. In the nine parental isolates, the PAP method showed colistin heteroresistance of colonies growing on plates with concentrations of up to 64 mg/L, corresponding to stable mutant subpopulations. The MICs of some mutants from the PAP plate containing 4×MIC of colistin had absolute values of ≤2 mg/L that were higher than the parental MICs and were defined as persistent variants. PCR of the mgr B gene identified an insertion sequence that inactivated the gene in 21 mutants. Other substitutions in the investigated mutants were found in PhoP, PhoQ, PmrB, PmrC, CrrA and CrrB proteins. Colistin heteroresistance in K. pneumoniae isolates was attributed mainly to insertions in the mgr B gene and point mutations in colistin resistance proteins. The results of this study will improve understanding regarding the mechanisms of colistin resistance in mutants of K. pneumoniae producing OXA-48.

Competing Interests: 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.

Background: Elizabethkingia anophelis is an emerging Gram-negative nonlactose fermenter in the health care setting, where it causes life-threatening infections in immunocompromised patients. We aimed to characterize the molecular mechanisms of antimicrobial resistance and evaluate the utility of contemporary antibiotics with the intent to offer targeted therapy against an uncommonly encountered pathogen., Methods: Whole-genome sequencing (WGS) was conducted to accurately identify isolate species and elucidate the determinants of β-lactam resistance. Antimicrobial susceptibility testing was performed using broth microdilution and disk diffusion assays. To assess the functional contribution of the major metallo-β-lactamase (MBL) encoding genes to the resistance profile, bla BlaB was cloned into pBCSK(-) phagemid vector and transformed into Escherichia coli DH10B., Results: WGS identified the organism as E. anophelis . MBL genes bla BlaB-1 and bla GOB-26 were identified, in addition to bla CME-2 , which encodes for an extended-spectrum β-lactamase (ESBL). Plasmids were not detected. The isolate was nonsusceptible to all commonly available β-lactams, carbapenems, newer β-lactam β-lactamase inhibitor combinations, and to the combination of aztreonam (ATM) with ceftazidime-avibactam (CAZ-AVI). Susceptibility to the novel siderophore cephalosporin cefiderocol was determined. A BlaB-1 transformant E. coli DH10B isolate was obtained and demonstrated increased minimum inhibitory concentrations to cephalosporins, carbapenems, and CAZ-AVI, but not ATM., Conclusions: Using WGS, we accurately identified and characterized an extensively drug-resistant E. anophelis in an immunocompromised patient. Rapid evaluation of the genetic background can guide accurate susceptibility testing to better inform antimicrobial therapy selection., Competing Interests: Potential conflicts of interest. All authors: no reported conflicts., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2023.)

The colibactin island ( pks ) of Escherichia coli formed by 19 genes (55-Kb), encodes non-ribosomal peptide (NRP) and polyketide (PK) synthases, which allow the synthesis of colibactin, a suspected hybrid PK-NRP compound that causes damage to DNA in eukaryotic cells. The clbP , an unusual essential gene, is found in the operon structure with the clbS gene in the pks -encoded machinery. Interestingly, the clbP gene has been annotated as a β-lactamase but no previous study has reported its β-lactamase characteristics. In this study, we (i) investigated the β-lactamase properties of the clbP gene in silico by analysing its phylogenetic relationship with bacterial β-lactamase and peptidase enzymes, (ii) compared its three-dimensional (3D) protein structure with those of bacterial β-lactamase proteins using the Phyr2 database and PyMOL software, and (iii) evaluated in vitro its putative enzymatic activities, including β-lactamase, nuclease, and ribonuclease using protein expression and purification from an E. coli BL21 strain. In this study, we reveal a structural configuration of toxin/antitoxin systems in this island. Thus, similar to the toxin/antitoxin systems, the role of the clbP gene within the pks -island gene group appears as an antitoxin, insofar as it is responsible for the activation of the toxin, which is colibactin. In silico, our analyses revealed that ClbP belonged to the superfamily of β-lactamase, class C. Furthermore, in vitro we were unable to demonstrate its β-lactamase activity, likely due to the fact that the clbP gene requires co-expression with other genes, such as the genes present in the pks -island (19 genes). More research is needed to better understand its actions, particularly with regards to antibiotics, and to discover whether it has any additional functions due to the importance of this gene and its toxicity.

Colistin is frequently used as a growth factor or treatment against infectious bacterial diseases in animals. The Veterinary Division of the European Medicines Agency (EMA) restricted colistin use as a second-line treatment to reduce colistin resistance. In 2020, 282 faecal samples were collected from chickens, cattle, sheep, goats, and pigs in the south of France. In order to track the emergence of mobilized colistin resistant (mcr) genes in pigs, 111 samples were re-collected in 2021 and included pig faeces, food, and water from the same location. All samples were cultured in a selective Lucie Bardet Jean-Marc Rolain (LBJMR) medium and colonies were identified using MALDI-TOF mass spectrometry and then antibiotic susceptibility tests were performed. PCR and Sanger sequencing were performed to screen for the presence of mcr genes. The selective culture revealed the presence of 397 bacteria corresponding to 35 different bacterial species including Gram-negative and Gram-positive. Pigs had the highest prevalence of colistin-resistant bacteria with an abundance of intrinsically colistin-resistant bacteria and from these samples one strain harbouring both mcr-1 and mcr-3 has been isolated. The second collection allowed us to identify 304 bacteria and revealed the spread of mcr-1 and mcr-3 in pigs. In the other samples, naturally, colistin-resistant bacteria were more frequent, nevertheless the mcr-1 variant was the most abundant gene found in chicken, sheep, and goat samples and one cattle sample was positive for the mcr-3 gene. Animals are potential reservoir of colistin-resistant bacteria which varies from one animal to another. Interventions and alternative options are required to reduce the emergence of colistin resistance and to avoid zoonotic transmissions., (© 2022. The Author(s).)

Background: Whole-genome sequencing is one of the best ways to investigate resistance mechanisms of clinical isolates as well as to detect and identify circulating multi-drug-resistant (MDR) clones or sub-clones in a given hospital setting., Methods: Here, we sequenced 37 isolates of Acinetobacter baumannii , 10 Klebsiella pneumoniae , and 5 Pseudomonas aeruginosa collected from the biobank of the hospital setting of the King Fahad Medical City. Complete phenotypic analyses were performed, including MALDI-TOF identification and antibiotic susceptibility testing. After the genome assembly of raw data, exhaustive genomic analysis was conducted including full resistome determination, genomic SNP (gSNP) analysis, and comparative genomics., Results: Almost all isolates were highly resistant to all tested antibiotics, including carbapenems and colistin. Resistome analysis revealed many antibiotic resistance genes, including those with resistance to β-lactams, aminoglycosides, macrolides, tetracyclines, sulfamids, quinolones, and phenicols. In A. baumannii isolates, the endemic carbapenemase bla OXA-23 gene was detected in 36 of the 37 isolates. Non-synonymous mutations in pmrB were detected in almost all of the isolates and likely mediated colistin resistance. Interestingly, while classical analyses, such as MLST, revealed the predominance of an ST2 clone in A. baumannii isolates, the genomic analysis revealed the presence of five circulating sub-clones and identified several isolate transmissions between patients. In the 10 K. pneumoniae isolates, several resistance genes were identified, and the observed carbapenem resistance was likely mediated by overexpression of the detected extended-spectrum-β-lactamase (ESBL) genes associated with low membrane permeability as few carbapenemase genes were detected with just bla OXA-48 in three isolates. Colistin resistance was mediated either by non-synonymous mutations in the MgrB regulator, PmrA, PmrB, and PhoQ proteins or the presence of the MCR-1 protein. Here, gSNP analysis also revealed the existence of bacterial clones and cases of isolate transmissions between patients. The five analyzed P. aeruginosa isolates were highly resistant to all tested antibiotics, including carbapenems mediated by loss or truncated OprD porin, and colistin resistance was associated with mutations in the genes encoding the PmrA, PmrB, or PhoQ proteins., Conclusion: We demonstrate here the usefulness of whole-genome sequencing to exhaustively investigate the dissemination of MDR isolates at the sub-clone level. Thus, we suggest implementing such an approach to monitor the emergence and spread of new clones or sub-clones, which classical molecular analyses cannot detect. Moreover, we recommend increasing the surveillance of the endemic and problematic colistin resistance mcr -1 gene to avoid extensive dissemination.

A prospective 3-month study carried out in 267 ICUs revealed an S. aureus nosocomial bacteremia in one admitted patient out of 110 in adult and pediatric sectors, and in one out of 230 newborns; 242 S. aureus bacteremias occurred during the study, including 7.9% MRSA-bacteremias. In one ICU out of ten, the molecular characteristics, antimicrobial susceptibility profiles and biofilm production of the strains responsible for S. aureus bacteremia were studied. Of the 53 strains studied, 9.4% were MRSA and 52.8% were resistant to erythromycin. MLST showed the predominance of CC398 (37.7% of the strains) followed by CC8 (17.0%), CC45 (13.2%) and CC30 (9.4%). The lukF/S genes were absent from our isolates and tst-1 was found in 9.4% of the strains. Under static conditions and without exposure to glucose, biofilm production was rare (9.4% of the strains, without any CC398). The percentage increased to 62.3% for strains grown in broth supplemented with 1% glucose (including 7 out of 9 CC8 and 17 out of the 20 CC398). Further study of the CC398, including whole genome sequencing, revealed (1) highly frequent patient death within seven days after CC398 bacteremia diagnosis (47.4%), (2) 95.0% of the strains producing biofilm when exposed to sub-inhibitory concentrations of cloxacillin, (3) a stronger biofilm production following exposure to cloxacillin than that observed in broth supplemented with glucose only (p < 0.001), (4) a high minimum biofilm eradication concentration of cloxacillin (128 mg/L) indicating a low cloxacillin susceptibility of biofilm-growing CC398, (5) 95.0% of the strains carrying a ϕSa-3 like prophage and its particular evasion cluster (i.e., yielding chp and scin genes), and (6) 30.0% of the strains carrying a ϕMR11-like prophage and yielding a higher ability to produce biofilm. Our results provide evidence that active surveillance is required to avoid spreading of this virulent staphylococcal clone.

Staphylococcus aureus causes a wide range of life-threatening infections. In this study, we determined its prevalence in the hospital environment and investigated nasal carriage among healthcare workers and patients admitted to a hospital in western Algeria. A total of 550 specimens were collected. An antibiogram was performed and the genes encoding resistance to methicillin, inducible clindamycin and toxins were sought among the 92 S. aureus isolates. The spread of clones with a methicillin- and/or clindamycin-resistance phenotype between these ecosystems was studied using genomic analysis. A prevalence of 27%, 30% and 13% of S. aureus (including 2.7%, 5% and 1.25% of MRSA) in patients, healthcare workers and the hospital environment were observed, respectively. The presence of the mecA , erm , pvl and tsst-1 genes was detected in 10.9%, 17.4%, 7.6% and 18.5% of samples, respectively. Sequencing allowed us to identify seven sequence types, including three MRSA-IV-ST6, two MRSA-IV-ST80-PVL+, two MRSA-IV-ST22-TSST-1, two MRSA-V-ST5, and one MRSA-IV-ST398, as well as many virulence genes. Here, we reported that both the hospital environment and nasal carriage may be reservoirs contributing to the spread of the same pathogenic clone persisting over time. The circulation of different pathogenic clones of MRSA, MSSA, and iMLSB, as well as the emergence of at-risk ST398 clones should be monitored.

Background : Pets, especially cats and dogs, represent a great potential for zoonotic transmission, leading to major health problems. The purpose of this systematic review was to present the latest developments concerning colistin resistance through mcr genes in pets. The current study also highlights the health risks of the transmission of colistin resistance between pets and humans. Methods : We conducted a systematic review on mcr -positive bacteria in pets and studies reporting their zoonotic transmission to humans. Bibliographic research queries were performed on the following databases: Google Scholar, PubMed, Scopus, Microsoft Academic, and Web of Science. Articles of interest were selected using the PRISMA guideline principles. Results : The analyzed articles from the investigated databases described the presence of mcr gene variants in pets including mcr-1 , mcr-2 , mcr-3 , mcr-4 , mcr-5 , mcr-8 , mcr-9, and mcr-10 . Among these articles, four studies reported potential zoonotic transmission of mcr genes between pets and humans. The epidemiological analysis revealed that dogs and cats can be colonized by mcr genes that are beginning to spread in different countries worldwide. Overall, reported articles on this subject highlight the high risk of zoonotic transmission of colistin resistance genes between pets and their owners. Conclusions : This review demonstrated the spread of mcr genes in pets and their transmission to humans, indicating the need for further measures to control this significant threat to public health. Therefore, we suggest here some strategies against this threat such as avoiding zoonotic transmission.

A multidrug-resistant (MDR) Campylobacter coli (C. coli) strain was isolated from a 2-month-old newborn who suffered from severe diarrhea in Lebanon. Here, whole-genome sequencing (WGS) analysis was deployed to determine the genetic basis of antimicrobial resistance and virulence in the C. coli isolate and to identify its epidemiological background (sequence type). The identity of the isolate was confirmed using API® Campy, MALDI-TOF, and 16S rRNA gene sequencing analysis. The antimicrobial susceptibility phenotype was determined using the disk diffusion assay. Our analysis showed that resistance to macrolide and quinolone was potentially associated with the presence of multiple point mutations in antibiotic targets on the chromosomal DNA. Furthermore, tetracycline and aminoglycoside resistance were encoded by genes on a pTet plasmid. The bla OXA-61 , which is associated with beta-lactam resistance, was also detected in the C. coli genome. A set of 30 genes associated with the virulence in C. coli was detected using WGS analysis. MLST analysis classified the isolate as belonging to a new sequence type (ST-9588), a member of ST-828 complex which is mainly associated with humans and chickens. Taking together, this study provides the first WGS analysis of Campylobacter isolated from Lebanon. The detection of a variety of AMR and virulence determinants strongly emphasizes the need for studying the burden of Campylobacter in Lebanon and the Middle East and North Africa (MENA) region, where information on campylobacteriosis is scant., (© 2021. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Background: Pets are the closest animals to humans with a considerable risk of zoonotic transmission. This study aimed to screen colistin-resistant bacteria from stools of dogs and cats from Marseille, France. Screening of mcr genes in pets has never been reported in France., Methods: Fecal samples ( n = 157) were cultivated on the selective Lucie-Bardet Jean-Marc-Rolain medium (LBJMR). Bacteria were identified using Microflex LS MALDI-TOF. The antibiotic resistance phenotype was investigated for several antibiotics (β-lactams, aminoside, cephalosporine, tetracycline, and sulfonamide). PCR techniques were performed to detect mcr genes., Results: A total of 218 bacteria were identified. For cats, intrinsically colistin-resistant bacteria were significantly higher than mcr-1 gene carriers ( n = 4). Dogs had more bacteria with the mcr-1 gene ( n = 10). Furthermore, cats had a high prevalence of Gram-positive bacteria (GPB), whereas dogs had GNB equal to GPB. The diversity of identified bacteria was due to the constitution of the pets' microorganisms. Even though colistin use is monitored in France, pets harbor various colistin-resistant bacteria. Additionally, in this geographical area, bacteria bearing mcr-1 gene from dogs and cats were detected for the first time., Conclusions: The current study opens a new perspective: the spread of colistin resistance is independent of colistin use. What are the most factors related to the emergence of colistin resistance? The surveillance of pets must be considered a priority to avoid the spread of mcr genes. It is important to know the contribution that pets make to the pool of multidrug-resistant mcr-1 -containing bacteria.

This study aimed to determine the incidence and the molecular mechanisms of carbapenem-resistant Enterobacteriaceae in patients from the Sétif University Hospital, Algeria. Nonduplicate clinical bacterial isolates recovered from patients attending the University Hospital of Sétif were collected between April and October 2018. Species identification was performed by MALDI-TOF/MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) method. The susceptibility of the isolates to carbapenems was determined using the disc diffusion method. The carbapenem resistant isolates were screened for the presence of common carbapenemase genes ( bla KPC , bla OXA-48 , bla VIM , bla IMP , and bla NDM ) and extended-spectrum β-lactamase ( bla CTX , bla TEM , and bla SHV ) using PCR and sequencing technique. A total of 123 nonrepetitive Enterobacteriaceae isolates were obtained. Klebsiella pneumoniae ( n  = 52/42.28%), Escherichia coli ( n  = 24/19.51%), and Enterobacter cloacae ( n  = 19/15.45%) were the most prevalent species. The Carba-NP test showed that 6 out of 123 isolates carried carbapenemase enzymes. OXA-48 was found in five isolates (four K. pneumoniae and one E. coli ) and NDM-5 in one E. cloacae isolate. We reported for the first time in Algeria the presence of NDM-5 carbapenemase enzyme in a clinical E. cloacae isolate.

Objectives: Colistin is considered a last-resort antibiotic against carbapenem-resistant isolates. Currently, this antibiotic is facing the emergence of mobilised colistin resistance (mcr) genes, which confer colistin resistance. This study conducted genomic characterisation of an atypical multidrug-resistant Escherichia coli harbouring two mcr genes in France. Samples collected from a pig farm in Avignon (Vaucluse department) were subjected to molecular screening targeting mcr variants., Methods: Samples were cultured on selective Lucie-Bardet-Jean-Marc-Rolain medium. Growing bacteria were identified using MALDI-TOF, followed by antibiotic susceptibility testing. Whole-genome sequencing and bioinformatic genome analysis were performed., Results: Selective culture of stools revealed the presence of an E. coli strain named Q4552 harbouring mcr-1.1 and mcr-3.5 genes, which is also resistant to 14 antibiotics. Genome sequencing and assembly yielded a complete and circular chromosome and eight different plasmids. Sequence analysis demonstrated an integration of a mobile genetic element carrying mcr-1.1 in the chromosome, whereas mcr-3.5 was in the plasmid and its resistome was composed of 22 resistance genes. The Q4552 strain was identified as an ST-843 clone that belonged to the clonal complex Cplx-568 and is the only ST type of this cplx-568 that has been isolated from animals, humans, and the environment., Conclusion: We report the first co-occurrence of mcr-1 and mcr-3 genes in France from a pathogenic E. coli isolated from a pig. Because this clone (ST-843) has been reported in zoonotic transmissions, programs to monitor the bacterium are urgently required to avoid its spread and zoonotic transmission to humans., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Empedobacter falsenii is an emerging opportunistic pathogen that has been occasionally implicated in various human infections. In this study, we described the genomic features of a multidrug resistant E. falsenii Q1655 obtained from a patient attending a public hospital in Sokoto, northwest Nigeria. The isolate, E. falsenii Q1655, was isolated from the stool sample of a patient in Sokoto, Nigeria. The identity of the isolate was confirmed by MALDITOF-MS. The disc diffusion test and modified Carba-NP test were used for phenotypic antibiotic susceptibility test and carbapenemase enzyme production test, respectively. The whole genome of the strain was sequenced using the Illumina MiSeq technique. Resistome analysis was done by annotation of the WGS against the ARG-ANNOT database. The isolate was resistant to all β-lactam antibiotics with the exception of cefepime. The MICs of imipenem and ertapenem as determined by E-test were 12 μg/ml and 2 μg/ml, respectively. Modified Carba NP test showed that the strain was carbapenemase producing. Resistome analysis revealed the presence of a novel metallo-β-lactamase, a chromosomal bla EBR-4 , which exhibited 94.92% and 97.02% nucleotide and protein sequence identities respectively with bla EBR-3 gene of E. falsenii 174,820. Seven and eight amino-acid substitutions were observed with the bla EBR-1 and bla EBR-2 , respectively. We reported the first isolation and genomic description of an extensively drug resistant isolate of Empedobacter falsenii in Nigeria. This report broadens our knowledge of carbapenem resistance in E. falsenii and it will serve as a useful guide in the development of antibiotic use policy., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Wautersiella falsenii is a rarely non-fermenting Gram-negative bacterium and belongs to the Flavobacteriaceae family. This nosocomial pathogen can cause several human infections, especially among immunocompromised patients. Here, we describe the whole genome sequence of a clinical W. falsenii strain isolated from a urine sample of a 35-year-old woman with a urinary tract infection in Tunisia. We investigated its phenotype and genotype. After bacterial identification by the MALDI-TOF method, the whole-genome sequencing of this strain was performed. This isolate was not susceptible to various antibiotics, including β-lactams, aminoglycosides, and quinolones. However, it remains susceptible to imipenem (MIC = 0.25 mg/l), ertapenem (MIC = 0.75 mg/l), and meropenem (MIC = 0.19 mg/l). Interestingly, the E-TEST ® (MP/MPI) showed a reduced MIC of meropenem +/- EDTA (0.064 μg/ml). Besides, the color change from yellow to red in the β CARBA test only after 24 hours of incubation can be interpreted in two ways. On the one hand, as a likely low expression of the gene encoding metallo-β-lactamase. On the other hand, and more likely, it may be a false-positive result because, according to the test manufacturer's recommendations, the test should be read after 30 minutes. Perhaps, therefore, this gene is not expressed in the tested strain. Moreover, the whole-genome sequence analysis demonstrated the presence of a novel chromosomally located subclass B1 metallo-β-lactamase EBR-like enzyme, sharing 94.92% amino acid identity with a previously described carbapenemase produced by Empedobacter brevis , EBR-1. The results also showed the detection of other antibiotic resistance genes and the absence of plasmids. So far, this study is the first report on the detection of W. falsenii in Tunisia. These findings prove that W. falsenii could be a potential reservoir of antibiotic resistance genes, e.g., β-lactamases. Collaborative efforts and effective hygiene measures should be established to prevent the emergence of this species in our health care settings., (© 2022 Raouaa Maaroufi et al., published by Sciendo.)

(1) Background: The purpose of this study was to determine the prevalence of clostridia strains in a hospital environment in Algeria and to evaluate their antimicrobial susceptibility to antibiotics and biocides. (2) Methods: Five hundred surface samples were collected from surfaces in the intensive care unit and surgical wards in the University Hospital of Tlemcen, Algeria. Bacterial identification was carried out using MALDI-TOF-MS, and then the minimum inhibitory concentrations (MICs) of various antimicrobial agents were determined by the E-test method. P. sordellii toxins were searched by enzymatic and PCR assays. Seven products intended for daily disinfection in the hospitals were tested against Clostridium spp. spore collections. (3) Results: Among 100 isolates, 90 P. sordellii were identified, and all strains were devoid of lethal and hemorrhagic toxin genes. Beta-lactam, linezolid, vancomycin, tigecycline, rifampicin, and chloramphenicol all proved effective against isolated strains. Among all strains tested, the spores of P. sordellii exhibited remarkable resistance to the tested biocides compared to other Clostridium species. The (chlorine-based 0.6%, 30 min), (glutaraldehyde solution 2.5%, 30 min), and (hydrogen peroxide/peracetic acid 3%, 15 min) products achieved the required reduction in spores. (4) Conclusions: Our hospital's current cleaning and disinfection methods need to be optimized to effectively remove spores from caregivers' hands, equipment, and surfaces.

Two novel bacteria species designated Marseille-Q1000 T and Marseille-Q0999 T were isolated from urine samples of patients in Sokoto, Northwest-Nigeria. They were Gram-positive bacteria and belong to two different genera, Bhargavaea and Dietzia. The genome size and G + C content of Marseille-Q1000 T and Marseille-Q0999 T were 3.07 and 3.51 Mbp with 53.8 and 71.0 mol% G + C content, respectively. The strains exhibited unique phenotypic and genomic features that are substantially different from previously known bacterial species with standing in nomenclature. On the basis of the phenotypic, phylogenetic and genomic characteristics, strains Marseille-Q0999 T (= CSURQ0999 = DSM 112394) and Marseille-Q1000 T (= CSURQ1000 = DSM 112384) were proposed as the type strains of Bhargavaea massiliensis sp. nov., and Dietzia massiliensis sp. nov., respectively., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The Shigella genus includes serious foodborne disease etiologic agents, with 4 species and 54 serotypes. Identification at species and serotype levels is a crucial task in microbiological laboratories. Nevertheless, the genetic similarity between Shigella spp. and Escherichia coli challenges the correct identification and serotyping of Shigella spp., with subsequent negative repercussions on surveillance, epidemiological investigations, and selection of appropriate treatments. For this purpose, multiple techniques have been developed historically ranging from phenotype-based methods and single or multilocus molecular techniques to whole-genome sequencing (WGS). To facilitate the selection of the most relevant method, we herein provide a global overview of historical and emerging identification and serotyping techniques with a particular focus on the WGS-based approaches. This review highlights the excellent discriminatory power of WGS to more accurately elucidate the epidemiology of Shigella spp., disclose novel promising genomic targets for surveillance methods, and validate previous well-established methods., (© 2021. Sociedade Brasileira de Microbiologia.)

In this paper, we investigate the epidemiology of infections-associated Staphylococcus aureus ( S. aureus ) from the Medical Intensive Care Unit (MICU) at University Hospital Center of Constantine (UHCC) in Algeria, with a special emphasis on methicillin-resistant strains (MRSA) revealed by cefoxitin disks (30 μg), then confirmed by penicillin-binding protein (PBP2a) agglutination and real-time polymerase chain reaction (RT-PCR) targeting mecA and mec C genes. Staphylococcal Cassette Chromosome mec ( SCCmec type), staphylococcal protein A ( spa -type), multilocus sequence type (MLST), Panton-Valentine Leucocidin (PVL), and toxic shock syndrome toxin-1 (TSST-1) were further investigated in all isolates, and whole genome sequencing was performed for a selected subset of three hospital-acquired MRSA (HA-MRSA) isolates. A measurement of 80% out of the 50 S. aureus isolates were identified as HA-MRSA harbouring the mecA gene, and 72.5% of them were multidrug resistant (MDR). Twelve STs, four different SCCmec cassettes, fourteen spa types, ten isolates Panton-Valentine Leukocidin (PVL)-positive, and three isolates TSST-1 were identified. Interestingly, there was a high prevalence (n = 29; 72.5%) of a worrisome emerging clone: the HA-MRSA ST239/241 SCCme c-III mercury with PVL negative, resistant to β-lactams, aminoglycosides, quinolones, and tetracyclines. Other clones of HA-MRSA isolates were also identified, including PVL-positive ST80 SCCmec -IV/ SCCmec -unknown (22.5%), ST34 SCCmec -V with TSST-1 positive (2.5%), and PVL-negative ST72 SCCmec -II (2.5%). Genome analysis enables us to describe the first detection of both PVL-negative HA-MRSA ST239/241 SCCmec-III mercury carrying ccrC , as well as SCCmec -V cassette, which dramatically changes the epidemiology of S. aureus infections in one of the hospitals in eastern Algeria.

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.

Strain Marseille-P3519T isolated from the fecal flora of a 25-year-old healthy French woman was a Gram-positive anaerobic bacterium, non-motile and non-spore forming. The 16S rRNA gene sequence of Marseille-P3519 showed 97.73% of sequence similarity with Limosilactobacillus reuteri DSM 20016, the closest species, phylogenetically. Furthermore, the average nucleotide identity of strain Marseille-3519 with its closest related species was 75.8% that was very below the recommended threshold (>95-96%). Its genome had 2 237 367 bp with 45.42 mol% of G + C content. Major fatty acids were C16:0 (50.8%), C18:1n9 (18.0%), C18:2n6 (9.8%) and C19:1n9 (8.9%). It was catalase negative and fermented glycerol, glucose, fructose, D-maltose, lactose and mannose. These findings support that strain Marseille-P3519 ( = CSURP3519 = CECT 30110) is a new member of the genus Limosilactobacillus for which the name Limosilactobacillus caccae sp. nov., is proposed., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Hospital environments constitute the main reservoir of multidrug-resistant bacteria. In this study we aimed to investigate the presence of Gram-negative bacteria in one Northwestern Tunisian hospital environment, and characterize the genes involved in bacterial resistance. A total of 152 environmental isolates were collected from various surfaces and isolated using MacConkey medium supplemented with cefotaxime or imipenem, with 81 fermenter bacteria (27 Escherichia coli, and 54 Enterobacter spp., including 46 Enterobacter cloacae ), and 71 non-fermenting bacteria (69 Pseudomonas spp., including 54 Pseudomonas aeruginosa, and 2 Stenotrophomonas maltophilia ) being identified by the MALDI-TOF-MS method. Antibiotic susceptibility testing was performed by disk diffusion method and E-Test was used to determine MICs for imipenem. Several genes implicated in beta-lactams resistance were characterized by PCR and sequencing. Carbapenem resistance was detected among 12 isolates; nine E. coli ( bla NDM-1 ( n = 8); bla NDM-1 + bla VIM-2 ( n = 1)) and three P. aeruginosa were carbapenem-resistant by loss of OprD porin. The whole-genome sequencing of P. aeruginosa 97H was determined using Illumina MiSeq sequencer, typed ST285, and harbored bla OXA-494 . Other genes were also detected, notably bla TEM ( n = 23), bla CTX-M-1 ( n = 10) and bla CTX-M-9 ( n = 6). These new epidemiological data imposed new surveillance strategies and strict hygiene rules to decrease the spread of multidrug-resistant bacteria in this area.

In this paper, we describe the first complete genome sequence of Providencia vermicola species, a clinical multidrug-resistant strain harboring the New Delhi Metallo-β-lactamase-1 (NDM-1) gene, isolated at the Kinshasa University Teaching Hospital, in Democratic Republic of the Congo. Whole genome sequencing of an imipenem-resistant clinical Gram-negative P. vermicola P8538 isolate was performed using MiSeq and Gridion, and then complete genome analysis, plasmid search, resistome analysis, and comparative genomics were performed. Genome assembly resulted in a circular chromosome sequence of 4,280,811-bp and 40.80% GC and a circular plasmid (pPV8538&#95;NDM-1) of 151,684-bp and 51.93%GC, which was identified in an Escherichia coli P8540 strain isolated in the same hospital. Interestingly, comparative genomic analysis revealed multiple sequences acquisition within the P. vermicola P8538 chromosome, including three complete prophages, a siderophore biosynthesis NRPS cluster, a Type VI secretion system (T6SS), a urease gene cluster, and a complete Type-I-F CRISPR-Cas3 system. Β-lactamase genes, including bla CMY-6 and bla NDM-1 , were found on the recombinant plasmid pPV8538&#95;NDM-1, in addition to other antibiotic resistance genes such as rmtC , aac6' - Ib3 , aacA4 , catA1 , sul1 , aac6' - Ib - cr , tetA , and tetB . Genome comparison with Providencia species revealed 82.95% of average nucleotide identity (ANI), with P. stuartii species exhibiting 90.79% of proteome similarity. We report the first complete genome of P. vermicola species and for the first time the presence of the bla NDM-1 gene in this species. This work highlights the need to improve surveillance and clinical practices in DR Congo in order to reduce or prevent the spread of such resistance.

The spread of vancomycin-resistant Enterococci (VRE) in Algerian hospital settings is poorly reported. Since the first report in 2006, few data have been available on the molecular mechanism of this resistance across the country. In this study, we investigate the frequency and antibiotic resistance mechanisms of Enterococci strains isolated from hospitalised patients in the Tlemcen university hospital. 191 Enterococcus spp. strains were collected from various clinical samples and were identified using MALDI-TOF-MS. The presence of van genes was investigated by standard PCR and sequencing. Results revealed that E. faecium and E. faecalis strains are the main pathogens identified in the study. Antibiotic susceptibility testing revealed that the resistance rate was high for the majority of antibiotic classes, including glycopeptides, and only linezolid was effective on all strains. Molecular analysis revealed that 52.2% of strains from intensive care unit (ICU) were positive for the vanA gene, including 44.44% E. faecium, 5.55% E. faecalis and 2.22% E. avium. 25.5% of these isolates co-harboured both the vanA and vanC genes, including E. gallinarum (n = 16) and E. faecium (n = 6). In surgical wards (SW) 29.70% of strains harboured the van genes, including 4.90% of E. faecalis harbouring the vanB gene, and of the rest of strains, (24.80%) harboured the vanC genes. Indeed, 9.90% E. gallinarum and 4.90% E. faecalis were positive for vanC1 and 9.90% of E. casseliflavus were positive for the vanC2/C3 gene. The glycopeptide resistance rate was higher among strains from the ICU and was mainly composed by E. faecium strains compared with surgical wards where resistant E. faecalis strains were predominant., (Copyright © 2021 Elsevier B.V. All rights reserved.)

CC17 Streptococcus agalactiae carrying group-A prophages is increasingly responsible for neonatal infections. To investigate the impact of the genetic features of a group-A prophage, we first conducted an in silico analysis of the genome of 12/111phiA, a group-A prophage carried by a strain responsible for a bloodstream infection in a parturient. This revealed a Restriction Modification system, suggesting a prophage maintenance strategy and five ORFs of interest for the host and encoding a type II toxin antitoxin system RelB/YafQ, an endonuclease, an S-adenosylmethionine synthetase MetK, and an StrP-like adhesin. Using the WT strain cured from 12/111phiA and constructing deleted mutants for the ORFs of interest, and their complemented mutants, we demonstrated an impact of prophage features on growth characteristics, cell morphology and biofilm formation. Our findings argue in favor of 12/111phiA domestication by the host and a role of prophage features in cell autoaggregation, glycocalyx and biofilm formation. We suggest that lysogeny may promote GBS adaptation to the acid environment of the vagina, consequently colonizing and infecting neonates.

Here, we performed a comparative genomic analysis of all available genomes of E. faecalis ( n = 1591) and E. faecium ( n = 1981) and investigated the association between the presence or absence of CRISPR-Cas systems, endonuclease/anti-endonuclease systems and the acquisition of antimicrobial resistance, especially vancomycin resistance genes. Most of the analysed Enterococci were isolated from humans and less than 14% of them were from foods and animals. We analysed and detected CRISPR-Cas systems in 75.36% of E. faecalis genomes and only 4.89% of E. faecium genomes with a significant difference ( p -value < 10 -5 ). We found a negative correlation between the number of CRISPR-Cas systems and genome size (r = -0.397, p -value < 10 -5 ) and a positive correlation between the genome %GC content and the number of CRISPR-Cas systems (r = 0.215, p -value < 10 -5 ). Our findings showed that the presence of the anti-endonuclease ardA gene may explain the decrease in the number of CRISPR-Cas systems in E. faecium , known to deactivate the endonucleases' protective activities and enable the E. faecium genome to be versatile in acquiring mobile genetic elements, including carriers of antimicrobial resistance genes, especially vanB . Most importantly, we observed that there was a direct association between the absence of CRISPR-Cas, the presence of the anti-CRISPR ardA gene and the acquisition of vancomycin resistance genes.

Thienamycin, the first representative of carbapenem antibiotics was discovered in the mid-1970s from soil microorganism, Streptomyces cattleya, during the race to discover inhibitors of bacterial peptidoglycan synthesis. Chemically modified into imipenem (N-formimidoyl thienamycin), now one of the most clinically important antibiotics, thienamycin is encoded by a thienamycin gene cluster composed of 22 genes (thnA to thnV) from S. cattleya NRRL 8057 genome. Interestingly, the role of all thn-genes has been experimentally demonstrated in the thienamycin biosynthesis, except thnS, despite its annotation as putative β-lactamase. Here, we expressed thnS gene and investigated its activities against various substrates. Our analyses revealed that ThnS belonged to the superfamily of metallo-β-lactamase fold proteins. Compared to known β-lactamases such as OXA-48 and NDM-1, ThnS exhibited a lower affinity and less efficiency toward penicillin G and cefotaxime, while imipenem is more actively hydrolysed. Moreover, like most MBL fold enzymes, additional enzymatic activities of ThnS were detected such as hydrolysis of ascorbic acid, single strand DNA, and ribosomal RNA. ThnS appears as a MBL enzyme with multiple activities including a specialised β-lactamase activity toward imipenem. Thus, like toxin/antitoxin systems, the role of thnS gene within the thienamycin gene cluster appears as an antidote against the produced thienamycin.

Here, we aim to determine the prevalence of Salmonella contamination of poultry meat from butcheries of the province of Skikda and to investigate antibiotic resistance. Salmonella spp. isolates were screened from 70 samples, including chicken breasts (n = 40 samples) and chicken thighs (n = 30 samples) collected from 14 butcheries. All suspected Salmonella colonies from selective media were confirmed by MALDI-TOF MS and serotyped. The susceptibility profile to 16 antibiotics was studied. According to the antibiotic susceptibility results, resistance genes were investigated by standard PCR targeting various genes such as blaSHV, blaTEM, aac3, aac6-Ibcr, aad, qnrA and qnrB. Of the 14 butcheries studied, samples from eight butcheries were contaminated with Salmonella (57.14%). 19 Salmonella strains were isolated, including five serotypes with a predominance of Kentucky serotype (n = 9), Enteridis (n = 3), followed by Heidelberg (n = 3), Virchow (n = 3), and Manhattan (n= 1). All isolates were resistant to Rifampicin (100%; n = 19), and to other antibiotics such as Ciprofloxacin (47.36%), Amoxicillin-clavulanic acid (47.36%; n = 9), Amoxicillin, (47.36%; n = 9), Ticarcillin-clavulanic acid (47.36%; n = 9), and Gentamycin (47.36%; n = 9). All isolates showing multidrug resistance (47.36%; n = 9) were positive by PCR to the blaTEM-1 β-lactamase gene, from which 8 strains carried the aminoglycoside resistance aad7 gene. However, none was positive for the tested blaSHV, Aac3, Aac6-Ibcr, qnrA, qnrB, ArmA and ArmB genes. Our findings show a worrying rate of Salmonella contamination of poultry meats., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2021 Djeffal Samia, Mamache Bakir , Elgroud Rachid , Bentchouala Chaffia , Bouaziz Omar , Jean-Marc Rolain, Seydina Mouhamadou Diene.)

The emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) represent a major clinical problem and raise serious health concerns. The present study aimed to investigate and ascertain the occurrence of CRE among hospitalized patients of Mohamed VI University Hospital, Marrakech, Morocco. Biological samples were collected over a one-year period (2018). The bacterial isolates were identified by MALDI-TOF-MS. Antibiotic susceptibility testing was performed using disc diffusion and Etest. The modified Hodge test and combined disc diffusion test were used for phenotypic detection. CRE hydrolyzing enzyme encoding genes: bla OXA-48, bla KPC, bla IMP, bla VIM, and bla NDM were characterized by PCR and DNA sequencing. In total, 131 non-duplicate CRE clinical strains resistant to Ertapenem were isolated out of 1603 initial Enterobacteriaceae . Klebsiella pneumoniae was the most common species (59%), followed by Enterobacter cloacae (24%), E. coli (10%), Citrobacter freundii (3%), Klebsiella oxycota (2%), Serratia marcescens (1%), and Citrobacter braakii (1%). Of these, 56.49%, 21.37%, 15.27%, 3.38%, and 3.05% were collected from blood, urine, pus, catheters and respiratory samples, respectively. Approximately 85.5% (112/131) of the isolates were carbapenemase producers (40 bla OXA-48, 27 bla NDM, 38 bla OXA-48 + bla NDM and 7 bla VIM). All metallo-β-lactamases isolates were NDM-1 and VIM-1 producers. This is the first documentation of bla OXA-48 genes from C. freundii and C. braakii in Morocco.

Fosfomycin is a decades-old antibiotic, currently reused because of its activity against multidrug-resistant bacteria. Here, we used a combined in vitro/in silico approach to search for fosfomycin resistance determinants in 25 new bacterial species isolated from the human microbiota. Putative resistance genes were cloned into a susceptible Escherichia coli strain. MIC values increased from 1 μg/ml to 1,024 μg/ml. Here, we report a new family of potential chromosomal fosfomycin resistance genes, named fosM ., (Copyright © 2021 American Society for Microbiology.)

The objective of this study was to investigate the occurrence of multidrug-resistant Escherichia coli in cows with clinical mastitis in 42 different dairy farms located in the Bordj Bou Arreridj region of Algeria. Milk samples were cultured on Columbia blood agar, and isolates were then identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. In total, 200 samples were screened and 52 E. coli strains confirmed as causative agents were obtained. The antimicrobial susceptibility testing was performed by disk diffusion method. Antibiotic resistance genes, including those conferring resistance to extended-spectrum β-lactamases (i.e., blaTEM, blaSHV, and blaCTX-M), tetracyclines (tetA, tetB, tetC, and tetJ), aminoglycosides [aph(3'), aac(3'), aac(6'), ant, aad, and armA], and quinolones (qnrA and qnrB) were amplified by standard PCR and sequenced when positive. Transferability of resistance genes has been investigated by conjugation experiments and multilocus sequence typing. The most frequently observed resistance was to amoxicillin (86.5%), followed by tetracycline (75%), amoxicillin-clavulanic acid (59.6%), trimethoprim-sulfamethoxazole (36.5%), doxycycline (13.5%), and ciprofloxacin (13.5%). Multidrug resistance was observed in 38.4% of isolates. Genotypic characterization showed that tetA (44.2%) and blaTEM-1 (30.7%) genes were the most prevalent. Screening for plasmid-mediated quinolone resistance genes demonstrated that seven isolates (13.5%) expressed qnrB and one isolate (1.9%) harbored qnrA. In addition, aminoglycoside resistance determinants including aadA1 and aac(3)-Id were detected in seven and two isolates, respectively. Moreover, blaTEM, tetA, tetB, qnrB, and aadA1 were successfully transferred horizontally to transconjugant strains. The multilocus sequence typing revealed the presence of three different sequence types (ST162, ST371, and ST 949)., (Copyright ©, International Association for Food Protection.)

β-lactam antibiotics have a well-known activity which disturbs the bacterial cell wall biosynthesis and may be cleaved by β-lactamases. However, these drugs are not active on archaea microorganisms, which are naturally resistant because of the lack of β-lactam target in their cell wall. Here, we describe that annotation of genes as β-lactamases in Archaea on the basis of homologous genes is a remnant of identification of the original activities of this group of enzymes, which in fact have multiple functions, including nuclease, ribonuclease, β-lactamase, or glyoxalase, which may specialized over time. We expressed class B β-lactamase enzyme from Methanosarcina barkeri that digest penicillin G. Moreover, while weak glyoxalase activity was detected, a significant ribonuclease activity on bacterial and synthetic RNAs was demonstrated. The β-lactamase activity was inhibited by β-lactamase inhibitor (sulbactam), but its RNAse activity was not. This gene appears to have been transferred to the Flavobacteriaceae group especially the Elizabethkingia genus, in which the expressed gene shows a more specialized activity on thienamycin, but no glyoxalase activity. The expressed class C-like β-lactamase gene, from Methanosarcina sp., also shows hydrolysis activity on nitrocefin and is more closely related to DD-peptidase enzymes. Our findings highlight the need to redefine the nomenclature of β-lactamase enzymes and the specification of multipotent enzymes in different ways in Archaea and bacteria over time.

Carbapenemase-producing Alcaligenes species has been described in only few studies, with none so far from the African continent. Here, we report the whole genome sequence of Peanalcaligenes suwonensis bearing bla VIM-5 metallo-β-lactamase and first detection of carbapenemase producing Alcaligenes faecalis isolated from patients attending tertiary healthcare facilities in Nigeria. The isolates were identified by MALDI-TOF Mass Spectrometry. Antibiotic susceptibility assay, modified Carba NP test and genomic investigation revealed that two isolates of Alcaligenes faecalis and an isolate of Paenalcaligenes suwonensis harboured bla VIM-5 gene. The genome sequence analysis of the P. suwonensis 191B isolate, responsible for acute gastroenteritis, reveal the presence of 18 antibiotic resistance genes coding for resistance to five different classes of antibiotics. Three of the genes (bla OXA-368 , bla CARB-4 and bla VIM-5 ) codes for resistance to β-lactam antibiotics. To our best knowledge, we describe here the first genome sequence of P. suwonensis species and the first detection of class B carbapenemase bla VIM-5 in a clinical isolate of P. suwonensis species and Alcaligenes faecalis in Nigeria. The finding of this study is of concern, as lateral dissemination of the genes into clinically important Gram-negative pathogens is highly likely., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Long considered to be a consequence of human antibiotics use by deduction, antibiotic resistance mechanisms appear to be in fact a much older phenomenon as antibiotic resistance genes have previously been detected from millions of year-old permafrost samples. As these specimens guarantee the viability of archaic bacteria, we herein propose to apply the culturomics approach to recover the bacterial content of a Siberian permafrost sample dated, using the in situ-produced cosmogenic nuclide chlorine36 ( 36 Cl), at 2.7 million years to study the dynamics of bacterial evolution in an evolutionary perspective. As a result, we cultured and sequenced the genomes of 28 ancient bacterial species including one new species. To perform genome comparison between permafrost strains and modern isolates we selected 7 of these species (i.e., Achromobacter insolitus, Bacillus idriensis, Brevundimonas aurantiaca, Janibacter melonis, Kocuria rhizophila, Microbacterium hydrocarbonoxydans and Paracoccus yeei ). We observed a high level of variability in genomic content with a percentage of shared genes in the core genomes ranging from 21.23% to 55.59%. In addition, the Single Nucleotide Polymorphism (SNP) comparison between permafrost and modern strains for the same species did not allow a dating of ancient strains based on genomic content. There were no significant differences in antibiotic resistance profiles between modern and ancient isolates of each species. Acquired resistance to antibiotics was phenotypically detected in all gram-negative bacterial species recovered from permafrost, with a significant number of genes coding for antibiotic resistance detected. Taken together, these findings confirm previously obtained data that antibiotic resistance predates humanity as most of antimicrobial agents are natural weapons used in inter-microbial conflicts within the biosphere.

A review of literature was conducted to assess the prevalence and mechanisms of antibiotic resistance to date, mainly to β-lactam antibiotics, cephalosporins, carbapenems, colistin, and tigecycline in the Democratic Republic of the Congo (DRC). English and French publications were listed and analysed using PubMed/Medline, Google Scholar, and African Journals database between 1 January 1990 and 31 December 2019. For the 30 published articles found: (1) bacterial resistance to antibiotics concerned both Gram-negative and Gram-positive bacteria; (2) multidrug resistance prevalence was the same in half of Streptococcus pneumoniae isolates; (3) a worrying prevalence of methicillin-resistant Staphylococcus aureus (MRSA) was noted, which is associated with co-resistance to several other antibiotics; and (4) resistance to third-generation cephalosporins was very high in Enterobacteriaceae, mainly because of bla CTX-M-1 group and bla SHV genes. Data on carbapenem and colistin resistance were not available in DRC until recently. Further work is required to set up a surveillance system for antibiotic resistance in DRC., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The probability of the evolution of a character depends on two factors: the probability of moving from one character state to another character state and the probability of the new character state fixation. The more the evolution of a character is probable, the more the convergent evolution will be witnessed, and consequently, convergent evolution could mean that the convergent character evolution results as a combination of these two factors. We investigated this phenomenon by studying the convergent evolution of biochemical functions. For the investigation we used the case of β-lactamases. β-lactamases hydrolyze β-lactams, which are antimicrobials able to block the DD-peptidases involved in bacterial cell wall synthesis. β-lactamase activity is present in two different superfamilies: the metallo-β-lactamase and the serine β-lactamase. The mechanism used to hydrolyze the β-lactam is different for the two superfamilies. We named this kind of evolution an allo-convergent evolution. We further showed that the β-lactamase activity evolved several times within each superfamily, a convergent evolution type that we named iso-convergent evolution. Both types of convergent evolution can be explained by the two evolutionary mechanisms discussed above. The probability of moving from one state to another is explained by the promiscuous β-lactamase activity present in the ancestral sequences of each superfamily, while the probability of fixation is explained in part by positive selection, as the organisms having β-lactamase activity allows them to resist organisms that secrete β-lactams. Indeed, an organism that has a mutation that increases the β-lactamase activity will be selected, as the organisms having this activity will have an advantage over the others.

Emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) are two of the major problems currently threatening global public health. In Nigeria, interest in CPE is recent. In Sokoto, northwest Nigeria, there are no data on the prevalence and mechanism underlying carbapenem resistance. In this study, we aimed to investigate the presence of clinical carbapenems-resistant Enterobacteriaceae isolates in two leading hospitals in Sokoto, northwest Nigeria. A total of 292 non-duplicate Enterobacteriaceae isolated from clinical specimens processed in the diagnostic laboratories of two hospitals between January and June 2019 were collected. Of these, 129 (44.2 %) and 19 (6.5%) were resistant to third-generation cephalosporin and carbapenems, respectively. RT-PCR revealed that 10 (7.8%), 19 (14.7%) and 46 (35.7%) of the third-generation cephalosporin-resistant isolates harboured bla SHV , bla TEM and bla CTX-M genes, respectively. The modified Carba NP test result showed that only 7 (36.8 %) of the 19 carbapenem-resistant isolates were carbapenemase producing; among them, bla NDM-5 and bla OXA-181 genes were identified in five and two isolates, respectively. However, none of the carbapenemase genes investigated, including bla VIM , bla KPC and bla IMP , was detected in the remaining carbapenem-resistant isolates, suggesting a non-enzymatic mechanism. This study reports for the first time, the emergence of CPE in Sokoto state and the detection of NDM-producing Citrobacter freundii in Nigeria. The observed CPE in this study is a concern in a country where alternative antibiotics are rarely available., (© 2020 The Authors.)