Algerian postcaesarean surgical site infections: A cross-sectional investigation of the epidemiology, bacteriology, and antibiotic resistance profile.

BACKGROUND: Surgical site infections (SSIs) are one of the most common health care-associated infections in low and middle-income countries. The aims of this cross-sectional descriptive study were to estimate the frequency of postcaesarean infection with associated clinical characteristics and the antibiotic resistance profile of bacterial isolates. METHODS: Patients who underwent a cesarean section at the obstetrics and gynecology department of the hospital in Annaba, Algeria were included. Each woman was followed postoperatively for 30 days and sociodemographic data were collected. Culture-based microbiological methods were used to identify the causative bacteria and determine their antibiotic resistance phenotype and molecular characterization. RESULTS: Among 1,810 patients, we recorded 36 (1.9%) SSIs. Most patients had undergone an emergency delivery (75%) and low educational level (72.2%). The most frequent maternal pathologies were Body Mass Index ≥ 30 (63.9%), scarred uteri (58.3%), anemia (55.6%), and an American Society of Anaesthesiologists score between II and III (33.3%). Of the 43 bacteria isolated, Enterobacteriaceae were the most frequent (62.8%), predominated by Escherichia coli strains (43.5%), a majority of which were extended-spectrum ß-lactamases carriers (62.9%). Although gram-positive cocci were less frequent (37.2%), a majority of Enterococcus faecalis (56.2%) were observed and 2 strains of vancomycin-resistant Enterococcus faecium harboring the vanA gene were identified. CONCLUSIONS: Extensive surveillance of at-risk populations should be integrated to prevent the occurrence of SSIs.

Prevalence of carbapenemases among Gram-negative bacteria in Tunisia: first report of KPC-2 producing Acinetobacter baumannii.

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.

Co-existence of NDM-, aminoglycoside- and fluoroquinolone-resistant genes in carbapenem-resistant Escherichia coli clinical isolates from Pakistan.

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 blaNDM; 69, 18 and 12% were NDM-1, NDM-5 and NDM-7, respectively, with 100% resistance to ß-lactams and ß-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.

Escherichia coli (E. coli) is a type of bacteria found in the gut of humans and animals that causes numerous illnesses such as infection of the blood or urinary tract, diarrhea and vomiting. New Delhi metallo-ß-lactamase (NDM) is a protein produced by E. coli that is capable of breaking down several important antibiotics including penicillins, cephalosporins and carbapenems. E. coli that produce this protein are known as 'resistant', and therefore treatments against these infections are limited. This study looked at how common NDM was found among E. coli taken from hospitalized patients in Lahore, Pakistan, to understand the risks of resistant bacteria in clinical settings and found a high number of a high-risk E. coli.

The Emergence of Carbapenem- and Colistin-Resistant Enterobacteria in Senegal.

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% blaTEM, 37.5% blaSHV, 83.75% blaCTX-A, and 0.42% blaCTX-B). The carbapenemase genes blaOXA-48 and blaNDM were found in 25 isolates, including 14 isolates with blaOXA-48, 13 isolates with blaNDM, 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.

Occurrence and characteristics of extended-spectrum-ß-lactamase producing Escherichia coli (blaTEM-128) isolated from Mus musculus captured from a veterinary clinic and houses in Tunis, Tunisia.

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 ß-lactamases-producing strains and determine the molecular mechanism of ß-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 blaTEM-128 gene. In addition, the other five strains were DDST negative and harbored the blaTEM gene, including three strains isolated from collective restaurant (n = 2: blaTEM-163; n = 1: blaTEM-1), one strain isolated from the veterinary clinic (blaTEM-82), and one strain isolated from a house (blaTEM-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.

Outbreak of carbapenem-resistant enterobacteria in a thoracic-oncology unit through clonal and plasmid-mediated transmission of the bla OXA-48 gene in Southern France.

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 Tn1999.2 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.

Genetic Diversity and Virulence Profile of Methicillin and Inducible Clindamycin-Resistant Staphylococcus aureus Isolates in Western Algeria.

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.

Detection by Whole-Genome Sequencing of a Novel Metallo-ß-Lactamase Produced by Wautersiella falsenii Causing Urinary Tract Infection in Tunisia.

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 ß-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 ß 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-ß-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-ß-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., ß-lactamases. Collaborative efforts and effective hygiene measures should be established to prevent the emergence of this species in our health care settings.

Population Diversity of Antibiotic Resistant Enterobacterales in Samples From Wildlife Origin in Senegal: Identification of a Multidrug Resistance Transposon Carrying bla CTX-M-15 in Escherichia coli.

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 TEM1B , 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.

In Vitro Screening of a 1280 FDA-Approved Drugs Library against Multidrug-Resistant and Extensively Drug-Resistant Bacteria.

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.

First Report of Extended-Spectrum ß-Lactamase (blaCTX-M1) and Colistin Resistance Gene mcr-1 in E. coli of Lineage ST648 from Cockroaches in Tunisia.

The emergence of multidrug-resistant bacteria has become a major problem. Cockroaches may play an important role in the spread of those bacteria between the environment and humans. This study was designed to screen extended-spectrum ß-lactamase (ESBL)-producing and colistin-resistant strains and to investigate the molecular support of multidrug-resistant Enterobacteriaceae in the external surface and gut homogenates of cockroaches collected from different locations in Tunisia. Between July 2017 and June 2018, 144 Enterobacteriaceae samples were isolated from 115 trapped cockroaches (collective catering, houses, and a hospital). Antibiotic susceptibility testing was performed using the disk diffusion method. Extended-spectrum ß-lactamase-encoding genes and the mcr-1 gene were investigated by real-time PCR (RT-PCR) and standard PCR. The genetic relationship among isolates was studied with the help of multilocus sequence type (MLST) analysis. Of the 144 Enterobacteriaceae isolates, 22 strains exhibited a positive ESBL-screening test (73.3%), including 17 Escherichia coli isolates and 5 Klebsiella pneumoniae isolates. Among them, 9 Escherichia coli isolates were resistant to colistin, with an MIC ranging from 8 to16 µg/L, all of which harbored the mcr-1 gene. Eight blaCTX-M-15 genes were detected; two among them were associated with blaTEM-117 and blaTEM-128, and seven blaCTX-M-1 genes were detected that also harbored the mcr-1 gene. Genotyping analysis revealed 7 different sequence types already described in humans and animals. We report the first survey of mcr-1 in ESBL-producing E. coli isolates from cockroaches. Our findings highlight cockroaches as a source of nosocomial infections, and they are a reservoir of colistin-resistant E. coli, which is a carrier of other additional risk genes such as blaESBL, especially in hospitals. IMPORTANCE Multidrug resistance in Enterobacteriaceae has become a major concern worldwide that is increasingly observed in human, animals, and also cockroaches. In our study, we found that cockroaches may play an important role as a potential vector of multidrug-resistant Enterobacteriaceae in the hospital environment and collective catering. Our study describes the first survey of mcr-1 in ESBL-producing E. coli isolates from hospital cockroaches. Our results further highlight the possibility that mcr-1 may enter humans via cockroach contamination and thereby threaten public health. Our results show that these cockroaches are an important reservoir of colistin-resistant E. coli and carriers of other additional risk genes such as blaESBL, hence the importance of strengthening prevention strategies and of strictly respecting hygiene measures in order to control their distribution and spread in Tunisia.

Real-Time PCR Assay for Rapid and Simultaneous Detection of vanA and vanB Genes in Clinical Strains.

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.

Emergence of Methicillin-Resistant Staphylococcus aureus ST239/241 SCCmec-III Mercury in Eastern Algeria.

In this paper, we investigate the epidemiology of infections-associated Staphylococcusaureus (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 mecC 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 SCCmec-III mercury with PVL negative, resistant to ß-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.

Occurrence of NDM-1 and VIM-2 Co-Producing Escherichia coli and OprD Alteration in Pseudomonas aeruginosa Isolated from Hospital Environment Samples in Northwestern Tunisia.

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 (blaNDM-1 (n = 8); blaNDM-1 + blaVIM-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 blaOXA-494. Other genes were also detected, notably blaTEM (n = 23), blaCTX-M-1 (n = 10) and blaCTX-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.

Rapid Detection of Imipenem Resistance in Gram-Negative Bacteria Using Tabletop Scanning Electron Microscopy: A Preliminary Evaluation.

Background: Enabling faster Antimicrobial Susceptibility Testing (AST) is critical, especially to detect antibiotic resistance, to provide rapid and appropriate therapy and to improve clinical outcomes. Although several standard and automated culture-based methods are available and widely used, these techniques take between 18 and 24 h to provide robust results. Faster techniques are needed to reduce the delay between test and results. Methods: Here we present a high throughput AST method using a new generation of tabletop scanning electron microscope, to evaluate bacterial ultra-structural modifications associated with susceptibilities to imipenem as a proof of concept. A total of 71 reference and clinical strains of Gram-negative bacteria were used to evaluate susceptibility toward imipenem after 30, 60, and 90 min of incubation. The length, width and electron density of bacteria were measured and compared between imipenem susceptible and resistant strains. Results: We correlated the presence of these morphological changes to the bacterial susceptibility and their absence to the bacterial resistance (e.g., Pseudomonas aeruginosa length without [2.24 ± 0.61 µm] and with [2.50 ± 0.68 µm] imipenem after 30 min [p = 3.032E-15]; Escherichia coli width without [0.92 ± 0.07 µm] and with [1.28 ± 0.19 µm] imipenem after 60 min [p = 1.242E-103]). We validated our method by a blind test on a series of 58 clinical isolates where all strains were correctly classified as susceptible or resistant toward imipenem. Conclusion: This method could be a potential tool for rapidly identifying carbapenem-resistance in Enterobacterales in clinical microbiology laboratories in <2 h, allowing the empirical treatment of patients to be rapidly adjusted.

Risk factors for acquisition of colistin-resistant Klebsiella pneumoniae and expansion of a colistin-resistant ST307 epidemic clone in hospitals in Marseille, France, 2014 to 2017.

BackgroundFrance is a low prevalence country for colistin resistance. Molecular and epidemiological events contributing to the emergence of resistance to colistin, one of the 'last-resort' antibiotics to treat multidrug-resistant Gram-negative infections, are important to investigate.AimThis retrospective (2014 to 2017) observational study aimed to identify risk factors associated with acquisition of colistin-resistant Klebsiella pneumoniae (CRKP) in hospitals in Marseille, France, and to molecularly characterise clinical isolates.MethodsTo identify risk factors for CRKP, a matched-case-control (1:2) study was performed in two groups of patients with CRKP or colistin-susceptible K. pneumoniae respectively. Whole-genome-sequences (WGS) of CRKP were compared with 6,412 K. pneumoniae genomes available at the National Center for Biotechnology Information (NCBI).ResultsMultivariate analysis identified male sex and contact with a patient carrying a CRKP as significant independent factors (p < 0.05) for CRKP acquisition, but not colistin administration. WGS of nine of 14 CRKP clinical isolates belonged to the same sequence type (ST)307. These isolates were from patients who had been hospitalised in the same wards, suggesting an outbreak. Comparison of the corresponding strains' WGS to K. pneumoniae genomes in NCBI revealed that in chromosomal genes likely playing a role in colistin resistance, a subset of five specific mutations were significantly associated with ST307 (p < 0.001).ConclusionA ST307 CRKP clone was identified in this study, with specific chromosomal mutations in genes potentially implicated in colistin resistance. ST307 might have a propensity to be or become resistant to colistin, however confirming this requires further investigations.

A metallo-ß-lactamase enzyme for internal detoxification of the antibiotic thienamycin.

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 ß-lactamase. Here, we expressed thnS gene and investigated its activities against various substrates. Our analyses revealed that ThnS belonged to the superfamily of metallo-ß-lactamase fold proteins. Compared to known ß-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 ß-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.

High frequency and diversity of Vancomycin-resistant Enterococci (VRE) in Algerian healthcare settings.

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.