Resistance phenotype and virulence potential of Leclercia adecarboxylata strains isolated from different sources.

Introduction. Leclercia adecarboxylata is a member of Enterobacterales, often considered an opportunistic pathogen. Recent reports have highlighted L. adecarboxylata as an emerging pathogen harbouring virulence and resistance determinants.Gap statement. Little information exists on virulence and resistance determinants in L. adecarboxylata strains isolated from environmental, food, and clinical samples.Aim. To determine the presence of resistance and virulence determinants and plasmid features in L. adecarboxylata strains isolated from environmental, food, and clinical samples, as well as their phylogenetic relationship.Results. All strains tested showed resistance to ß-lactams and quinolones but were sensitive to aminoglycosides and nitrofurans. However, even though fosfomycin resistance is considered a characteristic trait of L. adecarboxylata, the resistance phenotype was only observed in 50 % of the strains; bla TEM was the most prevalent BLEE gene (70 %), while the quinolone qnrB gene was observed in 60 % of the strains. Virulence genes were differentially observed in the strains, with adhesion-related genes being the most abundant, followed by toxin genes. Finally, all strains carried one to seven plasmid bands ranging from 7 to 125 kbps and harboured several plasmid addiction systems, such as ParDE, VagCD, and CcdAB in 80 % of the strains.Conclusions. L. adecarboxylata is an important emerging pathogen that may harbour resistance and virulence genes. Additionally, it has mobilizable genetic elements that may contribute to the dissemination of genetic determinants to other bacterial genera.

Transmission of blaNDM in Enterobacteriaceae among animals, food and human.

Despite carbapenems not being used in animals, carbapenem-resistant Enterobacterales (CRE), particularly New Delhi metallo-ß-lactamase-producing CRE (NDM-CRE), are prevalent in livestock. Concurrently, the incidence of human infections caused by NDM-CRE is rising, particularly in children. Although a positive association between livestock production and human NDM-CRE infections at the national level was identified, the evidence of direct transmission of NDM originating from livestock to humans remains largely unknown. Here, we conducted a cross-sectional study in Chengdu, Sichuan Province, to examine the prevalence of NDM-CRE in chickens and pigs along the breeding-slaughtering-retail chains, in pork in cafeterias of schools, and in colonizations and infections from children's hospital and examined the correlation of NDM-CRE among animals, foods and humans. Overall, the blaNDM increases gradually along the chicken and pig breeding (4.70%/2.0%) -slaughtering (7.60%/22.40%) -retail (65.56%/34.26%) chains. The slaughterhouse has become a hotspot for cross-contamination and amplifier of blaNDM. Notably, 63.11% of pork from the school cafeteria was positive for blaNDM. The prevalence of blaNDM in intestinal and infection samples from children's hospitals was 21.68% and 19.80%, respectively. whole genome sequencing (WGS) analysis revealed the sporadic, not large-scale, clonal spread of NDM-CRE along the chicken and pig breeding-slaughtering-retail chain, with further spreading via IncX3-blaNDM plasmid within each stage of whole chains. Clonal transmission of NDM-CRE is predominant in children's hospitals. The IncX3-blaNDM plasmid was highly prevalent among animals and humans and accounted for 57.7% of Escherichia coli and 91.3% of Klebsiella pneumoniae. Attention should be directed towards the IncX3 plasmid to control the transmission of blaNDM between animals and humans.

Molecular mechanisms of tigecycline-resistance among Enterobacterales.

The global emergence of antimicrobial resistance to multiple antibiotics has recently become a significant concern. Gram-negative bacteria, known for their ability to acquire mobile genetic elements such as plasmids, represent one of the most hazardous microorganisms. This phenomenon poses a serious threat to public health. Notably, the significance of tigecycline, a member of the antibiotic group glycylcyclines and derivative of tetracyclines has increased. Tigecycline is one of the last-resort antimicrobial drugs used to treat complicated infections caused by multidrug-resistant (MDR) bacteria, extensively drug-resistant (XDR) bacteria or even pan-drug-resistant (PDR) bacteria. The primary mechanisms of tigecycline resistance include efflux pumps' overexpression, tet genes and outer membrane porins. Efflux pumps are crucial in conferring multi-drug resistance by expelling antibiotics (such as tigecycline by direct expelling) and decreasing their concentration to sub-toxic levels. This review discusses the problem of tigecycline resistance, and provides important information for understanding the existing molecular mechanisms of tigecycline resistance in Enterobacterales. The emergence and spread of pathogens resistant to last-resort therapeutic options stands as a major global healthcare concern, especially when microorganisms are already resistant to carbapenems and/or colistin.

High prevalence of multidrug-resistant Enterobacterales carrying extended-spectrum beta-lactamase and AmpC genes isolated from neonatal sepsis in Ahvaz, Iran.

OBJECTIVES: In the recent years, multidrug resistant (MDR) neonatal septicemia-causing Enterobacterales has been dramatically increased due to the extended-spectrum beta-lactamases (ESBLs) and AmpC enzymes. This study aimed to assess the antibiotic resistance pattern, prevalence of ESBLs/AmpC beta-lactamase genes, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) fingerprints in Enterobacterales isolated from neonatal sepsis. RESULTS: In total, 59 Enterobacterales isolates including 41 (69.5%) Enterobacter species, 15 (25.4%) Klebsiella pneumoniae and 3 (5.1%) Escherichia coli were isolated respectively. Resistance to ceftazidime and cefotaxime was seen in all of isolates. Furthermore, all of them were multidrug-resistant (resistant to three different antibiotic categories). The phenotypic tests showed that 100% of isolates were ESBL-positive. Moreover, AmpC production was observed in 84.7% (n = 50/59) of isolates. Among 59 ESBL-positive isolates, the highest percentage belonged to blaCTX-M-15 gene (66.1%) followed by blaCTX-M (45.8%), blaCTX-M-14 (30.5%), blaSHV (28.8%), and blaTEM (13.6%). The frequency of blaDHA, blaEBC, blaMOX and blaCIT genes were 24%, 24%, 4%, and 2% respectively. ERIC-PCR analysis revealed that Enterobacterales isolates were genetically diverse. The remarkable prevalence of MDR Enterobacterales isolates carrying ESBL and AmpC beta-lactamase genes emphasizes that efficient surveillance measures are essential to avoid the more expansion of drug resistance amongst isolates.

A CRISPR/Cas12a-Based System for Sensitive Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales.

Antimicrobial-resistant (AMR) bacteria pose a significant global health threat, and bacteria that produce New Delhi metallo-ß-lactamase (NDM) are particularly concerning due to their resistance to most ß-lactam antibiotics, including carbapenems. The emergence and spread of NDM-producing genes in food-producing animals highlight the need for a fast and accurate method for detecting AMR bacteria. We therefore propose a PCR-coupled CRISPR/Cas12a-based fluorescence assay that can detect NDM-producing genes (blaNDM) in bacteria. Thanks to its designed gRNA, this CRISPR/Cas12a system was able to simultaneously cleave PCR amplicons and ssDNA-FQ reporters, generating fluorescence signals. Our method was found to be highly specific when tested against other foodborne pathogens that do not carry blaNDM and also demonstrated an excellent capability to distinguish single-nucleotide polymorphism. In the case of blaNDM-1 carrying E. coli, the assay performed exceptionally well, with a detection limit of 2.7 × 100 CFU/mL: 100 times better than conventional PCR with gel electrophoresis. Moreover, the developed assay detected AMR bacteria in food samples and exhibited enhanced performance compared to previously published real-time PCR assays. Thus, this novel PCR-coupled CRISPR/Cas12a-based fluorescence assay has considerable potential to improve current approaches to AMR gene detection and thereby contribute to mitigating the global threat of AMR.

Hi-C metagenomics facilitate comparative genome analysis of bacteria and yeast from spontaneous beer and cider.

Sequence-based analysis of fermented foods and beverages' microbiomes offers insights into their impact on taste and consumer health. High-throughput metagenomics provide detailed taxonomic and functional community profiling, but bacterial and yeast genome reconstruction and mobile genetic elements tracking are to be improved. We established a pipeline for exploring fermented foods microbiomes using metagenomics coupled with chromosome conformation capture (Hi-C metagenomics). The approach was applied to analyze a collection of spontaneously fermented beers and ciders (n = 12). The Hi-C reads were used to reconstruct the metagenome-assembled genomes (MAGs) of bacteria and yeasts facilitating subsequent comparative genomic analysis, assembly scaffolding and exploration of "plasmid-bacteria" links. For a subset of beverages, yeasts were isolated and characterized phenotypically. The reconstructed Hi-C MAGs primarily belonged to the Lactobacillaceae family in beers, along with Acetobacteraceae and Enterobacteriaceae in ciders, exhibiting improved quality compared to conventional metagenomic MAGs. Comparative genomic analysis of Lactobacillaceae Hi-C MAGs revealed clustering by niche and suggested genetic determinants of survival and probiotic potential. For Pediococcus damnosus, Hi-C-based networks of contigs enabled linking bacteria with plasmids. Analyzing phylogeny and accessory genes in the context of known reference genomes offered insights into the niche specialization of beer lactobacilli. The subspecies-level diversity of cider Tatumella spp. was disentangled using a Hi-C-based graph. We obtained highly complete yeast Hi-C MAGs primarily represented by Brettanomyces and Saccharomyces, with Hi-C-facilitated chromosome-level genome assembly for the former. Utilizing Hi-C metagenomics to unravel the genomic content of individual species can provide a deeper understanding of the ecological interactions within the food microbiome, aid in bioprospecting beneficial microorganisms, improving quality control and improving innovative fermented products.

Clinical distribution of carbapenem genotypes and resistance to ceftazidime-avibactam in Enterobacteriaceae bacteria.

Introduction: Bacterial resistance is a major threat to public health worldwide. To gain an understanding of the clinical infection distribution, drug resistance information, and genotype of CRE in Dongguan, China, as well as the resistance of relevant genotypes to CAZ-AVI, this research aims to improve drug resistance monitoring information in Dongguan and provide a reliable basis for the clinical control and treatment of CRE infection. Methods: VITEK-2 Compact automatic analyzer was utilized to identify 516 strains of CRE collected from January 2017 to June 2023. To determine drug sensitivity, the K-B method, E-test, and MIC methods were used. From June 2022 to June 2023, 80 CRE strains were selected, and GeneXpert Carba-R was used to detect and identify the genotype of the carbapenemase present in the collected CRE strains. An in-depth analysis was conducted on the CAZ-AVI in vitro drug sensitivity activity of various genotypes of CRE, and the results were statistically evaluated using SPSS 23.0 and WHONET 5.6 software. Results: This study identified 516 CRE strains, with the majority (70.16%) being K.pneumoniae, followed by E.coli (18.99%). Respiratory specimens had highest detection rate with 53.77% identified, whereas urine specimens had the second highest detection rate with 17.99%. From June 2022 to June 2023, 95% of the strains tested using the CRE GeneXpert Carba-R assay possessed carbapenemase genes, of which 32.5% were blaNDM strains and 61.25% blaKPC strains. The results showed that CRE strains containing blaKPC had a significantly higher rate of resistance to amikacin, cefepime, and aztreonam than those harboring blaNDM. Conclusions: The CRE strains isolated from Dongguan region demonstrated a high resistance rate to various antibiotics used in clinical practice but a low resistance rate to tigecycline. These strains produce Class A serine carbapenemases and Class B metals ß-lactamases, with the majority of them carrying blaNDM and blaKPC. Notably, CRE strains with blaKPC and blaNDM had significantly lower resistance rates to tigecycline. CAZ-AVI showed a good sensitivity rate with no resistance to CRE strains carrying blaKPC. Therefore, CAZ-AVI and tigecycline should be used as a guide for rational use of antibiotics in clinical practice to effectively treat CRE.

Apirhabdus apintestini gen. nov., sp. nov., a member of a novel genus of the family Enterobacteriaceae, isolated from the gut of the western honey bee Apis mellifera.

A Gram-negative, motile, rod-shaped bacterial strain, CA-0114T, was isolated from the midgut of a western honey bee, Apis mellifera. The isolate exhibited ≤96.43 % 16S rRNA gene sequence identity (1540 bp) to members of the families Enterobacteriaceae and Erwiniaceae. Phylogenetic trees based on genome blast distance phylogeny and concatenated protein sequences encoded by conserved genes atpD, fusA, gyrB, infB, leuS, pyrG and rpoB separated the isolate from other genera forming a distinct lineage in the Enterobacteriaceae. In both trees, the closest relatives were Tenebrionicola larvae YMB-R21T and Tenebrionibacter intestinalis BIT-L3T, which were isolated previously from Tenebrio molitor L., a plastic-eating mealworm. Digital DNA-DNA hybridization, orthologous average nucleotide identity and average amino acid identity values between strain CA-0114T and the closest related members within the Enterobacteriaceae were ≤23.1, 75.45 and 76.04 %, respectively. The complete genome of strain CA-0114T was 4 451669 bp with a G+C content of 52.12 mol%. Notably, the apparent inability of strain CA-0114T to ferment d-glucose, inositol and l-rhamnose in the API 20E system is unique among closely related members of the Enterobacteriaceae. Based on the results obtained through genotypic and phenotypic analysis, we propose that strain CA-0114T represents a novel species and genus within the family Enterobacteriaceae, for which we propose the name Apirhabdus apintestini gen. nov., sp. nov. (type strain CA-0114T=ATCC TSD-396T=DSM 116385T).

Genomic insights into the evolution, pathogenicity, and extensively drug-resistance of emerging pathogens Kluyvera and Phytobacter.

Introduction: Kluyvera is a Gram-negative, flagellated, motile bacillus within the Enterobacteriaceae. The case reports of clinical infections shed light on the importance of this organism as an emerging opportunistic pathogen. The genus Phytobacter, which often be misidentified with Kluyvera, is also an important clinically relevant member of the Enterobacteriaceae. However, the identification of Kluyvera and Phytobacter is problematic, and their phylogenetic relationship remains unclear. Methods: Here, 81 strains of Kluyvera and 16 strains of Phytobacter were collected. A series of comparative genomics approaches were applied to the phylogenetic relationship reconstruction, virulence related genes profiles description, and antibiotic resistance genes prediction. Results: Using average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH), we offered reliable species designations of 97 strains, in which 40 (41.24%) strains were incorrectly labeled. A new Phytobacter genomospecies-1 were defined. Phytobacter and Kluyvera show great genome plasticity and inclusiveness, which may be related to their diverse ecological niches. An intergenomic distances threshold of 0.15875 was used for taxonomy reassignments at the phylogenomic-group level. Further principal coordinates analysis (PCoA) revealed 11 core genes of Kluyvera (pelX, mdtL, bglC, pcak-1, uhpB, ddpA-2, pdxY, oppD-1, cptA, yidZ, csbX) that could be served as potential identification targets. Meanwhile, the Phytobacter specific virulence genes clbS, csgA-C, fliS, hsiB1_vipA and hsiC1_vipB, were found to differentiate from Kluyvera. We concluded that the evolution rate of Kluyvera was 5.25E-6, approximately three times higher than that of Phytobacter. Additionally, the co-existence of ESBLs and carbapenem resistance genes were present in approximately 40% strains, suggesting the potential development of extensively drug-resistant or even fully drug-resistant strains. Discussion: This work provided a better understanding of the differences between closely related species Kluyvera and Phytobacter. Their genomes exhibited great genome plasticity and inclusiveness. They not only possess a potential pathogenicity threat, but also a risk of multi-drug resistance. The emerging pathogens Kluyvera and Phytobacter warrant close attention.

Plasmid-encoded gene duplications of extended-spectrum ß-lactamases in clinical bacterial isolates.

Introduction: The emergence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is an urgent and alarming One Health problem. This study aimed to investigate duplications of plasmid-encoded ESBL genes and their impact on antimicrobial resistance (AMR) phenotypes in clinical and screening isolates. Methods: Multi-drug-resistant bacteria from hospitalized patients were collected during routine clinical surveillance from January 2022 to June 2023, and their antimicrobial susceptibility patterns were determined. Genotypes were extracted from long-read whole-genome sequencing data. Furthermore, plasmids and other mobile genetic elements associated with ESBL genes were characterized, and the ESBL genes were correlated to ceftazidime minimal inhibitory concentration (MIC). Results: In total, we identified four cases of plasmid-encoded ESBL gene duplications that match four genetically similar plasmids during the 18-month surveillance period: five Escherichia coli and three Klebsiella pneumoniae isolates. As the ESBL genes were part of transposable elements, the surrounding sequence regions were duplicated as well. In-depth analysis revealed insertion sequence (IS)-mediated transposition mechanisms. Isolates with duplicated ESBL genes exhibited a higher MIC for ceftazidime in comparison to isolates with a single gene copy (3-256 vs. 1.5-32 mg/L, respectively). Conclusion: ESBL gene duplications led to an increased phenotypic resistance against ceftazidime. Our data suggest that ESBL gene duplications by an IS-mediated transposition are a relevant mechanism for how AMR develops in the clinical setting and is part of the microevolution of plasmids.

Prevalence and antimicrobial resistance of Enterobacteriaceae in the north of Kazakhstan.

Background: An increasing number of drugs are used each year in the treatment of small pets (cats and dogs), including medicines (cephalosporins and fluoroquinolones) used in human therapy. Aim: The purpose of this study was to isolate and explore the antibiotic resistance of opportunistic Enterobacteriaceae (Escherichia coli, Klebsiella, Proteus, Ci trobacter, Enterobacter) from cats and dogs, and to isolate resistance genes in the microorganisms. Methods: In 2021, 808 samples of biological material from small domestic animals were collected in veterinary clinics in Kostanay. From these, 210 microorganisms were isolated and identified. Results: A large majority of the strains sampled belonged to E. coli-149 (70.9%), Enterobacter-11 (5.2%), Klebsiella-28 (13.3%), Proteus-12 (5.7%) and 10 Citrobacter isolates (4.8%). In all isolates identified, antibiotic resistance/sensitivity was determined by disc-diffusion method to ampicillin, cefoxitin, gentamicin, levomycetin, tetracycline, ciprofloxacin, norfloxacin, ofloxacin, cefoperazone, cefpodoxime, streptomycin, kanamycin, doxycycline, gemifloxacin, nalidixic acid, furazolidone, furadonine, amoxicillin, and enrofloxacin. Conclusion: The study has demonstrated that the greatest number of Enterobacteriaceae were sensitive to the action of meropenem, which belongs to the group of beta-lactam antibiotics; resistance was demonstrated against tetracycline, doxycycline, ampicillin, amoxicillin, ofloxacin, and cefpodoxime. The most common genes encoding antimicrobial resistance were as follows: BlaTEM and OXA in 41 and 28 isolates, respectively, encoding resistance to beta-lactams; StrA and StrB in 45 and 48 isolates encoding aminoglycosides; and tetA and tetB in 43 and 28 isolates encoding tetracyclines. Obtained data demonstrate that uncontrolled and frequent use of beta-lactam and tetracycline antibacterials, in cats and dogs, results in the spread of genotypic resistance among micro-organisms of the family Enterobacteriaceae.

Sanitation in urban areas may limit the spread of antimicrobial resistance via flies.

Synanthropic filth flies are common where sanitation is poor and fecal wastes are accessible to them. These flies have been proposed as mechanical vectors for the localized transport of fecal microbes including antimicrobial resistant (AMR) organisms and associated antimicrobial resistance genes (ARGs), increasing exposure risks. We evaluated whether an onsite sanitation intervention in Maputo, Mozambique reduced the concentration of enteric bacteria and the frequency of detection of ARGs carried by flies collected in household compounds of low-income neighborhoods. Additionally, we assessed the phenotypic resistance profile of Enterobacteriaceae isolates recovered from flies during the pre-intervention phase. After fly enumeration at study compounds, quantitative polymerase chain reaction was used to quantify an enteric 16S rRNA gene (i.e., specific to a cluster of phylotypes corresponding to 5% of the human fecal microflora), 28 ARGs, and Kirby Bauer Disk Diffusion of Enterobacteriaceae isolates was utilized to assess resistance to eleven clinically relevant antibiotics. The intervention was associated with a 1.5 log10 reduction (95% confidence interval: -0.73, -2.3) in the concentration of the enteric 16S gene and a 31% reduction (adjusted prevalence ratio = 0.69, [0.52, 0.92]) in the mean number of ARGs per fly compared to a control group with poor sanitation. This protective effect was consistent across the six ARG classes that we detected. Enterobacteriaceae isolates-only from the pre-intervention phase-were resistant to a mean of 3.4 antibiotics out of the eleven assessed. Improving onsite sanitation infrastructure in low-income informal settlements may help reduce fly-mediated transmission of enteric bacteria and the ARGs carried by them.

Genetic Loci of Plant Pathogenic Dickeya solani IPO 2222 Expressed in Contact with Weed-Host Bittersweet Nightshade (Solanum dulcamara L.) Plants.

Dickeya solani, belonging to the Soft Rot Pectobacteriaceae, are aggressive necrotrophs, exhibiting both a wide geographic distribution and a wide host range that includes many angiosperm orders, both dicot and monocot plants, cultivated under all climatic conditions. Little is known about the infection strategies D. solani employs to infect hosts other than potato (Solanum tuberosum L.). Our earlier study identified D. solani Tn5 mutants induced exclusively by the presence of the weed host S. dulcamara. The current study assessed the identity and virulence contribution of the selected genes mutated by the Tn5 insertions and induced by the presence of S. dulcamara. These genes encode proteins with functions linked to polyketide antibiotics and polysaccharide synthesis, membrane transport, stress response, and sugar and amino acid metabolism. Eight of these genes, encoding UvrY (GacA), tRNA guanosine transglycosylase Tgt, LPS-related WbeA, capsular biosynthesis protein VpsM, DltB alanine export protein, glycosyltransferase, putative transcription regulator YheO/PAS domain-containing protein, and a hypothetical protein, were required for virulence on S. dulcamara plants. The implications of D. solani interaction with a weed host, S. dulcamara, are discussed.

Exploring the genetic basis of natural resistance to microcins.

Enterobacteriaceae produce an arsenal of antimicrobial compounds including microcins, ribosomally produced antimicrobial peptides showing diverse structures and mechanisms of action. Microcins target close relatives of the producing strain to promote its survival. Their narrow spectrum of antibacterial activity makes them a promising alternative to conventional antibiotics, as it should decrease the probability of resistance dissemination and collateral damage to the host's microbiota. To assess the therapeutic potential of microcins, there is a need to understand the mechanisms of resistance to these molecules. In this study, we performed genomic analyses of the resistance to four microcins [microcin C, a nucleotide peptide; microcin J25, a lasso peptide; microcin B17, a linear azol(in)e-containing peptide; and microcin E492, a siderophore peptide] on a collection of 54 Enterobacteriaceae from three species: Escherichia coli, Salmonella enterica and Klebsiella pneumoniae. A gene-targeted analysis revealed that about half of the microcin-resistant strains presented mutations of genes involved in the microcin mechanism of action, especially those involved in their uptake (fhuA, fepA, cirA and ompF). A genome-wide association study did not reveal any significant correlations, yet relevant genetic elements were associated with microcin resistance. These were involved in stress responses, biofilm formation, transport systems and acquisition of immunity genes. Additionally, microcin-resistant strains exhibited several mutations within genes involved in specific metabolic pathways, especially for S. enterica and K. pneumoniae.

Evaluation of Phenotypic Tests for Carbapenemase Detection in Enterobacteriaceae in Tunisia.

Introduction: Resistance to carbapenems in Enterobacteriaceae is a challenge for public health. Carbapenemase production is the leading mechanism. This work aims to evaluate four phenotypic methods for carbapenemase detection in comparison with a molecular method. Materials and Methods: Thirty-seven nonrepeating Enterobacteriaceae strains with decreased susceptibility to ertapenem were included. Imipenem MIC, Modified Hodge Test (MHT), Neo-Rapid Carb Kit® and KPC, MBL, and OXA-48 Confirm Kit® were performed. Isolates were tested for blaOXA-48, blaNDM, and blaVIM genes by end-point polymerase chain reaction. The results of the molecular study were used as a reference test to determine the performances of the phenotypic tests. Results: Imipenem resistance does not seem to be a good marker for carbapenemase production with a sensitivity of 54% (95% CI: 38-71). MHT showed 82% sensitivity (95% CI: 65-91). Overall, the enzymatic test showed the best performances for carbapenemase detection with 100% sensitivity (95% CI: 89-100) and the best turnaround time. The characterization of carbapenemases classes by the combined discs test demonstrated 88% overall sensitivity (95% CI: 72-95). Conclusion: The results of this study support the combination of the enzymatic and the combined disc tests for carbapenemase detection in Enterobacteria.

Carbapenem-producing Enterobacteriaceae in mothers and newborns in southeast Gabon, 2022.

Introduction: Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) pose a significant threat, leading to severe morbidity and mortality among newborns. Methods: This study, conducted at Franceville hospital's maternity and neonatology wards from February 22nd to June 20th, 2022, investigated the prevalence of CPE in 197 parturients and 203 newborns. Rectal swabs were taken from parturients before delivery and from newborns 30 minutes after birth. Blood culture samples were collected if signs of infection were observed in newborns during a 28-day follow-up. A total of 152 environmental samples were obtained, comprising 18 from sinks, 14 from incubators, 27 from cradles, 39 from maternal beds, 14 from tables and desks, four from the two baby scales and 36 from bedside furniture. Results: None of the 203 newborns were found to be CPE carriers 30 minutes after delivery. CPE carriage was found in 4.6% of mothers. When comparing colonized and uncolonized parturients, well-established risk factors for CPE carriage, such as recent hospitalization and antibiotic therapy, were more frequently observed among CPE carriers (33.3 vs 10.6% for hospitalization in the past 15 days; 55.5 vs 30.3% for hospitalization during pregnancy, and 55.5 vs 35.1% for antibiotic therapy during pregnancy). Notably, the prevalence of treatment with amoxicillin and clavulanic acid was 44.4% in CPE carriers compared to 17.0% in non-carriers. The incidence density of CPE-associated bloodstream infection was 0.49 per 100 newborns, accounting for a fatal case of CPE-associated bacteremia identified in one of the 203 newborns. Seven environmental samples returned positive for CPE (5 sinks and two pieces of furniture). Whole genome sequencing, performed on the 25 CPE isolates, revealed isolates carrying blaNDM-7 (n=10), blaNDM-5 (n=3), blaOXA181 (n=10), blaOXA48 (n=2) or blaOXA244 (n=1), along with genetic traits associated with the ability to cause severe and difficult-to-treat infections in newborns. Core genome comparison revealed nine CPE belonging to three international high-risk clones: E. coli ST410 (four mothers and a sink), two E. coli ST167 (a mother and a piece of furniture), and K. pneumoniae ST307 (a sink and a piece of furniture), with highly similar genetic backgrounds shared by maternal and environmental isolates, suggesting maternal contamination originating from the environment. Discussion: Our study reveals key findings may guide the implementation of infection control measures to prevent nosocomial infections in newborns: the prevalence of CPE carriage in one out of 20 parturients, an infection occurring in one out of 400 newborns, substantial contamination of the care environment, clinical and environmental CPE isolates possessing genetic traits associated with the ability to cause severe and challenging infections, and clonal relationships between clinical and environmental isolates suggesting CPE spread within the wards, likely contributing to the acquisition and colonization of CPE by parturients during pregnancy.

Unraveling the diversity and dissemination dynamics of antimicrobial resistance genes in Enterobacteriaceae plasmids across diverse ecosystems.

AIM: The objective of this study was to investigate the antimicrobial resistance genes (ARGs) in plasmids of Enterobacteriaceae from soil, sewage, and feces of food-producing animals and humans. METHODS AND RESULTS: The plasmid sequences were obtained from the NCBI database. For the identification of ARG, comprehensive antibiotic resistance database (CARD), and ResFinder were used. Gene conservation and evolution were investigated using DnaSP v.6. The transfer potential of the plasmids was evaluated using oriTfinder and a MOB-based phylogenetic tree was reconstructed using Fastree. We identified a total of 1064 ARGs in all plasmids analyzed, conferring resistance to 15 groups of antibiotics, mostly aminoglycosides, beta-lactams, and sulfonamides. The greatest number of ARGs per plasmid was found in enterobacteria from chicken feces. Plasmids from Escherichia coli carrying multiple ARGs were found in all ecosystems. Some of the most abundant genes were shared among all ecosystems, including aph(6)-Id, aph(3'')-Ib, tet(A), and sul2. A high level of sequence conservation was found among these genes, and tet(A) and sul2 are under positive selective pressure. Approximately 62% of the plasmids carrying at least one ARG were potentially transferable. Phylogenetic analysis indicated a potential co-evolution of Enterobacteriaceae plasmids in nature. CONCLUSION: The high abundance of Enterobacteriaceae plasmids from diverse ecosystems carrying ARGs reveals their widespread distribution and importance.

A novel characterized multi-drug-resistant Pseudocitrobacter sp. isolated from a patient colonized while admitted to a tertiary teaching hospital.

BACKGROUND: Reports of nosocomial infections typically describe recognised microorganisms. Here, a novel bacterial species was isolated, based on rectal swab screening for carbapenemases post-admission, then phenotypically and genetically characterized. METHODS: Sensititre, Vitek and API kits, MALDI and Illumina MiSeq were employed before profiles and phylogeny were compared with other related species. FINDINGS: Determined to be a possible Enterobacterales, the isolate was found to have 99.7% 16s rRNA identity to Pseudocitrobacter corydidari; an Asian cockroach-associated species. Given the highly conserved/low variability of 16S rRNA genes in Enterobacterales, average nucleotide identity (ANI) analysis compared the new isolate's genome with those of 18 Enterobacteriaceae species, including confirmed species of Pseudocitrobacter and unnamed Pseudocitrobacter species in the SILVA database. Of these, Pseudocitrobactercorydidari had the highest ANI at 0.9562. The published genome of the only known isolate of P.corydidari does not include Antimicrobial Resistance Genes (ARGs), with exception of potential drug efflux transporters. In contrast, our clinical isolate bears recognised antimicrobial resistance genes, including Klebsiella pneumoniae carbapenemase. The associated genome suggests resistance to carbapenems, ß-lactams, sulfonamides, fluoroquinolones, macrolides, aminoglycosides and cephalosporins. Phenotypic antimicrobial resistance was confirmed. CONCLUSION: Evident variations in ARG profiles, human colonization and origin in a clinically relevant niche that is geographically, physically and chemically disparate lend credibility for divergent evolution or, less likely, parallel evolution with P. corydidari. Genome data for this new species have been submitted to GENBANK using the proposed nomenclature Pseudocitrobacter limerickensis. The patient was colonized, rather than infected, and did not require antimicrobial treatment.

Occurrence and characteristics of extended-spectrum-ß-lactamase- and pAmpC-producing Klebsiella pneumoniae isolated from companion animals with urinary tract infections.

This study examined 70 Klebsiella pneumoniae isolates derived from companion animals with urinary tract infections in Taiwan. Overall, 81% (57/70) of the isolates carried extended-spectrum ß-lactamase (ESBL) and/or plasmid-encoded AmpC (pAmpC) genes. ESBL genes were detected in 19 samples, with blaCTX-M-1, blaCTX-M-9, and blaSHV being the predominant groups. pAmpC genes were detected in 56 isolates, with blaCIT and blaDHA being the predominant groups. Multilocus sequence typing revealed that sequence types (ST)11, ST15, and ST655 were prevalent. wabG, uge, entB, mrkD, and fimH were identified as primary virulence genes. Two isolates demonstrated a hypermucoviscosity phenotype in the string test. Antimicrobial susceptibility testing exhibited high resistance to ß-lactams and fluoroquinolones in ESBL-positive isolates but low resistance to aminoglycosides, sulfonamides, and carbapenems. Isolates carrying pAmpC genes exhibited resistance to penicillin-class ß-lactams. These findings provide valuable insights into the role of K. pneumoniae in the context of the concept of One Health.