Antimicrobial Resistance Genes Research Articles

Genomic Analysis of Cronobacter condimenti s37: Identification of Resistance and Virulence Genes and Comparison with Other Cronobacter and Closely Related Species.

Cronobacter condimenti are environmental commensals that have not been associated with any clinical infections. To date, they are the least understood and described Cronobacter species within the genus. The objective of this study was to use a draft genome sequence (DGS) of the Cronobacter condimenti strain s37 to screen for genes encoding for antibiotic resistance, virulence, response to environmental stress, and biofilm formation. The strain was isolated in Poland from commercial small radish sprouts. This is the second genome of this species available in the GenBank database. The comparative genome analysis (cgMLST) of C. condimenti s37 with other Cronobacter spp. including the pathogenic species C. sakazakii and the plant-associated closely related genera Franconibacter and Siccibacter was also performed. The assembled and annotated genome of the C. condimenti s37 genome was 4,590,991 bp in length, with a total gene number of 4384, and a GC content of 55.7%. The s 37 genome encoded for genes associated with resistance to stressful environmental conditions (metal resistance genes: zinc, copper, osmotic regulation, and desiccation stress), 17 antimicrobial resistance genes encoding resistance to various classes of antibiotics and 50 genes encoding for the virulence factors. The latter were mainly genes associated with adhesion, chemotaxis, hemolysis, and biofilm formation. Cg-MLST analysis (3991 genes) revealed a greater similarity of C. condimenti s37 to S. turicensis, F. pulveris, and C. dublinensis than to other species of the genus Cronobacter. Studies on the diversity, pathogenicity, and virulence of Cronobacter species isolated from different sources are still insufficient and should certainly be continued. Especially the analysis of rare strains such as s37 is very important because it provides new information on the evolution of these bacteria. Comparative cgMLST analysis of s37 with other Cronobacter species, as well as closely related genera Franconibacter and Siccibacter, complements the knowledge on their adaptability to specific environments such as desiccation.

Unveiling the High Diversity of Clones and Antimicrobial Resistance Genes in Escherichia coli Originating from ST10 across Different Ecological Niches.

In this pioneering in silico study in Peru, we aimed to analyze Escherichia coli (E. coli) genomes for antimicrobial resistance genes (ARGs) diversity and virulence and for its mobilome. For this purpose, 469 assemblies from human, domestic, and wild animal hosts were investigated. Of these genomes, three were E. coli strains (pv05, pv06, and sf25) isolated from chickens in our previous study, characterized for antimicrobial susceptibility profile, and sequenced in this study. Three other genomes were included in our repertoire for having rare cgMLSTs. The phenotypic analysis for antimicrobial resistance revealed that pv05, pv06, and sf25 strains presented multidrug resistance to antibiotics belonging to at least three classes. Our in silico analysis indicated that many Peruvian genomes included resistance genes, mainly to the aminoglycoside class, ESBL-producing E. coli, sulfonamides, and tetracyclines. In addition, through Multi-locus Sequence Typing, we found more than 180 different STs, with ST10 being the most prevalent among the genomes. Pan-genome mapping revealed that, with new lineages, the repertoire of accessory genes in E. coli increased, especially genes related to resistance and persistence, which may be carried by plasmids. The results also demonstrated several genes related to adhesion, virulence, and pathogenesis, especially genes belonging to the high pathogenicity island (HPI) from Yersinia pestis, with a prevalence of 42.2% among the genomes. The complexity of the genetic profiles of resistance and virulence in our study highlights the adaptability of the pathogen to different environments and hosts. Therefore, our in silico analysis through genome sequencing enables tracking the epidemiology of E. coli from Peru and the future development of strategies to mitigate its survival.

Proximity to humans is associated with antimicrobial-resistant enteric pathogens in wild bird microbiomes.

Humans are radically altering global ecology, and one of the most apparent human-induced effects is urbanization, where high-density human habitats disrupt long-established ecotones. Changes to these transitional areas between organisms, especially enhanced contact among humans and wild animals, provide new opportunities for the spread of zoonotic pathogens. This poses a serious threat to global public health, but little is known about how habitat disruption impacts cross-species pathogen spread. Here, we investigated variation in the zoonotic enteric pathogen Campylobacter jejuni. The ubiquity of C.jejuni in wild bird gut microbiomes makes it an ideal organism for understanding how host behavior and ecology influence pathogen transition and spread. We analyzed 700 C.jejuni isolate genomes from 30 bird species in eight countries using a scalable generalized linear model approach. Comparing multiple behavioral and ecological traits showed that proximity to human habitation promotes lineage diversity and is associated with antimicrobial-resistant (AMR) strains in natural populations. Specifically, wild birds from urban areas harbored up to three times more C.jejuni genotypes and AMR genes. This study provides novel methodology and much-needed quantitative evidence linking urbanization to gene pool spread and zoonoses.

Dynamic antimicrobial resistance and phylogenomic structure of Salmonella Typhimurium from 2007 to 2019 in Shanghai, China.

Our study uncovered a widespread distribution of Salmonella enterica serovar Typhimurium isolates in Shanghai accompanied by the increase in antimicrobial resistance (AMR) especially MDR during a 10-year period, which filled in the gap about a long period of continuous monitoring of AMR in this pathogen in Shanghai. Meanwhile, we identified a new clade ST34C2 of S. Typhimurium with the acquisition of IncI (Gamma)-like plasmids mediated by extended-spectrum β-lactamase gene blaCTX-M-14 as well as gyrA 87 mutation, which had not been reported before. It was noted that IncI (Gamma)-like plasmids were reported in S. Typhimurium for the first time and conjugation could accelerate the spread of antimicrobial resistance gene blaCTX-M-14. These findings on the epidemic, antimicrobial resistance, and phylogenomic characterization for S. Typhimurium provide valuable insights into its potential risk to public health and also the basis for AMR prevention and control strategies in Shanghai in the future.

Genomic Evaluation of Multidrug-Resistant Extended-Spectrum β-Lactamase (ESBL)-Producing Escherichia coli from Irrigation Water and Fresh Produce in South Africa: A Cross-Sectional Analysis.

Escherichia coli, both commensal and pathogenic, can colonize plants and persist in various environments. It indicates fecal contamination in water and food and serves as a marker of antimicrobial resistance. In this context, 61 extended-spectrum β-lactamase (ESBL)-producing E. coli from irrigation water and fresh produce from previous studies were characterized using whole genome sequencing (Illumina MiSeq). The Center for Genomic Epidemiology and Galaxy platforms were used to determine antimicrobial resistance genes, virulence genes, plasmid typing, mobile genetic elements, multilocus sequence typing (MLST), and pathogenicity prediction. In total, 19 known MLST groups were detected among the 61 isolates. Phylogroup B1 (ST58) and Phylogroup E (ST9583) were the most common sequence types. The six ST10 (serotype O101:H9) isolates carried the most resistance genes, spanning eight antibiotic classes. Overall, 95.1% of the isolates carried resistance genes from three or more classes. The blaCTX-M-1, blaCTX-M-14, and blaCTX-M-15 ESBL genes were associated with mobile genetic elements, and all of the E. coli isolates showed a >90% predicted probability of being a human pathogen. This study provided novel genomic information on environmental multidrug-resistant ESBL-producing E. coli from fresh produce and irrigation water, highlighting the environment as a reservoir for multidrug-resistant strains and emphasizing the need for ongoing pathogen surveillance within a One Health context.

Integrative genome-centric metagenomics for surface water surveillance: Elucidating microbiomes, antimicrobial resistance, and their associations

Surface water ecosystems are intimately intertwined with anthropogenic activities and have significant public health implications as primary sources of irrigation water in agricultural production. Our extensive metagenomic analysis examined 404 surface water samples from four different geological regions in Chile and Brazil, spanning irrigation canals (n = 135), rivers (n = 121), creeks (n = 74), reservoirs (n = 66), and ponds (n = 8). Overall, 50.25 % of the surface water samples contained at least one of the pathogenic or contaminant bacterial genera (Salmonella: 29.21 %; Listeria: 6.19 %; Escherichia: 35.64 %). Furthermore, a total of 1,582 antimicrobial resistance (AMR) gene clusters encoding resistance to 25 antimicrobial classes were identified, with samples from Brazil exhibiting an elevated AMR burden. Samples from stagnant water sources were characterized by dominant Cyanobacteriota populations, resulting in significantly reduced biodiversity and more uniform community compositions. A significant association between taxonomic composition and the resistome was supported by a Procrustes analysis (p < 0.001). Notably, regional signatures were observed regarding the taxonomic and resistome profiles, as samples from the same region clustered together on both ordinates. Additionally, network analysis illuminated the intricate links between taxonomy and AMR at the contig level. Our deep sequencing efforts not only mapped the microbial landscape but also expanded the genomic catalog with newly characterized metagenome-assembled genomes (MAGs), boosting the classification of reads by 12.85 %. In conclusion, this study underscores the value of metagenomic approaches in surveillance of surface waters, enhancing our understanding of microbial and AMR dynamics with far-reaching public health and ecological ramifications.

Dissemination and persistence of antimicrobial resistance (AMR) along the wastewater-river continuum

Antimicrobial resistance (AMR) is a global health hazard. Although clinical and agricultural environments are well-established contributors to the evolution and dissemination of AMR, research on wastewater treatment works (WwTWs) has highlighted their potential role as disseminators of AMR in freshwater environments. Using metagenomic sequencing and analysis, we investigated the changes in resistomes and associated mobile genetic elements within untreated wastewater influents and treated effluents of five WwTWs, and sediments collected from corresponding river environments in Oxfordshire, UK, across three seasonal periods within a year. Our analysis demonstrated a high diversity and abundance of antimicrobial resistance genes (ARGs) in untreated wastewater influents, reflecting the varied anthropogenic and environmental origins of wastewater. WwTWs effectively reduced AMR in the final effluent, with an average 87 % reduction in normalised ARG abundance and an average 63 % reduction in richness. However, wastewater effluents significantly impacted the antimicrobial resistome of the receiving rivers, with an average 543 % increase in ARG abundance and a 164 % increase in richness from upstream sediments to downstream sediments. The normalised abundance of the human gut-associated bacteriophage crAssphage was highly associated with both ARG abundance and richness. We observed seasonal variation in the resistome of raw influent which was not found in the effluent-receiving sediments. We illustrate the potential of WwTWs as focal points for disseminating ARGs and resistance-selecting chemicals, contributing to the elevation of environmental AMR. Our study emphasises the need for a comprehensive understanding of the anthropogenic impacts on AMR evolution and dissemination in wastewater and river environments, informing efforts to mitigate this growing public health crisis.

Comparison of microbiological and molecular diagnosis for identification of respiratory secondary infections in COVID-19 patients and their antimicrobial resistance patterns

We report the use of a new multiplex Real-Time PCR platform to simultaneously identify 24 pathogens and 3 antimicrobial-resistance genes directly from respiratory samples of COVID-19 patients. Results were compared to culture-based diagnosis.Secondary infections were detected in 60% of COVID-19 patients by molecular analysis and 73% by microbiological assays, with no significant differences in accuracy, indicating Gram-negative bacteria as the predominant species. Among fungal superinfections, Aspergillus spp. were detected by both methods in more than 7% of COVID-19 patients. Oxacillin-resistant S. aureus and carbapenem-resistant K. pneumoniae were highlighted by both methods.Secondary microbial infections in SARS-CoV-2 patients are associated with poor outcomes and an increased risk of death. Since PCR-based tests significantly reduce the turnaround time to 4 hours and 30 minutes (compared to 48 hours for microbial culture), we strongly support the routine use of molecular techniques, in conjunction with microbiological analysis, to identify co/secondary infections.

Microplastic-Mediated Transfer of Tetracycline Resistance: Unveiling the Role of Mussels in Marine Ecosystems.

The global threat of antimicrobial resistance (AMR) is exacerbated by the mobilization of antimicrobial resistance genes (ARGs) occurring in different environmental niches, including seawater. Marine environments serve as reservoirs for resistant bacteria and ARGs, further complicated by the ubiquity of microplastics (MPs). MPs can adsorb pollutants and promote bacterial biofilm formation, creating conditions favorable to the dissemination of ARGs. This study explores the dynamics of ARG transfer in the marine bivalve Mytilus galloprovincialis within a seawater model, focusing on the influence of polyethylene MPs on the mobilization of the Tn916-carrying tetM gene and plasmid-encoded ermB. Experiments revealed that biofilm formation on MPs by Enterococcus faecium and Listeria monocytogenes facilitated the transfer of the tetM resistance gene, but not the ermB gene. Furthermore, the presence of MPs significantly increased the conjugation frequency of tetM within mussels, indicating that MPs enhance the potential for ARG mobilization in marine environments. These findings highlight the role of MPs and marine organisms in ARG spread, underscoring the ecological and public health implications.

Unravelling the Complex Mechanisms of Multidrug Resistance in Bovine Mastitis Pathogens: Insights into Antimicrobial Resistance Genes, Biofilm Dynamics, and Efflux Systems.

Mastitis remains a paramount economic threat to dairy livestock, with antibiotic resistance severely compromising treatment efficacy. This study provides an in-depth investigation into the multidrug resistance (MDR) mechanisms in bacterial isolates from bovine mastitis, emphasizing the roles of antimicrobial resistance genes (ARGs), biofilm formation, and active efflux systems. A total of 162 Staphylococci, eight Escherichia coli, and seven Klebsiella spp. isolates were obtained from 215 milk samples of clinical and subclinical mastitis cases. Antibiotic susceptibility testing identified Twenty Staphylococci (12.35%), six E. coli (75%) and seven Klebsiella (100%) identified as MDR displaying significant resistance to β-lactams and tetracyclines The Multiple Antibiotic Resistance (MAR) index of these isolates ranged from 0.375 to 1.0, highlighting extensive resistance. Notably, 29 of the 33 MDR isolates produced biofilms on Congo red agar, while all exhibited biofilm formation in the Microtitre Plate assay. Critical ARGs (blaZ, blaTEM, blaCTX-M, tetM, tetA, tetB, tetC, strA/B, aadA) and efflux pump genes (acrB, acrE, acrF, emrB, norB) regulating active efflux were identified. This pioneering study elucidates the synergistic contribution of ARGs, biofilm production, and efflux pump activity to MDR in bovine mastitis pathogens. To our knowledge, this comprehensive study is the first of its kind, offering novel insights into the complex resistance mechanisms. The findings underscore the imperative need for advanced antibiotic stewardship and strategic interventions in dairy farming to curb the rise of antibiotic-resistant infections, thereby protecting both animal and public health.

Sensitivity and specificity of Nanopore sequencing for detecting carbapenem and 3rd-generation cephalosporin-resistant Enterobacteriaceae in urine samples: Real-time simulation with public antimicrobial resistance gene database

ObjectivesTo evaluate the accuracy of beta-lactamase gene detection directly from urine samples by Nanopore sequencing. MethodsDNA was extracted from bacterial pellets in spun urine. The purified DNA was then sequenced in native form by a Nanopore sequencer (MinION) to identify the organisms and beta-lactamase genes. Results were compared to routine urine cultures and standard antimicrobial susceptibility tests (AST). ResultsWe processed 60 urine samples of which routine cultures grew Enterobacteriaceae, including 28 carbapenem-resistant (CRE), 17 extended-spectrum beta-lactamase (ESBL) or AmpC producing, and 15 non-ESBL/AmpC phenotypes. We excluded 7 samples with extremely low DNA amounts (<1 ng/μl) for a final case of 53 in total. The sensitivity of antimicrobial resistance gene detection within 6 h, the optimal duration from real-time simulation, of Nanopore sequencing for the diagnosis of carbapenem-resistant and ceftriaxone-resistant phenotypes was 73.9 % (95%CI 56.0–91.9 %) and 81.1 % (95%CI 68.5–93.7 %), while the specificity was 96.7 % (95%CI 90.2–100.0 %) and 56.3 % (95%CI 31.9–80.6 %), respectively. The median times for MinION to generate DNA reads containing carbapenemase and ESBL/AmpC genes were 93 min (IQR 17–245.5) and 99 min (IQR 31.25–269.75) after sequencing commencement, respectively. ConclusionsNanopore sequencing can identify bacterial genotypic resistance in urine and may enable clinicians to adjust antimicrobial therapy earlier than routine AST.

COMPARATIVE WHOLE METAGENOME ANALYSIS IN LESIONAL AND NON-LESIONAL SCALP AREAS OF PSORIASIS CAPITIS PATIENTS AND HEALTHY INDIVIDUALS

Psoriasis is an immune-mediated inflammatory disorder, where the majority of the patients suffer from psoriasis capitis or scalp psoriasis. Current therapeutics remain ineffective to treat scalp lesions. Here, we present a whole-metagenome characterisation of the scalp microbiome in psoriasis capitis. We investigated how changes in the homeostatic cutaneous microbiome correlate with the condition and identified metagenomic biomarkers (taxonomic, functional, virulence factors, antimicrobial resistance genes) that could partly explain its emergence. Within this study, 83 top and back scalp samples from healthy individuals and 64 lesional and non-lesional scalp samples from untreated psoriasis capitis subjects were analysed. Using qPCR targeting the 16S and 18S rRNA genes, we found a significant decrease in microbial load within scalp regions affected by psoriasis compared to their non-lesional counterparts. Metagenomic analysis revealed that psoriatic lesions displayed significant lower Cutibacterium species (incl. C. modestum, C. namnetense, C. granulosum, C. porci), along with an elevation in Staphylococcus aureus. A heightened relative presence of efflux pump protein-encoding genes was detected, suggesting potential antimicrobial resistance mechanisms. These mechanisms are known to specifically target human antimicrobial peptides (incl. cathelicidin LL-37) which are frequently encountered within psoriasis lesions. These shifts in microbial community dynamics may contribute to psoriasis disease pathogenesis.

An increased prevalence of carbapenem-resistant hypervirulent Klebsiella pneumoniae associated with the COVID-19 pandemic

BackgroundKlebsiella pneumoniae (Kp) is a common community-acquired and nosocomial pathogen. Carbapenem-resistant and hypervirulent (CR-hvKp) variants can emerge rapidly within healthcare facilities and impacted by other infectious agents such as COVID-19 virus. MethodsTo understand the impact of COVID-19 virus on the prevalence of CR-hvKp, we accessed Kp genomes with corresponding metadata from GenBank. Sequence types (STs), antimicrobial resistance genes, and virulence genes, and those scores and CR-hvKp were identified. We analyzed population diversity and phylogenetic characteristics of five most common STs, measured the prevalence of CR-hvKp, identified CR-hvKp subtypes, and determined associations between carbapenem resistance gene subtypes with STs and plasmid types. These variables were compared pre- and during the COVID-19 pandemic. FindingsThe proportion of CR-hvKp isolates increased within multiple STs in different continents during the COVID-19 pandemic and persistent CR-hvKp subtypes were found in common STs. blaKPC was dominant in CG258, blaKPC-2 was detected in 97% of the ST11 CR-hvKp, blaNDM subtypes were prominent in ST147 (87.4%) and ST307 (70.8%); blaOXA-48 and its subtypes were prevalent in ST15 (80.5%). The possession of carbapenemase genes was different among subclades from different origins in different periods of time within each ST. IncFIB/IncHI1B hybrid plasmids contained virulence genes and carbapenemase genes and were predominant in ST147 (67.37%) and ST307 (56.25%). InterpretationThe prevalence of CR-hvKp increased during the COVID-19 pandemic, which was evident by an increase in local endemic clones. This process was facilitated by the convergence of plasmids containing carbapenemase genes and virulence genes. These findings have implications for the appropriate use of antimicrobials and infection prevention and control during outbreaks of respiratory viruses and pandemic management.

Characterization of a ST137 multidrug-resistant Campylobacter jejuni strain with a tet(O)-positive genomic island from a bloodstream infection patient

Campylobacter jejuni (C. jejuni) is a major cause of gastroenteritis and rarely cause bloodstream infection. Herein, we characterized a multidrug-resistant C. jejuni strain LZCJ isolated from a tumor patient with bloodstream infection. LZCJ was resistant to norfloxacin, ampicillin, ceftriaxone, ciprofloxacin and tetracycline. It showed high survival rate in serum and acidic environment. Whole genome sequencing (WGS) analysis revealed that strain LZCJ had a single chromosome of 1,629,078 bp (30.6% G+C content) and belonged to the ST137 lineage. LZCJ shared the highest identity of 99.66% with the chicken-derived C. jejuni MTVDSCj20. Four antimicrobial resistance genes (ARGs) were detected, blaOXA-61, tet(O), gyrA (T86I), and cmeR (G144D and S207G). In addition, a 12,746 bp genomic island GI_LZCJ carrying 15 open reading frames (ORFs) including the resistance gene tet(O) was identified. Sequence analysis found that the GI_LZCJ was highly similar to the duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. 137 non-synonymous mutations in motility related genes (flgF, fapR, flgS), capsular polysaccharide (CPS) coding genes (kpsE, kpsF, kpsM, kpsT), metabolism associated genes (nuoF, nuoG, epsJ, holB), and transporter related genes (comEA, gene0911) were confirmed in LZCJ compared with the best closed chicken-derived strain MTVDSCj20. Our study showed that C. jejuni strain LZCJ was highly similar to the chicken-derived strain MTVDSCj20 but with a lot of SNPs involved in motility, CPS and metabolism coding genes. This strain possessed a tet(O)-positive genomic island GI_LZCJ, which was closed to duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. The above data indicated that the LZCJ strain may originate from foodborne bacteria on animals and the importance of continuous surveillance for the spread of foodborne bacteria.

Population genomics uncovers global distribution, antimicrobial resistance, and virulence genes of the opportunistic pathogen Klebsiella aerogenes

Klebsiella aerogenes is an understudied and clinically important pathogen. We therefore investigate its population structure by genome analysis aligned with metadata. We sequence 130 non-duplicated K. aerogenes clinical isolates and identify two inter-patient transmission events. We then retrieve all publicly available K. aerogenes genomes (n= 1,026, accessed by January 1, 2023) and analyze them with our 130 genomes. We develop a core-genome multi-locus sequence-typing scheme. We find that K. aerogenes is a species complex comprising four phylogroups undergoing evolutionary divergence, likely forming three species. We delineate remarkable clonal diversity and identify three worldwide-distributed carbapenemase-encoding clonal clusters, representing high-risk lineages. We uncover that K. aerogenes has an open genome equippedby a large arsenal of antimicrobial resistance genes. We identify two genetic regions specific for K. aerogenes, encoding a type VI secretion system and flagella/chemotaxis for motility, respectively, both contributing to the virulence. These results provide much-needed insights into the population structure and pan-genomes of K. aerogenes.

Genomic insights of the emerging human pathogen Proteus appendicitidis sp. nov.

ObjectivesProteus are known as opportunistic human pathogens that can cause a variety of infections. Proteus appendicitidis is a novel Proteus species associated with appendicitis, whereas their genomic characteristics and virulence potential remain understudied. This study aims to compare the genomic features of P. appendicitidis to that of the close Proteus species, and to assess its virulence-factor encoding capacity as an emerging pathogen. MethodsGenomes similar to that of P. appendicitidis HZ0627T were retrieved from the PATRIC-v3.6.10 web-server using the implanted Similar Genome Finder tool. Average nucleotide identity (ANI) between HZ0627T and the retrieved genomes was calculated using FastANI-v1.33. Core-genome sequences were extracted using Roary-v3.13.0, and core-genomic tree was constructed using FastTree-v2.1.11. Virulence-factor encoding capacity was predicted using PathoFact-v1.0. ResultsTwo previously unclassified Proteus sp. strains were reclassified as P. appendicitidis. Strains phylogenomically close to P. appendicitidis were clustered into five species, three of which were previously categorized under P. vulgaris biogroup 3. Remarkably, Proteus genomosp. 6 was identified as the closest species to P. appendicitidis, exhibiting ANI values ranging from 94.45% to 94.95% against HZ0627T. Genome annotation revealed shared genomic features and antimicrobial resistance (AMR) genes between P. appendicitidis and its phylogenetic neighbors. Additionally, P. appendicitidis is hypothesized to share infection mechanisms with Proteus genomosp. 6, as evidenced by the encoding of numerous virulence factors implicated in cell lysis and membrane pore-formation in the genome of both species. ConclusionsThis study provides genomic insights of P. appendicitidis sp. nov. and its taxonomic relatives, shedding light on their evolutionary relationships, pathogenic mechanisms, and AMR profiles. The findings are significant for the development of targeted therapeutic interventions against infections caused by this emerging pathogen.

Capturing clinically relevant Campylobacter attributes through direct whole genome sequencing of stool.

Campylobacter is the leading bacterial cause of infectious intestinal disease, but the pathogen typically accounts for a very small proportion of the overall stool microbiome in each patient. Diagnosis is even more difficult due to the fastidious nature of Campylobacter in the laboratory setting. This has, in part, driven a change in recent years, from culture-based to rapid PCR-based diagnostic assays which have improved diagnostic detection, whilst creating a knowledge gap in our clinical and epidemiological understanding of Campylobacter genotypes - no isolates to sequence. In this study, direct metagenomic sequencing approaches were used to assess the possibility of replacing genome sequences with metagenome sequences; metagenomic sequencing outputs were used to describe clinically relevant attributes of Campylobacter genotypes. A total of 37 diarrhoeal stool samples with Campylobacter and five samples with an unknown pathogen result were collected and processed with and without filtration, DNA was extracted, and metagenomes were sequenced by short-read sequencing. Culture-based methods were used to validate Campylobacter metagenome-derived genome (MDG) results. Sequence output metrics were assessed for Campylobacter genome quality and accuracy of characterization. Of the 42 samples passing quality checks for analysis, identification of Campylobacter to the genus and species level was dependent on Campylobacter genome read count, coverage and genome completeness. A total of 65% (24/37) of samples were reliably identified to the genus level through Campylobacter MDG, 73% (27/37) by culture and 97% (36/37) by qPCR. The Campylobacter genomes with a genome completeness of over 60% (n=21) were all accurately identified at the species level (100%). Of those, 72% (15/21) were identified to sequence types (STs), and 95% (20/21) accurately identified antimicrobial resistance (AMR) gene determinants. Filtration of stool samples enhanced Campylobacter MDG recovery and genome quality metrics compared to the corresponding unfiltered samples, which improved the identification of STs and AMR profiles. The phylogenetic analysis in this study demonstrated the clustering of the metagenome-derived with culture-derived genomes and revealed the reliability of genomes from direct stool sequencing. Furthermore, Campylobacter genome spiking percentages ranging from 0 to 2% total metagenome abundance in the ONT MinION sequencer, configured to adaptive sequencing, exhibited better assembly quality and accurate identification of STs, particularly in the analysis of metagenomes containing 2 and 1% of Campylobacter jejuni genomes. Direct sequencing of Campylobacter from stool samples provides clinically relevant and epidemiologically important genomic information without the reliance on cultured genomes.

Safety and efficacy of a feed additive consisting of Loigolactobacillus coryniformisDSM 34345 as a silage additive for all animal species (Lactosan GmbH & Co.KG).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on Loigolactobacillus coryniformis DSM 34345 when used as a technological additive to improve ensiling of fresh plant material. The additive is intended for use with all fresh plant material for all animal species at a proposed minimum concentration of 1 × 108 colony forming units (CFU)/kg fresh plant material. The bacterial species L. coryniformis is considered by EFSA to be suitable for the qualified presumption of safety approach to safety assessment. The identity of the strain was established and no acquired antimicrobial resistance genes of concern were detected. Therefore, the FEEDAP Panel concluded that the use of the strain as a silage additive is considered safe for all the animal species, for consumers of products from animals fed the treated silage and for the environment. Regarding user safety, the additive containing Loigolactobacillus coryniformis DSM 34345 should be considered as a potential skin and respiratory sensitiser, and any exposure through skin and respiratory tract is considered a risk. One preparation was shown not to be irritant to skin or eyes. However, the Panel cannot assess the irritation potential of other possible preparations. The FEEDAP Panel concluded that Loigolactobacillus coryniformis DSM 34345 has the potential to improve the production of silages prepared from all fresh plant materials at a minimum concentration of 1 × 108 CFU/kg fresh material.

Antibiotic Resistance Profiles of Escherichia coli and Salmonella spp. Isolated From Dairy Farms and Surroundings in a Rural Area of Western Anatolia, Turkey.

Background Antibiotic resistance is a significant public health issue worldwide. Antibiotic-resistant zoonotic bacteria such as Escherichia coli (E. coli), Campylobacter, Salmonella, Listeria, Coxiella, and Mycobacterium can be particularly isolated from biofertilizers. Epidemiological studies have shown that cases of foodborne infections and intoxications are significantly related to animal-derived foods. The presence of these species in aquatic environments indicates areas or organisms contaminated with animal or human feces. Especially, the presence of E. coli in aquatic environments has become a serious problem worldwide. Pathogenic strains of E. coli cause waterborne and foodborne diseases. Materials and methods This study included a total of 290 samples collected from five different dairy farms between April and September 2023 which comprised 20 samples of cow manure, 20 samples of milk, three samples of dairy workers' hand washing water, five samples ofsoil, five samples of water, and five samples of vegetables. The samples taken from the farms were homogenized with 0.1% peptone water at a ratio of 1/10. They were then cultured on xylose lysine deoxycholate (XLD), eosin methylene blue agar (EMB), and blood agar media, and gram-negative colonies were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the VITEK2 automated system (BioMerieux Inc., Durham, NC). Amplification of the isolated DNA extracts was performed with A.B.T.™ 2X HS-PCR MasterMix (A.B.T Laboratory Industry, Arnavutköy, Turkey) in the SimpliAmp™ thermal cycler (Thermo Fischer Scientific Inc., Waltham, MA) and visualized by agarose gel electrophoresis. Results Among the 52E. coli strains isolated in our study, the highest antibiotic sensitivity rate was observed in meropenem, while the lowest sensitivity rates were determined in cefazolin and cefuroxime. While two of the Salmonella spp. (n = 2) isolates were found to be resistant to tetracycline, and one was found to be resistant to penicillin and ampicillin. No resistance to trimethoprim/sulfamethoxazole was detected in either isolate. Extended-spectrum beta-lactamases (ESBLs) were detected in only four (7.7%) E. coli strains. While tetA, tetB, and TEM genes were seen in almost all E. coli strains, they were not found in Salmonella spp. Conclusion In conclusion, our study revealed the presence of antimicrobial resistance genes in E. coli and Salmonella spp. isolates collected from various farms and environmental samples, which render the antimicrobials used for disease treatment ineffective. Consequently, research should be undertaken to prevent the development of new resistance genes in our country, as creating new medications and treatment strategies for these diseases is costly and time-intensive.

Hypermucoviscous Multidrug-Resistant Klebsiella variicola Strain LL2208 Isolated from Chinese Longsnout Catfish (Leiocassis longirostris): Highly Similar to Human K. variicola Strains.

Outbreaks of bacterial diseases occur in farmed Chinese longsnout catfish (Leiocassis longirostris). Due to limited information on aquatic Klebsiella variicola-infected animals, this study aimed to identify strain LL2208 isolated from diseased L. longirostris, determine its biological features, and evaluate its risk to public health. Strain LL2208 was tested for molecular identification, challenge, string, biofilm formation, and antimicrobial susceptibility. Furthermore, the whole genome of the strain was sequenced and analyzed. Based on molecular identification, strain LL2208 was identified as K. variicola. Artificial infection showed that this strain was moderately virulent to L. longirostris with an LD50 = 7.92 × 107 CFU/mL. Antibiotic sensitivity tests showed that this strain was resistant to penicillins, macrolides, aminoglycosides, amphenicols, glycopeptides, and lincosamide, indicating multidrug resistance. Strain LL2208 has a genome size of 5,557,050 bp, with a GC content of 57.38%, harboring 30 antimicrobial resistance genes and numerous virulence-related genes. Its molecular type was ST595-KL16-O5. Collinearity analysis showed that strain LL2208 was highly similar to the human-derived K. variicola strain. In conclusion, the multidrug-resistant and virulent K. variicola strain LL2208 was isolated from fish and may have originated from humans. These results provide a foundation for further studies on the transmission of K. variicola between humans and aquatic animals.