Antimicrobial Resistance Determinants Research Articles

Comparison of Oral Microbial Composition and Determinants Encoding Antimicrobial Resistance in Dogs and Their Owners.

Consolidated studies on animal, human, and environmental health have become very important for understanding emerging zoonotic diseases and the spread of antimicrobial resistance (AMR). The aim of this study was to analyse the oral microbiomes of healthy dogs and their owners, including determinants of AMR. Shotgun metagenomic sequencing detected 299 bacterial species in pets and their owners, from which 70 species were carried by dogs and 229 species by humans. Results demonstrated a unique microbial composition of dogs and their owners. At an order level, Bacteroidales were the most prevalent oral microbiota of dogs with significantly lower prevalence in their owners where Actinomycetales and Lactobacillales predominated. Porphyromonas and Corynebacterium were the most prevalent genera in dogs, whereas Streptococcus and Actinomyces were in animal owners. The resistances to macrolides, tetracyclines, lincosamides and Cfx family A class broad-spectrum β-lactamase were detected in both animal and human microbiomes. Resistance determinants to amphenicols, aminoglycosides, sulphonamides, and quaternary ammonium compounds were detected exceptionally in dogs. In conclusion, the study demonstrated different bacterial composition in oral microbiomes of healthy dogs without clinical signs of periodontal disease and their owners. Due to the low numbers of the samples tested, further investigations with an increased number of samples should be performed.

Comparison of contemporary invasive and non-invasive Streptococcus pneumoniae isolates reveals new insights into circulating anti-microbial resistance determinants.

Streptococcus pneumoniae is a major human pathogen with a high burden of disease. Non-invasive isolates (those found in non-sterile sites) are thought to be a key source of invasive isolates (those found in sterile sites) and a reservoir of anti-microbial resistance (AMR) determinants. Despite this, pneumococcal surveillance has almost exclusively focused on invasive isolates. We aimed to compare contemporaneous invasive and non-invasive isolate populations to understand how they interact and identify differences in AMR gene distribution. We used a combination of whole-genome sequencing and phenotypic anti-microbial susceptibility testing and a data set of invasive (n = 1,288) and non-invasive (n = 186) pneumococcal isolates, collected in Victoria, Australia, between 2018 and 2022. The non-invasive population had increased levels of antibiotic resistance to multiple classes of antibiotics including beta-lactam antibiotics penicillin and ceftriaxone. We identified genomic intersections between the invasive and non-invasive populations and no distinct phylogenetic clustering of the two populations. However, this analysis revealed sub-populations overrepresented in each population. The sub-populations that had high levels of AMR were overrepresented in the non-invasive population. We determined that WamR-Pneumo was the most accurate in silico tool for predicting resistance to the antibiotics tested. This tool was then used to assess the allelic diversity of the penicillin-binding protein genes, which acquire mutations leading to beta-lactam antibiotic resistance, and found that they were highly conserved (≥80% shared) between the two populations. These findings show the potential of non-invasive isolates to serve as reservoirs of AMR determinants.

Characterization of a Tigecycline-, Linezolid- and Vancomycin-Resistant Clinical Enteroccoccus faecium Isolate, Carrying vanA and vanB Genes.

Increasing incidence of Enterococcus faecium resistant to key antimicrobials used in therapy of hospitalized patients is a worrisome phenomenon observed worldwide. Our aim was to characterize a tigecycline-, linezolid- and vancomycin-resistant E. faecium isolate with the vanA and vanB genes, originating from a hematoma of a patient hospitalized in an intensive care unit in Poland. Antimicrobial susceptibility (a broad panel) was tested using gradient tests with predefined antibiotic concentrations. The complete genome sequence was obtained from a mixed assembly of Illumina MiSeq and Oxford Nanopore's MinION reads. The genome was analyzed with appropriate tools available at the Center for Genomic Epidemiology, PubMLST and GenBank. Transferability of oxazolidinone, tigecycline and vancomycin resistance genes was investigated by conjugation, followed by PCR screen of transconjugants for antimicrobial resistance genes and plasmid rep genes characteristic for the donor and genomic sequencing of selected transconjugants. The isolate was resistant to most antimicrobials tested; susceptibility to daptomycin, erythromycin and chloramphenicol was significantly reduced, and only oritavancin retained the full activity. The isolate represented sequence type 18 (ST18) and carried vanA, vanB, poxtA, fexB, tet(L), tet(M), aac(6')-aph(2''), ant(6)-Ia and ant(6')-Ii. The vanA, poxtA and tet(M) genes located on ~ 40-kb plasmids were transferable by conjugation yielding transconjugants resistant to vancomycin, linezolid and tigecycline. The substitutions in LiaS, putative histidine kinase, SulP, putative sulfate transporter, RpoB and RpoC were potential determinants of an elevated daptomycin MIC. Comparative analyses of the studied isolate with E. faecium isolates from other countries revealed its similarity to ST18 isolates from Ireland and Uganda from human infections. We provide the detailed characteristics of the genomic determinants of antimicrobial resistance of a clinical E. faecium demonstrating the concomitant presence of both vanA and vanB and resistance to vancomycin, linezolid, tigecycline and several other compounds and decreased daptomycin susceptibility. This isolate is a striking example of an accumulation of resistance determinants involving various mechanisms by a single hospital strain.

A genomic survey of Clostridioides difficile isolates from hospitalized patients in Melbourne, Australia.

There has been a decrease in healthcare-associated Clostridioides difficile infection (CDI) in Australia, coupled with an increase in the genetic diversity of strains isolated in these settings, and an increase in community-associated cases. To explore this changing epidemiology, we studied the genetic relatedness of C. difficile isolated from patients at a major hospital in Melbourne, Australia. Whole-genome sequencing of C. difficile isolates from symptomatic (n = 61) and asymptomatic (n = 10) hospital patients was performed. Genomic comparisons were made using single-nucleotide polymorphism (SNP) analysis, ribotyping, and toxin, resistome, and mobilome profiling. C. difficle clade 1 strains were found to be predominant (64/71), with most strains (63/71) encoding both toxins A and B (A+B+). Despite these similarities, only two isolates were genetically related (≤2 SNPs) and a diverse range of ribotypes was detected, with those predominating including ribotypes commonly found in community-associated cases. Five non-toxigenic (A-B-CDT-) clade 1 strains were identified, all in asymptomatic patients. Three clade 4 (A-B+CDT-) and four clade 5 (A+B+CDT+) strains were detected also, with these strains more likely to carry antimicrobial resistance determinants, many of which were associated with mobile genetic elements. Overall, within a single hospital, C. difficile-associated disease was caused by a diverse range of strains, including many strain types associated with community and environmental sources. While strains carried asymptomatically were more likely to be non-toxigenic, toxigenic strains were isolated also from asymptomatic patients, which together suggest the presence of diverse sources of transmission, potentially including asymptomatic patients. IMPORTANCE There has been a decrease in healthcare-associated Clostridioides difficile infection in Australia, but an increase in the genetic diversity of infecting strains, and an increase in community-associated cases. Here, we studied the genetic relatedness of C. difficile isolated from patients at a major hospital in Melbourne, Australia. Diverse ribotypes were detected, including those associated with community and environmental sources. Some types of isolates were more likely to carry antimicrobial resistance determinants, and many of these were associated with mobile genetic elements. These results correlate with those of other recent investigations, supporting the observed increase in genetic diversity and prevalence of community-associated C. difficile, and consequently the importance of sources of transmission other than symptomatic patients. Thus, they reinforce the importance of surveillance for in both hospital and community settings, including asymptomatic carriage, food, animals, and other environmental sources to identify and circumvent important sources of C. difficile transmission.

Pathognomonic features of Pasteurella multocida isolates among various avian species in Sharkia Governorate, Egypt

The present study aimed to isolate Pasteurella multocida (P. multocida) from pulmonary cases in several avian species and then investigate the histopathological features, antimicrobial resistance determinants, virulence characteristics, and risk factors analysis of the isolates in each species in correlation with epidemiological mapping of pasteurellosis in Sharkia Governorate, Egypt. The obtained data revealed a total occurrence of 9.4% (30/317) of P. multocida among the examined birds (chickens, ducks, quails, and turkeys). The incidence rate was influenced by avian species, climate, breed, age, clinical signs, and sample type. Antimicrobial susceptibility testing revealed that all isolates were sensitive to florfenicol and enrofloxacin, while 86.6 and 73.3% of the isolates displayed resistance to amoxicillin-clavulanic acid and erythromycin, respectively. All of the P. multocida isolates showed a multiple-drug resistant pattern with an average index of 0.43. Molecular characterization revealed that the oma87, sodA, and ptfA virulence genes were detected in the all examined P. multocida isolates. The ermX (erythromycin), blaROB-1 (β-lactam), and mcr-1(colistin) resistance genes were present in 60, 46.6, and 40% of the isolates, respectively. Ducks and quails were the most virulent and harbored species of antimicrobial-resistant genes. These results were in parallel with postmortem and histopathological examinations which detected more severe interstitial pneumonia lesions in the trachea and lung, congestion, and cellular infiltration especially in ducks. Epidemiological mapping revealed that the Fakous district was the most susceptible to pasteurellosis infection. Thus, farmers are recommended to monitor their flocks for signs of respiratory disease, seek veterinary care promptly if any birds are sick, and avoid the random usage of antibiotics. In conclusion, this study presents a comprehensive picture of the risk factors in correlation to the pathognomonic characteristics of P. multocida infection in poultry sectors to help in developing more effective strategies for prevention and control.

A One Health approach based on genomics for enhancing the Salmonella enterica surveillance in Colombia

ObjectivesThis study aimed to provide evidence of the domestic benefits of introducing an integrative genomic analysis from the One Health approach in the national surveillance of Salmonella enterica between 1997-2017 in Colombia. MethodsData on Salmonella from clinical laboratory-based surveillance between 1997-2017 and from a national cross-sectional study at chicken retail stores in Colombia were compared using a phenotypic, molecular, and genomic approaches. Additional analysis by serovar using single nucleotide polymorphism was developed to increase the resolution of the relatedness between the interfaces. ResultsLocally, the diversity and pathogenic factors of the prevalent S. enterica serovars associated with foodborne disease in Colombia were described using laboratory, pulse field gel electrophoresis, and whole genome sequencing data. For example, the resolution of pulse field gel electrophoresis allowed the description of two main foodborne clusters of Salmonella Enteritidis isolates, which were expanded to eight foodborne clades using whole genome sequencing. Likewise, virulence factors and antimicrobial resistance determinants, and mobile genetic elements that converged in the foodborne clades should be considered a public health concern in Colombia. All results by serovar were compiled in an interactive easy to share report. ConclusionWhole genome sequencing is a technology that provides a precise assessment of emerging foodborne risks such as the Salmonella foodborne clades, but it requires an integrative and continued collaboration between the stakeholders across the One Health sectors to promote appropriated actions and policies in public health.

Rapid and Accurate Antimicrobial Susceptibility Testing Using Label-Free Electrical Impedance-Based Microfluidic Platform.

Antimicrobial resistance has become a serious threat to the global public health. Accurate and rapid antimicrobial susceptibility testing (AST) allows evidence-based prescribing of antibiotics to improve patient care and clinical outcomes. Current culture-based AST assays are inherently limited by the doubling time of bacterial reproduction, which require at least 24 h to have a decisive result. Herein, a label-free electrical impedance-based microfluidic platform designed to expedite and streamline AST procedure for clinical practice is presented. Following a 30-min exposure of bacterial samples to antibiotics, the presented high-throughput, single-bacterium level impedance characterization platform enables a rapid 2-min AST assay. The platform facilitates accurate analysis of individual bacterial viability, as indicated by changes in electrical characteristics, thereby enabling the determination of antimicrobial resistance. Moreover, the potential clinical applicability of this platform is demonstrated by testing different E. coli strains against five antibiotics, yielding 100% categorical agreements compared to standard culture methods.

Nosocomial infections by diverse carbapenemase-producing Aeromonas hydrophila associated with combination of plumbing issues and heat waves

Aquatic opportunistic pathogen Aeromonas hydrophila, known to persist in low-nutrient chlorinated waters, can cause life-threatening infections. Two intensive care units experienced a cluster of Aeromonas infections following outdoor temperature spikes coinciding with recurrent plumbing issues, with fatalities due to severe underlying comorbidities co-occurring with extensively-drug resistant (XDR) Aeromonas. We investigated this cluster using whole genome sequencing to assess genetic relatedness of isolates and identify antimicrobial resistance determinants. Three A. hydrophila were isolated from patients staying in or adjacent to rooms with plumbing issues during or immediately after periods of elevated outdoor temperatures. Sinks and faucets were swabbed for culture. All A. hydrophila clinical isolates exhibited carbapenem resistance but were not genetically related. Diverse resistance determinants corresponding to extensively-drug resistant were found, including co-occurring KPC-3 and VIM-2, OXA-232, and chromosomal CphA-like carbapenemase genes, contributing to major treatment challenges. All 3 patients were treated with multiple antibiotic regimens to overcome various carbapenemase classes and expired due to underlying comorbidities. Environmental culture yielded no Aeromonas. While the investigation revealed no singular source of contamination, it supports a possible link between plumbing issues, elevated outdoor temperatures and incidence of nosocomial Aeromonas infections. The diversity of carbapenemase genes detected in these wastewater-derived Aeromonas warrants heightened infection prevention precautions during periods of plumbing problems especially with heat waves.

The dynamic evolution and IS26-mediated interspecies transfer of a blaNDM-1-bearing fusion plasmid leading to a hypervirulent carbapenem-resistant Klebsiella pneumoniae strain harbouring blaKPC-2 in a single patient

To characterize the evolution and interspecies transfer of plasmids between Klebsiella pneumoniae and Escherichia coli within a single patient. Minimum inhibitory concentrations were measured using broth microdilution assays. Conjugation assays, string tests, and Galleria mellonella infection model experiments were also conducted. Whole-genome sequencing was performed on the Illumina and Nanopore platforms. Antimicrobial resistance determinants, insertion sequences, and virulence factors were identified using ABRicate/ResFinder database, ISFinder, and virulence factor database. Wzi and capsular polysaccharide (KL) were typed using Kleborate and Kaptive. Multi-locus sequence typing (MLST), replicon typing, and single nucleotide polymorphism analyses were conducted using the BacWGSTdb server. The carbapenem-resistant K. pneumoniae 2111KP was characterized as ST11, wzi64, and KL64, with a positive string test result and a relatively high virulence phenotype. Analysis of the 2111KP genome revealed that blaNDM-1 was located in a 268,400-bp IncFIB/IncHI1B/IncX3 conjugative plasmid (p2111KP-1), regulated by IS26, IS5, and ISKox3. p2111KP-1 was also a rmpA2-associated virulence plasmid with an iutA-iucABCD gene cluster and a IS26-mediated multidrug-resistant fusion plasmid, which contained 8-bp (AGCTGCAC or GGCCTTTG) target site duplications. Segments flanked by IS26 of p2111KP-1 were 99.99% identical to a 49,016-bp E. coli plasmid. This study provided direct evidence of plasmid fusion via IS26 between two different bacterial species within one patient and revealed the process by which genetic elements conferring carbapenem resistance and virulence were simultaneously transferred between these species. It highlights the need for strategic antibiotic use and rigorous monitoring to prevent the plasmid-mediated fusion and transmission of drug-resistance/virulence factors.

A Whole-Genome Sequencing-Based Approach for the Characterization of Klebsiella pneumoniae Co-Producing KPC and OXA-48-like Carbapenemases Circulating in Sardinia, Italy.

Whole-genome sequencing (WGS) provides important information for the characterization, surveillance, and monitoring of antimicrobial resistance (AMR) determinants, particularly in cases of multi- and extensively drug-resistant microorganisms. We reported the results of a WGS analysis carried out on carbapenemases-producing Klebsiella pneumoniae, which causes hospital-acquired infections (HAIs) and is characterized by a marked resistance profile. Clinical, phenotypic, and genotypic data were collected for the AMR surveillance screening program of the University Hospital of Sassari (Italy) during 2020-2021. Genomic DNA was sequenced using the Illumina Nova Seq 6000 platform. Final assemblies were manually curated and carefully verified for the detection of antimicrobial resistance genes, porin mutations, and virulence factors. A phylogenetic analysis was performed using the maximum likelihood method. All 17 strains analyzed belonged to ST512, and most of them carried the blaKPC-31 variant blaOXA-48-like, an OmpK35 truncation, and an OmpK36 mutation. Phenotypic analysis showed a marked resistance profile to all antibiotic classes, including β-lactams, carbapenems, aminoglycosides, fluoroquinolone, sulphonamides, and novel β-lactam/β-lactamase inhibitors (BL/BLI). WGS characterization revealed the presence of several antibiotic resistance determinants and porin mutations in highly resistant K. pneumoniae strains responsible for HAIs. The detection of blaKPC-31 in our hospital wards highlights the importance of genomic surveillance in hospital settings to monitor the emergence of new clones and the need to improve control and preventive strategies to efficiently contrast AMR.

Whole genome sequence-based characterization of virulence and antimicrobial resistance gene profiles of Staphylococcus aureus isolated from food poisoning incidents in eastern China.

Staphylococcus aureus is an opportunistic foodborne pathogen occasionally isolated from diarrhea patients. In recent years, increasing studies have reported the detection of S. aureus in food poisoning incidents due to food contamination in the North and South of China. However, the epidemiology and genetic characteristics of S. aureus from food poisoning incidents in Eastern China remain unknown. The present study examined the genetic characteristics, antimicrobial resistance, and virulent factors of multidrug-resistant S. aureus isolated from 22 food poisoning incidents reported by the hospitals and health centers in Eastern China from 2011 to 2021. A total of 117 resistant and enterotoxigenic S. aureus isolates were collected and sequenced, among which 20 isolates were identified as methicillin resistant. Genetic analysis revealed 19 distinct CC/ST types, with CC6, CC22, CC59, CC88, and CC398 being the most frequent variants in methicillin-resistant S. aureus (MRSA). A considerable shift in CC types from CC1 to CC398 between 2011 and 2021 was observed in this study, indicating that CC398 may be the main epidemic strain circulating in the current food poisoning incidents. Additionally, genes for enterotoxins were detected in 55 isolates, with a prevalence of 27.8% (27/97) for methicillin-sensitive variants and 35.0% (7/20) for MRSA. The scn gene was detected in 59.0% of the isolates, demonstrating diverse contaminations of S. aureus among livestock-to-human transmission. Of the 117 isolates, only ten isolates displayed multi-drug resistance (MDR) to penicillin, tetracycline, and macrolides. None of the 117 foodborne S. aureus isolates tested positive for vanA in this study. Together, the present study provided phylogenetic characteristics of S. aureus from food poisoning incidents that emerged in Eastern China from 2011 to 2021. Our results suggested that these diarrhea episodes were hypotonic and merely transient low-MDR infections, however, further research for continued surveillance given the detection of virulence and antimicrobial resistance determinants is required to elucidate the genomic characteristics of pathogenic S. aureus in food poisoning incidents in the context of public health.

Global diversity and antimicrobial resistance of typhoid fever pathogens: Insights from a meta-analysis of 13,000 Salmonella Typhi genomes.

The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000). This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch. Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show the potential of travel-associated sequences to provide informal 'sentinel' surveillance for such locations. The data indicate that ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level ciprofloxacin resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020) but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes. The consortium's aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies. No specific funding was awarded for this meta-analysis. Coordinators were supported by fellowships from the European Union (ZAD received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 845681), the Wellcome Trust (SB, Wellcome Trust Senior Fellowship), and the National Health and Medical Research Council (DJI is supported by an NHMRC Investigator Grant [GNT1195210]).

Sequencing-based analysis of the microbiomes of Spanish food processing facilities reveals environment-specific variation in the dominant taxa and antibiotic resistance genes

In the last years, advances in high throughput sequencing technologies have opened the possibility to broaden environmental monitoring activities in facilities processing food, offering expanded opportunities for characterizing in an untargeted manner the microbiome and resistome of foods and food processing environments (FPE) with huge potential benefits in food safety management systems. Here the microbiome and resistome of FPE from slaughterhouses (n = 3), dairy (n = 12) and meat (n = 10) processing plants were assessed through whole metagenome sequencing of 2 composite samples for each facility, comprising 10 FPE swabs taken from food contact surfaces and 10 FPE samples from non-food contact surfaces, respectively. FPE from slaughterhouses had more diverse microbiomes and resistomes, while FPE from dairy processing plants showed the highest β-dispersion, consistent with a more heterogeneous microbiome and resistome composition. The predominant bacterial genera depended on the industry type, with Pseudomonas and Psychrobacter being highly dominant in surfaces from slaughterhouses and meat industries, while different lactic acid bacteria predominated in dairy industries. The most abundant antimicrobial resistance genes (ARG) found were associated with resistance to aminoglycosides, tetracyclines and quaternary ammonium compounds (QAC). ARGs relating to resistance to aminoglycosides and tetracyclines were significantly more prevalent in slaughterhouses than in food processing plants, while QAC resistance genes were particularly abundant in some food contact surfaces from dairy and meat processing plants, suggesting that daily sanitation under suboptimal conditions may be selecting for persistent microbiota tolerant to these biocides in some facilities. The taxonomic mapping of ARG pointed to specific bacterial genera, such as Escherichia, Bacillus, or Staphylococcus, as carriers of the most relevant resistance determinants. About 63% of all ARG reads were assigned to contigs classified as plasmid-associated, indicating that the resistome of FPE may be strongly shaped through the spread of mobile genetic elements. Overall, the relevance of FPE as reservoirs of ARG was confirmed and it was demonstrated that next generation sequencing technologies allowing a deep characterisation of sources and routes of spread of microorganisms and antimicrobial resistance determinants in food industry settings hold promise to be integrated in monitoring and food safety management programmes.

Evaluation of metagenomic assembly methods for the detection and characterization of antimicrobial resistance determinants and associated mobilizable elements

Antimicrobial resistance genes (ARGs) can be transferred between members of a bacterial population by mobile genetic elements (MGE). Understanding the risk of these transfer events is important in monitoring and predicting antimicrobial resistance (AMR), especially in the context of a One Health Continuum. However, there is no universally accepted method for detection of ARGs and MGEs, and especially for determining their linkages. This study used publicly available shotgun metagenomic DNA short-read (Illumina, 100 bp paired-end) sequence data from samples across the One Health Continuum (including beef cattle composite feces from feedlots, catch basin water at feedlots, agricultural soil from feedlot manured surrounding fields, and urban/municipal sewage influent from two municipal wastewater treatment plants) to develop a workflow to identify and associate ARGs and MGEs. ARG- and MGE-based targeted-assemblies with available short-read data were unable to meet this analysis goal. In contrast, de novo assembly of contigs provided enough sequence context to associate ARGs and MGEs, without compromising discovery rate. However, to estimate the relative abundance of these elements, unassembled sequence data must still be used.

Differential effects of wastewater treatment plant effluents on the antibiotic resistomes of diverse river habitats.

Wastewater treatment plants (WWTPs) are key sources of antimicrobial resistance genes (ARGs) that could influence the resistomes of microbial communities in various habitats of the receiving river ecosystem. However, it is currently unknown which habitats are most impacted and whether ARGs, like certain chemical contaminants, could be accumulated or enriched in the river ecosystem. We conducted a systematic metagenomic survey on the antibiotic resistomes of WWTP effluent, four riverine habitats (water, suspended particles, sediment, epilithic biofilm), and freshwater amphipod gut microbiomes. The impact of WWTP effluent on the downstream habitats was assessed in nine Swiss rivers. While there were significant differences in resistomes across habitats, the wastewater resistome was more similar to the resistome of receiving river water than to the resistomes of other habitats, and river water was the habitat most strongly impacted by the WWTPs effluent. The sulfonamide, beta-lactam, and aminoglycoside resistance genes were among the most abundant ARGs in the WWTP effluents, and especially aadA, sul1, and class A beta-lactamase genes showed significantly increased abundance in the river water of downstream compared to upstream locations (p < 0.05). However, this was not the case for the sediment, biofilm, and amphipod gut habitats. Accordingly, evidence for accumulation or enrichment of ARGs through the riverine food web was not identified. Our study suggests that monitoring riverine antimicrobial resistance determinants could be conducted using "co-occurrence" of aadA, sul1, and class A beta-lactamase genes as an indicator of wastewater-related pollution and should focus on the water as the most affected habitat.

Phylogenetic lineages and antimicrobial resistance determinants of clinical Klebsiella oxytoca spanning local to global scales.

Klebsiella oxytoca is an opportunistic pathogen causing serious nosocomial infections. Knowledge about the population structure and diversity of healthcare-associated K. oxytoca from a genomic standpoint remains limited. Here, we characterized the phylogenetic relationships and genomic characteristics of 20 K. oxytoca sensu stricto isolates recovered from bloodstream infections at the Dartmouth-Hitchcock Medical Center, New Hampshire, USA from 2017 to 2021. Results revealed a diverse population consisting of 15 sequence types (STs) that together harbored 10 variants of the intrinsic beta-lactamase gene bla OXY-2, conferring resistance to penicillins. Similar sets of antimicrobial resistance (AMR) determinants reside in multiple distinct lineages, with no one lineage dominating the local population. To place the New Hampshire K. oxytoca in a broader context, we compared them to 304 publicly available genomes of clinical isolates from 18 countries. This global clinical K. oxytoca sensu stricto population is represented by over 65 STs that together harbored resistance genes against 14 antimicrobial classes, including eight bla OXY-2 variants. Three dominant STs in the global population (ST2, ST176, ST199) circulate across multiple countries and were also present in the New Hampshire population. The global K. oxytoca population is genetically diverse, but there is evidence for broad dissemination of a few lineages carrying distinct set of AMR determinants. Our findings reveal the clinical diversity of K. oxytoca sensu stricto and its importance in surveillance efforts aimed at monitoring the evolution of this drug-resistant nosocomial pathogen. IMPORTANCE The opportunistic pathogen Klebsiella oxytoca has been increasingly implicated in patient morbidity and mortality worldwide, including several outbreaks in healthcare settings. The emergence and spread of antimicrobial resistant strains exacerbate the disease burden caused by this species. Our study showed that clinical K. oxytoca sensu stricto is phylogenetically diverse, harboring various antimicrobial resistance determinants and bla OXY-2 variants. Understanding the genomic and population structure of K. oxytoca is important for international initiatives and local epidemiological efforts for surveillance and control of drug-resistant K. oxytoca.

In silico and in vitro safety assessment of a fungal biomass from Rhizomucor pusillus for use as a novel food ingredient.

To address the growing world population and reduce the impact of environmental changes on the global food supply, ingredients are being produced using microorganisms to yield sustainable and innovative products. Food ingredients manufactured using modern biotechnology must be produced by non-toxigenic and nonpathogenic production organisms that do not harbor antimicrobial resistance (AMR). Several fungal species represent attractive targets as sources of alternative food products. One such product is a fungal biomass obtained from the fermentation of Rhizomucor pusillus strain CBS 143028. The whole genome sequence of this strain was annotated and subjected to sequence homology searches and in silico phenotype prediction tools to identify genetic elements encoding for protein toxins active via oral consumption, virulence factors associated with pathogenicity, and determinants of AMR. The in silico investigation revealed no genetic elements sharing significant sequence homology with putative virulence factors, protein toxins, or AMR determinants, including the absence of mucoricin, an essential toxin in the pathogenesis of mucormycosis. These in silico findings were corroborated in vitro based on the absence of clinically relevant mycotoxin or antibacterial secondary metabolites. Consequently, it is unlikely that R. pusillis strain CBS 143028 would pose a safety concern for use in food for human consumption.

Antimicrobial Resistance, Biofilm Formation, and Virulence Determinants in Enterococcus faecalis Isolated from Cultured and Wild Fish.

Fish has always been an integral part of Bengali cuisine and economy. Fish could also be a potential reservoir of pathogens. This study aimed to inquisite the distribution of virulence, biofilm formation, and antimicrobial resistance of Enterococcus faecalis isolated from wild and cultivated fish in Bangladesh. A total of 132 koi fish (Anabas scandens) and catfish (Heteropneustes fossilis) were collected from different markets in the Mymensingh district and analyzed to detect E. faecalis. E. faecalis was detected by conventional culture and polymerase chain reaction (PCR), followed by the detection of virulence genes by PCR. Antibiotic susceptibility was determined using the disk diffusion method, and biofilm-forming ability was investigated by crystal violet microtiter plate (CVMP) methods. A total of 47 wild and 40 cultured fish samples were confirmed positive for E. faecalis by PCR. The CVMP method revealed four per cent of isolates from cultured fish as strong biofilm formers, but no strong producers were found from the wild fish. In the PCR test, 45% of the isolates from the wild and cultivated fish samples were found to be positive for at least one biofilm-producing virulence gene, where agg, ace, gelE, pil, and fsrC genes were detected in 80, 95, 100, 93, and 100% of the isolates, respectively. Many of the isolates from both types of samples were multidrug resistant (MDR) (73% in local fish and 100% in cultured fish), with 100% resistance to erythromycin, linezolid, penicillin, and rifampicin in E. faecalis from cultured fish and 73.08, 69.23, 69.23, and 76.92%, respectively, in E. faecalis from wild fish. This study shows that E. faecalis from wild fish have a higher frequency of virulence genes and biofilm-forming genes than cultivated fish. However, compared to wild fish, cultured fish were found to carry E. faecalis that was more highly multidrug resistant. Present findings suggest that both wild and cultured fish could be potential sources for MDR E. faecalis, having potential public health implications.

Characteristics and genomic epidemiology of colistin-resistant Enterobacterales from farmers, swine, and hospitalized patients in Thailand, 2014–2017

BackgroundColistin is one of the last resort therapeutic options for treating carbapenemase-producing Enterobacterales, which are resistant to a broad range of beta-lactam antibiotics. However, the increased use of colistin in clinical and livestock farming settings in Thailand and China, has led to the inevitable emergence of colistin resistance. To better understand the rise of colistin-resistant strains in each of these settings, we characterized colistin-resistant Enterobacterales isolated from farmers, swine, and hospitalized patients in Thailand.MethodsEnterobacterales were isolated from 149 stool samples or rectal swabs collected from farmers, pigs, and hospitalized patients in Thailand between November 2014–December 2017. Confirmed colistin-resistant isolates were sequenced. Genomic analyses included species identification, multilocus sequence typing, and detection of antimicrobial resistance determinants and plasmids.ResultsThe overall colistin-resistant Enterobacterales colonization rate was 26.2% (n = 39/149). The plasmid-mediated colistin-resistance gene (mcr) was detected in all 25 Escherichia coli isolates and 9 of 14 (64.3%) Klebsiella spp. isolates. Five novel mcr allelic variants were also identified: mcr-2.3, mcr-3.21, mcr-3.22, mcr-3.23, and mcr-3.24, that were only detected in E. coli and Klebsiella spp. isolates from farmed pigs.ConclusionOur data confirmed the presence of colistin-resistance genes in combination with extended spectrum beta-lactamase genes in bacterial isolates from farmers, swine, and patients in Thailand. Differences between the colistin-resistance mechanisms of Escherichia coli and Klebsiella pneumoniae in hospitalized patients were observed, as expected. Additionally, we identified mobile colistin-resistance mcr-1.1 genes from swine and patient isolates belonging to plasmids of the same incompatibility group. This supported the possibility that horizontal transmission of bacterial strains or plasmid-mediated colistin-resistance genes occurs between humans and swine.

Isolation and characterization of multidrug resistant Gallibacterium anatis biovar haemolytica strains from Polish geese and hens

Gallibacterium anatis biovar haemolytica is a bacterium that is frequently associated with infections of the reproductive tract and respiratory system in poultry. To assess the current prevalence and resistance profile of these bacteria in Poland, we collected and investigated 63 strains of Gallibacterium from diseased domestic poultry flocks including geese, laying hens, breeding hens and an ornamental hen. Detailed characterization of the isolates included the analysis of phenotypic antimicrobial resistance profiles and biofilm formation ability. Furthermore, the genetic background of 40 selected isolates regarding the presence of virulence and antimicrobial resistance genes and mobile genetic elements was determined. All investigated isolates were multidrug resistant, most prominently to β-lactams, fluoroquinolones, sulfonamides and macrolides. A total of 48 different resistance profiles were detected. Of all isolates, 50.8% formed a strong biofilm, where strains isolated from geese appeared to be better at biofilm formation than strains isolated from laying and breeding hens. Single-nucleotide polymorphism genotyping revealed that G. anatis bv. haemolytica strains are restricted in host and geographical distribution, and the geese isolates showed greater phylogenetic similarity. Whole genome sequencing enabled identification of 25 different antimicrobial resistance determinants. The most common resistance genes were tetB, blaROB-1, and blaTEM-1 which may be located on mobile genetic elements. All isolates possessed the toxin gene gtxA, and the fimbrial gene flfA was identified in 95% of strains. Our results indicated that all G. anatis bv. haemolytica isolates showed multidrug resistant phenotypes. Strains isolated from geese were characterized by the highest percentage of isolates resistant to selected antimicrobials, probably reflecting host-related adaptations.