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We describe the whole-genome sequencing and comparative genomic analysis of 22 mastitis-associated NASM strains isolated from India. The mean genome size of the strains was 2.55 Mbp, with an average GC content of 32.2%. We identified 14 different sequence types (STs) among the 22 NASM strains. Of these, ST1 and ST6 of S. chromogenes were exclusively associated with bovine mastitis. Genome-wide SNP-based minimum spanning tree revealed the intricate phylogenetic relationships among NASM strains from India, categorizing them into five major clades. Interestingly, mastitis-associated strains formed separate subclades in all the NASM species studied, indicating distinct host-specific co-evolution. The study identified 32 antimicrobial resistance (AMR) genes and 53 virulence-associated genes, providing insights into the genetic factors that could contribute to the pathogenicity of NASM species. Some virulence and AMR genes were found in the predicted genomic islands, suggesting possible horizontal transfer events. [ABSTRACT FROM AUTHOR]

Background: Processing environments can be an important source of pathogenic and spoilage microorganisms that cross contaminate meat and meat products. The aim of this study was to characterize the microbiome of raw materials, processing environments and end products from 19 facilities producing different meat products. Results: The taxonomic profiles of the microbial communities evolved along processing, from raw materials to end products, suggesting that food contact (FC) surfaces play an important role in modulating the microbiome of final products. Some species persisted with the highest relative abundance in raw materials, food processing environments and/or in the final product, including species from the genera Pseudomonas, Staphylococcus, Brochothrix, Acinetobacter and Psychrobacter. Processing environments showed a very diverse core microbiota, partially shared with the products. Pseudomonas fragi and Pseudomonas sp. Lz4W (in all sample and facility types) and Brochothrix thermosphacta, Psychrobacter sp. and Psychrobacter sp. P11F6 (in raw materials, FC surfaces and end products) were prominent members of the core microbiota for all facilities, while Latilactobacillus sakei was found as a dominant species exclusively in end products from the facilities producing fermented sausages. Processing environments showed a higher amount of antimicrobial resistance genes and virulence factors than raw materials and end products. One thousand four hundred twenty-one medium/high-quality metagenome-assembled genomes (MAGs) were reconstructed. Of these, 274 high-quality MAGs (completeness > 90%) corresponded to 210 putative new species, mostly found in processing environments. For two relevant taxa in meat curing and fermentation processes (S. equorum and L. sakei, respectively), phylogenetic variation was observed associated with the specific processing facility under study, which suggests that specific strains of these taxa may be selected in different meat processing plants, likely contributing to the peculiar sensorial traits of the end products produced in them. Conclusions: Overall, our findings provide the most detailed metagenomics-based perspective up to now of the microbes that thrive in meat, meat products and associated environments and open avenues for future research activities to better understand the microbiome functionality and potential contribution to meat quality and safety. BW5-yJYgBWXLb4-yN7W47n Video Abstract [ABSTRACT FROM AUTHOR]

Prosthetic joint infections caused by Campylobacter are uncommon, with the majority of cases being attributed to C. fetus. This case report represents the third instance of a prosthetic hip infection caused by C. coli following an episode of gastroenteritis and, notably, in an immunocompetent patient. The infection was successfully managed by surgical debridement and lavage with retention of the prosthesis and 12 weeks of antibiotics. Furthermore, we present the first whole-genome sequence of a Campylobacter strain responsible for prosthetic joint infection and offer a comprehensive review of the literature on such infections. [ABSTRACT FROM AUTHOR]

Abstract We describe the whole-genome sequencing and comparative genomic analysis of 22 mastitis-associated NASM strains isolated from India. The mean genome size of the strains was 2.55 Mbp, with an average GC content of 32.2%. We identified 14 different sequence types (STs) among the 22 NASM strains. Of these, ST1 and ST6 of S. chromogenes were exclusively associated with bovine mastitis. Genome-wide SNP-based minimum spanning tree revealed the intricate phylogenetic relationships among NASM strains from India, categorizing them into five major clades. Interestingly, mastitis-associated strains formed separate subclades in all the NASM species studied, indicating distinct host-specific co-evolution. The study identified 32 antimicrobial resistance (AMR) genes and 53 virulence-associated genes, providing insights into the genetic factors that could contribute to the pathogenicity of NASM species. Some virulence and AMR genes were found in the predicted genomic islands, suggesting possible horizontal transfer events.

Abstract Background Processing environments can be an important source of pathogenic and spoilage microorganisms that cross contaminate meat and meat products. The aim of this study was to characterize the microbiome of raw materials, processing environments and end products from 19 facilities producing different meat products. Results The taxonomic profiles of the microbial communities evolved along processing, from raw materials to end products, suggesting that food contact (FC) surfaces play an important role in modulating the microbiome of final products. Some species persisted with the highest relative abundance in raw materials, food processing environments and/or in the final product, including species from the genera Pseudomonas, Staphylococcus, Brochothrix, Acinetobacter and Psychrobacter. Processing environments showed a very diverse core microbiota, partially shared with the products. Pseudomonas fragi and Pseudomonas sp. Lz4W (in all sample and facility types) and Brochothrix thermosphacta, Psychrobacter sp. and Psychrobacter sp. P11F6 (in raw materials, FC surfaces and end products) were prominent members of the core microbiota for all facilities, while Latilactobacillus sakei was found as a dominant species exclusively in end products from the facilities producing fermented sausages. Processing environments showed a higher amount of antimicrobial resistance genes and virulence factors than raw materials and end products. One thousand four hundred twenty-one medium/high-quality metagenome-assembled genomes (MAGs) were reconstructed. Of these, 274 high-quality MAGs (completeness > 90%) corresponded to 210 putative new species, mostly found in processing environments. For two relevant taxa in meat curing and fermentation processes (S. equorum and L. sakei, respectively), phylogenetic variation was observed associated with the specific processing facility under study, which suggests that specific strains of these taxa may be selected in different meat processing plants, likely contributing to the peculiar sensorial traits of the end products produced in them. Conclusions Overall, our findings provide the most detailed metagenomics-based perspective up to now of the microbes that thrive in meat, meat products and associated environments and open avenues for future research activities to better understand the microbiome functionality and potential contribution to meat quality and safety. Video Abstract

Abstract The whole genome sequence (WGS) of prevalent MRSA strains harboring mecA gene obtained from skin and soft tissue infections (SSTIs) in Nigerian hospitals were profiled for pathogenomic structure and evaluated for clonal diversity. The two MRSA strains identified among 66 isolated multi-drug resistant S. aureus from a collection of 256 clinical samples were phenotyped for antibiotic resistance and genotyped for mecA, SCCmec, and spa types. The mecA positive MRSA was analysed using whole-genome sequencing for resistomes, virulomes, phylogenomic profiles and clonal diversity. The identified MRSA-CC7-ST789-t091-SCCmecV strains from a female child (aged 1 year) with severe otorrhea and an adult male (aged 23) with purulent wound abscess showed high-level resistance to streptomycin, vancomycin, kanamycin, sulfamethoxazole and ciprofloxacin. Both strains harbored abundant resistomes, inherent plasmids, chromosomal replicons and typical seven housekeeping genes (arc3, aroE4, glpF1, gmk4, pta4, tpi6, yqiL3). The most abundant putative virulomes were pathogenesis-associated proteins (included hemolysin gamma, leucocidins, proteases, staphylococcal superantigen/enterotoxin-like genes (Set/Ssl), capsule- and biofilm-associated genes, and hyaluronate lyase). Comparative phylogenomic analysis revealed the relatedness of the two clonal strains with prevalent MRSA-CC7 pathotypes observed in Italy (2013 and 2014), Denmark (2014), Thailand (2015 and 2016), USA (2018), and Nigeria (2016 and 2020); and share high genetic similarities with livestock strains from cow milk and cattle. Identified MRSA-CC7-ST789-t091-SCCmecV pathotypes implicated in SSTIs from Nigeria harboring repertoires of antibiotic resistance and virulence genes, and genetic relatedness with livestock strains; show the possibility of gene transfer between animal and human. Adequate hospital MRSA infection control and geno-epidemiological surveillance for animal and human transfer is required.

Abstract The incidence of Pseudomonas aeruginosa infections in healthcare environments, particularly in low-and middle-income countries, is on the rise. The purpose of this study was to provide comprehensive genomic insights into thirteen P. aeruginosa isolates obtained from Egyptian healthcare settings. Phenotypic analysis of the antimicrobial resistance profile and biofilm formation were performed using minimum inhibitory concentration and microtiter plate assay, respectively. Whole genome sequencing was employed to identify sequence typing, resistome, virulome, and mobile genetic elements. Our findings indicate that 92.3% of the isolates were classified as extensively drug-resistant, with 53.85% of these demonstrating strong biofilm production capabilities. The predominant clone observed in the study was ST773, followed by ST235, both of which were associated with the O11 serotype. Core genome multi-locus sequence typing comparison of these clones with global isolates suggested their potential global expansion and adaptation. A significant portion of the isolates harbored Col plasmids and various MGEs, all of which were linked to antimicrobial resistance genes. Single nucleotide polymorphisms in different genes were associated with the development of antimicrobial resistance in these isolates. In conclusion, this pilot study underscores the prevalence of extensively drug-resistant P. aeruginosa isolates and emphasizes the role of horizontal gene transfer facilitated by a diverse array of mobile genetic elements within various clones. Furthermore, specific insertion sequences and mutations were found to be associated with antibiotic resistance.

Antimicrobial resistance (AMR) is a growing global health challenge, compromising bacterial infection treatments and necessitating robust surveillance and mitigation strategies. The overuse of antimicrobials in humans and farm animals has made them hotspots for AMR. However, the spread of AMR genes in wildlife and the environment represents an additional challenge, turning these areas into new AMR hotspots. Among the AMR bacteria considered to be of high concern for public health, Campylobacter has been the leading cause of foodborne infections in the European Union since 2005. This study examines the prevalence of AMR genes and virulence factors in Campylobacter isolates from wild birds and surface waters in Luxembourg. The findings reveal a significant prevalence of resistant Campylobacter strains, with 12% of C. jejuni from wild birds and 37% of C. coli from surface waters carrying resistance genes, mainly against key antibiotics like quinolones and tetracycline. This study underscores the crucial role of the environment in the spread of AMR bacteria and genes, highlighting the urgent need for enhanced surveillance and control measures to curb AMR in wildlife and environmental reservoirs and reduce transmission risks to humans. This research supports One Health approaches to tackling antimicrobial resistance and protecting human, animal, and environmental health. [ABSTRACT FROM AUTHOR]

The incidence of Pseudomonas aeruginosa infections in healthcare environments, particularly in low-and middle-income countries, is on the rise. The purpose of this study was to provide comprehensive genomic insights into thirteen P. aeruginosa isolates obtained from Egyptian healthcare settings. Phenotypic analysis of the antimicrobial resistance profile and biofilm formation were performed using minimum inhibitory concentration and microtiter plate assay, respectively. Whole genome sequencing was employed to identify sequence typing, resistome, virulome, and mobile genetic elements. Our findings indicate that 92.3% of the isolates were classified as extensively drug-resistant, with 53.85% of these demonstrating strong biofilm production capabilities. The predominant clone observed in the study was ST773, followed by ST235, both of which were associated with the O11 serotype. Core genome multi-locus sequence typing comparison of these clones with global isolates suggested their potential global expansion and adaptation. A significant portion of the isolates harbored Col plasmids and various MGEs, all of which were linked to antimicrobial resistance genes. Single nucleotide polymorphisms in different genes were associated with the development of antimicrobial resistance in these isolates. In conclusion, this pilot study underscores the prevalence of extensively drug-resistant P. aeruginosa isolates and emphasizes the role of horizontal gene transfer facilitated by a diverse array of mobile genetic elements within various clones. Furthermore, specific insertion sequences and mutations were found to be associated with antibiotic resistance. [ABSTRACT FROM AUTHOR]

ABSTRACT Campylobacter jejuni is the main cause of bacterial gastroenteritis and a public health problem worldwide. Little information is available on the genotypic characteristics of human C. jejuni in Spain. This study is based on an analysis of the resistome, virulome, and phylogenetic relationship, antibiogram prediction, and antimicrobial susceptibility of 114 human isolates of C. jejuni from a tertiary hospital in southern Spain from October 2020 to June 2023. The isolates were sequenced using Illumina technology, and a bioinformatic analysis was subsequently performed. The susceptibility of C. jejuni isolates to ciprofloxacin, tetracycline, and erythromycin was also tested. The resistance rates for each antibiotic were 90.3% for ciprofloxacin, 66.7% for tetracycline, and 0.88% for erythromycin. The fluoroquinolone resistance rate obtained is well above the European average (69.1%). CC-21 (n = 23), ST-572 (n = 13), and ST-6532 (n = 13) were the most prevalent clonal complexes (CCs) and sequence types (STs). In the virulome, the cadF, ciaB, and cdtABC genes were detected in all the isolates. A prevalence of 20.1% was obtained for the genes wlaN and cstIII, which are related to the pathogenesis of Guillain-Barré syndrome (GBS). The prevalence of the main antimicrobial resistance markers detected were CmeABC (92.1%), RE-cmeABC (7.9%), the T86I substitution in gyrA (88.9%), blaOXA-61 (72.6%), tet(O) (65.8%), and ant (6)-Ia (17.1%). High antibiogram prediction rates (>97%) were obtained, except for in the case of the erythromycin-resistant phenotype. This study contributes significantly to the knowledge of C. jejuni genomics for the prevention, treatment, and control of infections caused by this pathogen.IMPORTANCEDespite being the pathogen with the greatest number of gastroenteritis cases worldwide, Campylobacter jejuni remains a poorly studied microorganism. A sustained increase in fluoroquinolone resistance in human isolates is a problem when treating Campylobacter infections. The development of whole genome sequencing (WGS) techniques has allowed us to better understand the genotypic characteristics of this pathogen and relate them to antibiotic resistance phenotypes. These techniques complement the data obtained from the phenotypic analysis of C. jejuni isolates. The zoonotic transmission of C. jejuni through the consumption of contaminated poultry supports approaching the study of this pathogen through “One Health” approach. In addition, due to the limited information on the genomic characteristics of C. jejuni in Spain, this study provides important data and allows us to compare the results with those obtained in other countries.

This study presents a comprehensive analysis of samples from urban wastewater treatment plants using anoxic/oxic processes in Slovakia and Taiwan, focusing on microbiome, resistome, mobilome, and virulome, which were analyzed using a shotgun metagenomic approach. Distinct characteristics were observed; in Taiwan, a higher abundance and diversity of antibiotic resistance genes were found in both influent and effluent samples, while there was a higher prevalence of mobile genetic elements and virulence factor genes in Slovakia. Variations were noted in microbial community structures; influent samples in Taiwan were reflected from fecal and hospital sources, and those in Slovakia were derived from environmental elements. At the genus level, the samples from Taiwan's sewage treatment plants were dominated by Cloacibacterium and Bacteroides, while Acinetobacter was predominant in samples from Slovakia. Despite similar antibiotic usage patterns, distinct wastewater characteristics and operational disparities influenced microbiome, resistome, mobilome, and virulome compositions, with limited reduction of most resistance genes by the studied anoxic/oxic processes. These findings underscore the importance of region-specific insights into microbial communities for understanding the dynamics of antimicrobial resistance and pathogenicity in urban wastewater treatment systems. Such insights may lay the groundwork for optimizing treatment processes and reducing the dissemination of antibiotic resistance and pathogenicity genes for safeguarding public health.

Klebsiella pneumoniae is a ubiquitous nosocomial pathogen associated with various types of infections in hospitalized patients and different animal species. In the current study, 49 Klebsiella strains isolated from humans, dogs, and cats were investigated using NGS technology. MALDI-TOF failed to identify newly discovered K. variicola and K. quasipneumoniae isolates correctly. MLST analysis revealed different sequence types among K. pneumoniae isolates, and the most frequent STs were ST29, ST219, and ST37. Three ST23 that are generally known as hypervirulent type were identified but they lacked major discriminatory determinants for hypervirulent K. pneumoniae (hvKp). K. pneumoniae isolates showed high diversity, and several isolates from humans and animals were assigned to the same ST and were almost identical. Isolates from humans exhibited more pronounced resistance patterns compared to the animal isolates. High levels of resistance were observed for piperacillin, trimethoprim/sulfamethoxazole, and cephalosporins, and resistance to carbapenem compounds was only found in isolates of human origin. Three strains of human origin were extensively drug-resistant (XDR). A diverse range of resistance genes primarily confer resistance to beta-lactams., phenicol/quinolone, aminoglycoside, macrolide, sulfonamides, and fosfomycin were identified in silico. However, there were inconsistencies between the phenotypic characterization of isolates and the set of resistance genes detected in silico in this set of Klebsiella isolates. Further research using a larger number of isolates from various sources is necessary to fully comprehend the relationship between the presence of antimicrobial resistance determinants and phenotypic data. It is also necessary to monitor the spread of K. pneumoniae from a One Health perspective in Egypt.

Background: Klebsiella pneumoniae, a notorious pathogen for causing nosocomial infections has become a major cause of neonatal septicemia, leading to high morbidity and mortality worldwide. This opportunistic bacterium has become highly resistant to antibiotics due to the widespread acquisition of genes encoding a variety of enzymes such as extended-spectrum beta-lactamases (ESBLs) and carbapenemases. We collected Klebsiella pneumoniae isolates from a local tertiary care hospital from February 2019–February 2021. To gain molecular insight into the resistome, virulome, and genetic environment of significant genes of multidrug-resistant K. pneumoniae isolates, we performed the short-read whole-genome sequencing of 10 K. pneumoniae isolates recovered from adult patients, neonates, and hospital tap water samples. Results: The draft genomes of the isolates varied in size, ranging from 5.48 to 5.96 Mbp suggesting the genome plasticity of this pathogen. Various genes conferring resistance to different classes of antibiotics e.g., aminoglycosides, quinolones, sulfonamides, tetracycline, and trimethoprim were identified in all sequenced isolates. The highest resistance was observed towards carbapenems, which has been putatively linked to the presence of both class B and class D carbapenemases, blaNDM, and blaOXA, respectively. Moreover, the biocide resistance gene qacEdelta1 was found in 6/10 of the sequenced strains. The sequenced isolates exhibited a broad range of sequence types and capsular types. The significant antibiotic resistance genes (ARGs) were bracketed by a variety of mobile genetic elements (MGEs). Various spontaneous mutations in genes other than the acquired antibiotic-resistance genes were observed, which play an indirect role in making these bugs resistant to antibiotics. Loss or deficiency of outer membrane porins, combined with ESBL production, played a significant role in carbapenem resistance in our sequenced isolates. Phylogenetic analysis revealed that the study isolates exhibited evolutionary relationships with strains from China, India, and the USA suggesting a shared evolutionary history and potential dissemination of similar genes amongst the isolates of different origins. Conclusions: This study provides valuable insight into the presence of multiple mechanisms of carbapenem resistance in K. pneumoniae strains including the acquisition of multiple antibiotic-resistance genes through mobile genetic elements. Identification of rich mobilome yielded insightful information regarding the crucial role of insertion sequences, transposons, and integrons in shaping the genome of bacteria for the transmission of various resistance-associated genes. Multi-drug resistant isolates that had the fewest resistance genes exhibited a significant number of mutations. K. pneumoniae isolate from water source displayed comparable antibiotic resistance determinants to clinical isolates and the highest number of virulence-associated genes suggesting the possible interplay of ARGs amongst bacteria from different sources. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii, a notorious opportunistic pathogen, presents a formidable challenge in both clinical and environmental fields due to its resilience and ability to acquire resistance. This study undertook a comprehensive analysis of 183 A. baumannii isolates collected between 2019 and 2022 from intra-hospital infections (IHI), hospital sewages (Hs), wastewater treatment plants (WWTP), and adjacent river waters from two Southern cities, focusing on their resistome, virulome, and mobilome through isolation on chromogenic media, identification by MALDI-TOF-MS and antibiotic susceptibility testing by disk diffusion) followed by genotypic characterization [Whole Genome Sequencing (WGS), 3rd generation sequencing through the MinION (ONT) platform, pangenome description, and respectively horizontal gene transfer through conjugation assays]. Our findings reveal significant genomic plasticity and the prevalence of high-risk international clones, underlining the potential of these isolates to act as reservoirs for antibiotic resistance genes (ARGs) that could be dynamically exchanged between clinical and environmental settings through mobile genetic elements (MGEs) such as the pMAL1 plasmids and the critical role of WWTPs in the persistence and spread of A. baumannii. Moreover, our study presents the first report of the co-occurrence of blaOXA-23 and blaOXA-72 in A. baumannii ST2 clone. Thus, our research underscores the necessity for integrated surveillance and targeted interventions across healthcare and environmental sectors to mitigate the risk posed by this adaptable pathogen.

Prosthetic joint infections caused by Campylobacter are uncommon, with the majority of cases being attributed to C. fetus. This case report represents the third instance of a prosthetic hip infection caused by C. coli following an episode of gastroenteritis and, notably, in an immunocompetent patient. The infection was successfully managed by surgical debridement and lavage with retention of the prosthesis and 12 weeks of antibiotics. Furthermore, we present the first whole-genome sequence of a Campylobacter strain responsible for prosthetic joint infection and offer a comprehensive review of the literature on such infections.

Antimicrobial resistance (AMR) is a global health threat requiring urgent attention and effective strategies for containment. AMR is fueled by wastewater mismanagement and global mobility, disseminating multidrug-resistant (MDR) strains worldwide. While global estimates of AMR burden have been informative, community-level understanding has received little attention despite reports of high AMR prevalence in healthy communities. We assessed the "invasion" of antibiotic resistance genes (ARGs) into the normal human flora by characterizing AMR Escherichia coli in local wastewaters contributed by a healthy youth population. This study estimated 26% (out of 300 isolates) resistant and 59% plasmid-bearing E. coli in local wastewater. Of the 78 AMR isolates, the frequency of mono-resistance was higher against tetracycline (32%), followed by kanamycin (17%) and chloramphenicol (9%). Five isolates were potentially MDR. We further sequenced four MDRs and four sensitive strains to comprehend the genome and resistome diversity in comparison to the global wastewater E. coli (genomes from the PATRIC database). The whole-genome analysis revealed extensive genome similarity among global isolates, suggesting global dissemination and colonization of E. coli. Global wastewater resistome majorly comprised ARGs against aminoglycosides (26%), beta-lactam (17%), sulfonamide (11%), and trimethoprim (8%). Resistance to colistin, a last-resort antibiotic, was prevalent in MDRs of European and South Asian isolates. A systems approach is required to address the AMR crisis on a global scale, reduce antibiotic usage, and increase the efficiency of wastewater management and disinfection. [ABSTRACT FROM AUTHOR]

The emergence of Klebsiella (K.) pneumoniae as a leading cause of nosocomial infections in Southeast Asia is of concern. Vietnam has an outstanding position with regard to antimicrobialresistant (AMR) pathogens and is a hotspot for carbapenem-resistant K. pneumoniae. In the current study, 19 clinical K. pneumoniae strains isolated from patients in Vietnam were tested for antibiotic susceptibility, and their genome sequences were analyzed to investigate potential resistance profiles, genotypes, AMR determinants, and virulence-associated genes. More than half of the isolates were multidrug-resistant (MDR), displaying resistance to ciprofloxacin, levofloxacin, chloramphenicol, piperacillin, cefotaxime, ceftazidime, and cefepime. Carbapenem- resistance was detected in 47% (n=9) of isolates. Five isolates were assigned to sequence type (ST) 23 by multi-locus sequence typing (MLST). These ST23 strains exhibited determinants characteristic of hypervirulent strains and were sensitive to all antibiotics tested except for one strain, which was resistant to chloramphenicol and trimethoprim/sulfamethoxazole. All isolates had AMR determinants that conferred resistance to aminoglycosides and ß-lactams. Single nucleotide variants of oqxA and oqxB conferring resistance to phenicol/quinolone, ompK37 and ompA conferring resistance to beta-lactams, and lptD conferring resistance to rifamycin, were found in all isolates. Additionally, 95% of the isolates (n=18) carried fosA genes; however, only two were resistant to fosfomycin. Five isolates carried genes conferring resistance to colistin; only one carried mcr-1.1 and showed resistance to colistin. One hundred thirty virulence-associated genes were identified. The current study demonstrated an inverse relationship between the number of detected virulence determinants and antibiotic resistance. Several well-known resistance genes have been identified but did not mediate resistance, which could be due to insufficient levels or lack of expression. A comprehensive study of the genotypic findings of AMR determinants and virulence with phenotypic data is required. The presence of hypervirulent K. pneumoniae carrying specific virulomes is a growing problem, and monitoring of virulence characteristics of K. pneumoniae is important. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is a growing global health challenge, compromising bacterial infection treatments and necessitating robust surveillance and mitigation strategies. The overuse of antimicrobials in humans and farm animals has made them hotspots for AMR. However, the spread of AMR genes in wildlife and the environment represents an additional challenge, turning these areas into new AMR hotspots. Among the AMR bacteria considered to be of high concern for public health, Campylobacter has been the leading cause of foodborne infections in the European Union since 2005. This study examines the prevalence of AMR genes and virulence factors in Campylobacter isolates from wild birds and surface waters in Luxembourg. The findings reveal a significant prevalence of resistant Campylobacter strains, with 12% of C. jejuni from wild birds and 37% of C. coli from surface waters carrying resistance genes, mainly against key antibiotics like quinolones and tetracycline. This study underscores the crucial role of the environment in the spread of AMR bacteria and genes, highlighting the urgent need for enhanced surveillance and control measures to curb AMR in wildlife and environmental reservoirs and reduce transmission risks to humans. This research supports One Health approaches to tackling antimicrobial resistance and protecting human, animal, and environmental health.

ABSTRACT Foodborne Salmonella infection remains a major public health concern worldwide. With rising antimicrobial resistance, genomic surveillance is key to tracking outbreaks and monitoring transmission, but there is no comprehensive national surveillance scheme for Salmonella involving humans, food, animals, and the environment in China. Moreover, the association between antimicrobial resistance and climate, social, and economic factors has rarely been investigated. Here, we use 1,962 Salmonella isolates collected from 22 Chinese provinces and add 6,035 publicly available genomes to build a Chinese local Salmonella genome database version 2 (CLSGDB v2) representing 30 Chinese provinces, covering 1905–2022. Using the CLSGDB v2, we mapped the landscape and spatiotemporal dynamics of antimicrobial resistance markers, virulome, and mobilome in Salmonella. We identified 317 mcr positive and 745 azithromycin resistance genes positive Salmonella isolates out of 7,997 isolates. We further uncovered the geographic distribution veil of mcr-1, fosA7, fosA3, mph(A), and bla CTX-M-55 genes in China, all of them resistant to the critically important antimicrobials including colistin, fosfomycin, azithromycin, and the third-generation cephalosporins. Interestingly, economic, climatic, and social factors can drive the rise of antimicrobial resistance was observed. Finally, we release the CLSGDB v2 as an open-access database and thus can assist surveillance studies tracking 164 Salmonella enterica serovars and 295 sequence types across the globe. The CLSGDB v2 is freely available at https://nmdc.cn/clsgdbv2. IMPORTANCE We established the largest Salmonella genome database from China and presented the landscape and spatiotemporal dynamics of antimicrobial resistance genes. We also found that economic, climatic, and social factors can drive the rise of antimicrobial resistance. The Chinese local Salmonella genome database version 2 was released as an open-access database (https://nmdc.cn/clsgdbv2) and thus can assist surveillance studies across the globe. This database will help inform interventions for AMR, food safety, and public health.

The spread of carbapenemase-producing Klebsiella pneumoniae beyond hospital settings is a global critical issue within a public health and One Health perspective. Another worrisome concern is the convergence of virulence and resistance in healthcare-associated lineages of K. pneumoniae leading to unfavorable clinical outcomes. During a surveillance study of WHO critical priority pathogens circulating in an impacted urban river in São Paulo, Brazil, we isolate two hypermucoviscous and multidrug-resistant K. pneumoniae strains (PINH-4250 and PINH-4900) from two different locations near to medical centers. Genomic investigation revealed that both strains belonged to the global high-risk sequence type (ST) ST11, carrying the blaKPC-2 carbapenemase gene, besides other medically important antimicrobial resistance determinants. A broad virulome was predicted and associated with hypervirulent behavior in the Galleria mellonella infection model. Comparative phylogenomic analysis of PINH-4250 and PINH-4900 along to an international collection of publicly available genomes of K. pneumoniae ST11 revealed that both environmental strains were closely related to hospital-associated K. pneumoniae strains recovered from clinical samples between 2006 and 2018, in São Paulo city. Our findings support that healthcare-associated KPC-2-positive K. pneumoniae of ST11 clone has successfully expanded beyond hospital settings. In summary, aquatic environments can become potential sources of international clones of K. pneumoniae displaying carbapenem resistance and hypervirulent behaviors, which is a critical issue within a One Health perspective.

Engine oil spills have been associated with a wide range of human health problems. However, little is known about the effects of petroleum hydrocarbon pollution on soil microbial communities. In this study, three samples were collected from oil-polluted soils (OPS), and one control soil (CS) from Taolin town, China, near the old engine's scrapes was used. The aims of this study were to conduct metagenomic sequencing and subsequently perform resistome and virulome analysis. We also aimed to validate anti-microbial resistance and virulence genes and anti-bacterial sensitivity profiles among the isolates from oil-polluted soils. The OPS microbial community was dominated by bacterial species compared to the control samples which were dominated by metazoans and other organisms. Secondly, the resistosome and virulome analysis showed that ARGs and virulence factors were higher among OPS microbial communities. Antibiotic susceptibility assay and qPCR analysis for ARGs and virulence factors showed that the oil-polluted soil samples had remarkably enhanced expression of these ARGs and some virulence genes. Our study suggests that oil pollution contributes to shifting microbial communities to more resilient types that could survive the toxicity of oil pollution and subsequently become more resilient in terms of higher resistance and virulence potential. [ABSTRACT FROM AUTHOR]

Background: The Andean condor (Vultur gryphus) is the largest scavenger in South America. This predatory bird plays a crucial role in their ecological niche by removing carcasses. We report the first metagenomic analysis of the Andean condor gut microbiome. Methods: This work analyzed shotgun metagenomics data from a mixture of fifteen captive Chilean Andean condors. To filter eukaryote contamination, we employed BWA-MEM v0.7. Taxonomy assignment was performed using Kraken2 and MetaPhlAn v2.0 and all filtered reads were assembled using IDBA-UD v1.1.3. The two most abundant species were used to perform a genome reference-guided assembly using MetaCompass. Finally, we performed a gene prediction using Prodigal and each gene predicted was functionally annotated. InterproScan v5.31-70.0 was additionally used to detect homology based on protein domains and KEGG mapper software for reconstructing metabolic pathways. Results: Our results demonstrate concordance with the other gut microbiome data from New World vultures. In the Andean condor, Firmicutes was the most abundant phylum present, with Clostridium perfringens, a potentially pathogenic bacterium for other animals, as dominating species in the gut microbiome. We assembled all reads corresponding to the top two species found in the condor gut microbiome, finding between 94% to 98% of completeness for Clostridium perfringens and Plesiomonas shigelloides, respectively. Our work highlights the ability of the Andean condor to act as an environmental reservoir and potential vector for critical priority pathogens which contain relevant genetic elements. Among these genetic elements, we found 71 antimicrobial resistance genes and 1,786 virulence factors that we associated with several adaptation processes. [ABSTRACT FROM AUTHOR]

Abstract Background Bovine mastitis accounts for significant economic losses to the dairy industry worldwide. Staphylococcus aureus is the most common causative agent of bovine mastitis. Investigating the prevalence of virulence factors and antimicrobial resistance would provide insight into the molecular epidemiology of mastitis-associated S. aureus strains. The present study is focused on the whole genome sequencing and comparative genomic analysis of 41 mastitis-associated S. aureus strains isolated from India. Results The results elucidate explicit knowledge of 15 diverse sequence types (STs) and five clonal complexes (CCs). The clonal complexes CC8 and CC97 were found to be the predominant genotypes comprising 21 and 10 isolates, respectively. The mean genome size was 2.7 Mbp with a 32.7% average GC content. The pan-genome of the Indian strains of mastitis-associated S. aureus is almost closed. The genome-wide SNP-based phylogenetic analysis differentiated 41 strains into six major clades. Sixteen different spa types were identified, and eight isolates were untypeable. The cgMLST analysis of all S. aureus genome sequences reported from India revealed that S. aureus strain MUF256, isolated from wound fluids of a diabetic patient, was the common ancestor. Further, we observed that all the Indian mastitis-associated S. aureus isolates belonging to the CC97 are mastitis-associated. We identified 17 different antimicrobial resistance (AMR) genes among these isolates, and all the isolates used in this study were susceptible to methicillin. We also identified 108 virulence-associated genes and discuss their associations with different genotypes. Conclusion This is the first study presenting a comprehensive whole genome analysis of bovine mastitis-associated S. aureus isolates from India. Comparative genomic analysis revealed the genome diversity, major genotypes, antimicrobial resistome, and virulome of clinical and subclinical mastitis-associated S. aureus strains.

American cranberry (Vaccinium macrocarpon) and lowbush/wild blueberry (V. angustifolium) pomace are polyphenol-rich products having potentially beneficial effects in broiler chickens. This study investigated the cecal microbiome of broiler-vaccinated or non-vaccinated birds against coccidiosis. Birds in each of the two groups (vaccinated or non-vaccinated) were fed a basal non-supplemented diet (NC), a basal diet supplemented with bacitracin (BAC), American cranberry (CP), and lowbush blueberry (BP) pomace alone or in combination (CP + BP). At 21 days of age, cecal DNA samples were extracted and analyzed using both whole-metagenome shotgun sequencing and targeted-resistome sequencing approaches. Ceca from vaccinated birds showed a lower abundance of Lactobacillus and a higher abundance of Escherichia coli than non-vaccinated birds (p < 0.05). The highest and lowest abundance of L. crispatus and E. coli, respectively, were observed in birds fed CP, BP, and CP + BP compared to those from NC or BAC treatments (p < 0.05). Coccidiosis vaccination affected the abundance of virulence genes (VGs) related to adherence, flagella, iron utilization, and secretion system. Toxin-related genes were observed in vaccinated birds (p < 0.05) in general, with less prevalence in birds fed CP, BP, and CP + BP than NC and BAC (p < 0.05). More than 75 antimicrobial resistance genes (ARGs) detected by the shotgun metagenomics sequencing were impacted by vaccination. Ceca from birds fed CP, BP, and CP + BP showed the lowest (p < 0.05) abundances of ARGs related to multi-drug efflux pumps, modifying/hydrolyzing enzyme and target-mediated mutation, when compared to ceca from birds fed BAC. Targeted metagenomics showed that resistome from BP treatment was distant to other groups for antimicrobials, such as aminoglycosides (p < 0.05). Significant differences in the richness were observed between the vaccinated and non-vaccinated groups for aminoglycosides, β-lactams, lincosamides, and trimethoprim resistance genes (p < 0.05). Overall, this study demonstrated that dietary berry pomaces and coccidiosis vaccination significantly impacted cecal microbiota, virulome, resistome, and metabolic pathways in broiler chickens. [ABSTRACT FROM AUTHOR]

Introduction: With more public interest in consuming locally grown produce, small specialty crop farms (SSCF) are a viable and growing segment of the food production chain in the United States. Methods: The goal of this study was to investigate the genomic diversity of Campylobacter isolated from dairy manure (n = 69) collected from 10 SSCF in Northeast Ohio between 2018 and 2020. Results: A total of 56 C. jejuni and 13 C. coli isolates were sequenced. Multi-locus sequence typing (MLST) identified 22 sequence types (STs), with ST-922 (18%) and ST-61 (13%) predominant in C. jejuni and ST-829 (62%) and ST-1068 (38%) predominant in C. coli. Interestingly, isolates with similar genomic and gene contents were detected within and between SSCF over time, suggesting that Campylobacter could be transmitted between farms and may persist in a given SSCF over time. Virulenceassociated genes (n = 35) involved in the uptake and utilization of potassium and organic compounds (succinate, gluconate, oxoglutarate, and malate) were detected only in the C. jejuni isolates, while 45 genes associated with increased resistance to environmental stresses (capsule production, cell envelope integrity, and iron uptake) were detected only in the C. coli isolates. Campylobacter coli isolates were also subdivided into two distinct clusters based on the presence of unique prophages (n = 21) or IncQ conjugative plasmid/type-IV secretion system genes (n = 15). Campylobacter coli isolates harbored genes associated with resistance to streptomycin (aadE-Cc; 54%) and quinolone (gyrA-T86I; 77%), while C. jejuni had resistance genes for kanamycin (aph3'-IIIa; 20%). Both species harbored resistance genes associated with β-lactam (especially, blaOXA-193; up to 100%) and tetracycline (tetO; up to 59%). Discussion/Conclusion: Our study demonstrated that Campylobacter genome plasticity associated with conjugative transfer might provide resistance to certain antimicrobials and viral infections via the acquisition of protein-encoding genes involved in mechanisms such as ribosomal protection and capsule modification. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus ST398 can cause diseases in several different animals. In this study we analyzed ten S. aureus ST398 previously collected in three different reservoirs in Portugal (humans, gilthead seabream from aquaculture and dolphin from a zoo). Strains tested against sixteen antibiotics, by disk diffusion or minimum inhibitory concentration, showed decreased susceptibility to benzylpenicillin (all strains from gilthead seabream and dolphin) and to erythromycin with an iMLSb phenotype (nine strains), and susceptibility to cefoxitin (methicillin-susceptible S. aureus, MSSA). All strains from aquaculture belonged to the same spa type, t2383, whereas strains from the dolphin and humans belonged to spa type t571. A more detailed analysis using single nucleotide polymorphisms (SNPs)-based tree and a heat map, showed that all strains from aquaculture origin were highly related with each other and the strains from dolphin and humans were more distinct, although they were very similar in ARG, VF and MGE content. Mutations F3I and A100V in glpT gene and D278E and E291D in murA gene were identified in nine fosfomycin susceptible strains. The blaZ gene was also detected in six of the seven animal strains. The study of the genetic environment of erm(T)-type (found in nine S. aureus strains) allowed the identification of MGE (rep13-type plasmids and IS431R-type), presumably involved in the mobilization of this gene. All strains showed genes encoding efflux pumps from major facilitator superfamily (e.g., arlR, lmrS-type and norA/B-type), ATP-binding cassettes (ABC; mgrA) and multidrug and toxic compound extrusion (MATE; mepA/R-type) families, all associated to decreased susceptibility to antibiotics/disinfectants. Moreover, genes related with tolerance to heavy metals (cadD), and several VF (e.g., scn, aur, hlgA/B/C and hlb) were also identified. Insertion sequences, prophages, and plasmids made up the mobilome, some of them associated with ARG, VF and genes related with tolerance to heavy metals. This study highlights that S. aureus ST398 can be a reservoir of several ARG, heavy metals resistance genes and VF, which are essential in the adaption and survival of the bacterium in the different environments and an active agent in its dissemination. It makes an important contribution to understanding the extent of the spread of antimicrobial resistance, as well as the virulome, mobilome and resistome of this dangerous lineage. [ABSTRACT FROM AUTHOR]

Background The Andean condor (Vultur gryphus) is the largest scavenger in South America. This predatory bird plays a crucial role in their ecological niche by removing carcasses. We report the first metagenomic analysis of the Andean condor gut microbiome. Methods This work analyzed shotgun metagenomics data from a mixture of fifteen captive Chilean Andean condors. To filter eukaryote contamination, we employed BWA-MEM v0.7. Taxonomy assignment was performed using Kraken2 and MetaPhlAn v2.0 and all filtered reads were assembled using IDBA-UD v1.1.3. The two most abundant species were used to perform a genome reference-guided assembly using MetaCompass. Finally, we performed a gene prediction using Prodigal and each gene predicted was functionally annotated. InterproScan v5.31-70.0 was additionally used to detect homology based on protein domains and KEGG mapper software for reconstructing metabolic pathways. Results Our results demonstrate concordance with the other gut microbiome data from New World vultures. In the Andean condor, Firmicutes was the most abundant phylum present, with Clostridium perfringens, a potentially pathogenic bacterium for other animals, as dominating species in the gut microbiome. We assembled all reads corresponding to the top two species found in the condor gut microbiome, finding between 94% to 98% of completeness for Clostridium perfringens and Plesiomonas shigelloides, respectively. Our work highlights the ability of the Andean condor to act as an environmental reservoir and potential vector for critical priority pathogens which contain relevant genetic elements. Among these genetic elements, we found 71 antimicrobial resistance genes and 1,786 virulence factors that we associated with several adaptation processes.

Shewanella spp. are Gram-negative rods widely disseminated in aquatic niches that can also be found in human-associated environments. In recent years, reports of infections caused by these bacteria have increased significantly. Mobilome and resistome analysis of a few species showed that they are versatile; however, comprehensive comparative studies in the genus are lacking. Here, we analyzed the genetic traits of 144 genomes from Shewanella spp. isolates focusing on the mobilome, resistome, and virulome to establish their evolutionary relationship and detect unique features based on their genome content and habitat. Shewanella spp. showed a great diversity of mobile genetic elements (MGEs), most of them associated with monophyletic lineages of clinical isolates. Furthermore, 79/144 genomes encoded at least one antimicrobial resistant gene with their highest occurrence in clinical-related lineages. CRISPR-Cas systems, which confer immunity against MGEs, were found in 41 genomes being I-E and I-F the more frequent ones. Virulome analysis showed that all Shewanella spp. encoded different virulence genes (motility, quorum sensing, biofilm, adherence, etc.) that may confer adaptive advantages for survival against hosts. Our data revealed that key accessory genes are frequently found in two major clinical-related groups, which encompass the opportunistic pathogens Shewanella algae and Shewanella xiamenensis together with several other species. This work highlights the evolutionary nature of Shewanella spp. genomes, capable of acquiring different key genetic traits that contribute to their adaptation to different niches and facilitate the emergence of more resistant and virulent isolates that impact directly on human and animal health. [ABSTRACT FROM AUTHOR]

Simple Summary: The bacterium Streptomyces scabiei is the main causative agent of common scab disease on economically important root and tuber crops. Our work investigated if and how this pathogen uses multiple sugar transport systems to sense the presence of a living plant host through perception and import of cello-oligosaccharides, the elicitors that trigger the onset of the pathogenic lifestyle of S. scabiei. Plant colonization by Streptomyces scabiei, the main cause of common scab disease on root and tuber crops, is triggered by cello-oligosaccharides, cellotriose being the most efficient elicitor. The import of cello-oligosaccharides via the ATP-binding cassette (ABC) transporter CebEFG-MsiK induces the production of thaxtomin phytotoxins, the central virulence determinants of this species, as well as many other metabolites that compose the 'virulome' of S. scabiei. Homology searches revealed paralogues of the CebEFG proteins, encoded by the cebEFG2 cluster, while another ABC-type transporter, PitEFG, is encoded on the pathogenicity island (PAI). We investigated the gene expression of these candidate alternative elicitor importers in S. scabiei 87-22 upon cello-oligosaccharide supply by transcriptomic analysis, which revealed that cebEFG2 expression is highly activated by both cellobiose and cellotriose, while pitEFG expression was barely induced. Accordingly, deletion of pitE had no impact on virulence and thaxtomin production under the conditions tested, while the deletion of cebEFG2 reduced virulence and thaxtomin production, though not as strong as the mutants of the main cello-oligosaccharide transporter cebEFG1. Our results thus suggest that both ceb clusters participate, at different levels, in importing the virulence elicitors, while PitEFG plays no role in this process under the conditions tested. Interestingly, under more complex culture conditions, the addition of cellobiose restored thaxtomin production when both ceb clusters were disabled, suggesting the existence of an additional mechanism that is involved in sensing or importing the elicitor of the onset of the pathogenic lifestyle of S. scabiei. [ABSTRACT FROM AUTHOR]

Background: Bovine mastitis accounts for significant economic losses to the dairy industry worldwide. Staphylococcus aureus is the most common causative agent of bovine mastitis. Investigating the prevalence of virulence factors and antimicrobial resistance would provide insight into the molecular epidemiology of mastitis-associated S. aureus strains. The present study is focused on the whole genome sequencing and comparative genomic analysis of 41 mastitis-associated S. aureus strains isolated from India. Results: The results elucidate explicit knowledge of 15 diverse sequence types (STs) and five clonal complexes (CCs). The clonal complexes CC8 and CC97 were found to be the predominant genotypes comprising 21 and 10 isolates, respectively. The mean genome size was 2.7 Mbp with a 32.7% average GC content. The pan-genome of the Indian strains of mastitis-associated S. aureus is almost closed. The genome-wide SNP-based phylogenetic analysis differentiated 41 strains into six major clades. Sixteen different spa types were identified, and eight isolates were untypeable. The cgMLST analysis of all S. aureus genome sequences reported from India revealed that S. aureus strain MUF256, isolated from wound fluids of a diabetic patient, was the common ancestor. Further, we observed that all the Indian mastitis-associated S. aureus isolates belonging to the CC97 are mastitis-associated. We identified 17 different antimicrobial resistance (AMR) genes among these isolates, and all the isolates used in this study were susceptible to methicillin. We also identified 108 virulence-associated genes and discuss their associations with different genotypes. Conclusion: This is the first study presenting a comprehensive whole genome analysis of bovine mastitis-associated S. aureus isolates from India. Comparative genomic analysis revealed the genome diversity, major genotypes, antimicrobial resistome, and virulome of clinical and subclinical mastitis-associated S. aureus strains. [ABSTRACT FROM AUTHOR]

Background: Perturbation of gut symbiosis has been linked to childhood allergic diseases. However, the underlying host–microbe interaction connected with specific phenotypes is poorly understood. Methods: To address this, integrative analyses of stool metagenomic and metabolomic profiles associated with IgE reactions in 56 children with mite‐sensitized airway allergies (25 with rhinitis and 31 with asthma) and 28 nonallergic healthy controls were conducted. Results: We noted a decrease in the number and abundance of gut microbiome‐encoded carbohydrate‐active enzyme (CAZyme) genes, accompanied with a reduction in species richness, in the asthmatic gut microflora but not in that from allergic rhinitis. Such loss of CAZymes was consistent with the observation that a CAZyme‐linked decrease in fecal butyrate was found in asthmatics and negatively correlated with mite‐specific IgE responses. Different from the CAZymes, we demonstrated an increase in α diversity at the virulome levels in asthmatic gut microbiota and identified phenotype‐specific variations of gut virulome. Moreover, use of fecal metagenomic and metabolomic signatures resulted in distinct effects on differentiating rhinitis and asthma from nonallergic healthy controls. Conclusion: Overall, our integrative analyses reveal several signatures of systems‐level gut microbiome in robust associations with fecal metabolites and disease phenotypes, which may be of etiological and diagnostic implications in childhood airway allergies. [ABSTRACT FROM AUTHOR]

Shewanella spp. are Gram-negative rods widely disseminated in aquatic niches that can also be found in human-associated environments. In recent years, reports of infections caused by these bacteria have increased significantly. Mobilome and resistome analysis of a few species showed that they are versatile; however, comprehensive comparative studies in the genus are lacking. Here, we analyzed the genetic traits of 144 genomes from Shewanella spp. isolates focusing on the mobilome, resistome, and virulome to establish their evolutionary relationship and detect unique features based on their genome content and habitat. Shewanella spp. showed a great diversity of mobile genetic elements (MGEs), most of them associated with monophyletic lineages of clinical isolates. Furthermore, 79/144 genomes encoded at least one antimicrobial resistant gene with their highest occurrence in clinical-related lineages. CRISPR-Cas systems, which confer immunity against MGEs, were found in 41 genomes being I-E and I-F the more frequent ones. Virulome analysis showed that all Shewanella spp. encoded different virulence genes (motility, quorum sensing, biofilm, adherence, etc.) that may confer adaptive advantages for survival against hosts. Our data revealed that key accessory genes are frequently found in two major clinical-related groups, which encompass the opportunistic pathogens Shewanella algae and Shewanella xiamenensis together with several other species. This work highlights the evolutionary nature of Shewanella spp. genomes, capable of acquiring different key genetic traits that contribute to their adaptation to different niches and facilitate the emergence of more resistant and virulent isolates that impact directly on human and animal health.

Ceftazidime-avibactam (CZA) is one of the best therapeutic options available for infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria. However, sporadic reports of CZA-resistant strains have been rapidly increasing in patients. Herein, we provide detailed case reports of the emergence of ceftazidime-avibactam resistance to identify their resistome and virulome using genomic molecular approaches. Sixteen isolates were collected from 13 patients at three hospitals in Catania and Catanzaro (Italy) between 2020-2021. Antimicrobial susceptibility was determined by broth microdiluition. The samples included in study were analyzed for resistome, virulome and Sequence Type (ST) using Whole Genome Sequencing (WGS). All strains were resistant to ceftazidime/avibactam, ciprofloxacin, extended-spectrum cephalosporins and aztreonam, 13/16 to meropenem, 8/16 to colistin and 7/16 to fosfomycin; 15/16 were susceptible to meropenem/vaborbactam; all strains were susceptible to cefiderocol. Molecular analysis showed circulation of three major clones: ST101, ST307 and ST512. In 10/16 strains, we found a blaKPC-3 gene; in 6/16 strains, four different blaKPC variants (blaKPC28-31-34-50) were detected. A plethora of other beta-lactam genes (blaSHV28-45-55-100-106-187-205-212, blaOXA1-9-48, blaTEM-181 and blaCTX-M-15) was observed; blaOXA-9 was found in ST307 and ST512, instead blaOXA48 in one out four ST101 strains. With regard to membrane permeability, ompK35 and ompK36 harbored frameshift mutations in 15/16 strains; analysis of ompK37 gene revealed that all strains harbored a non-functional protein and carry wild-type PBP3. There is an urgent need to characterize the mechanisms underlying carbapenem resistance and the intrinsic bacterial factors that facilitate the rapid emergence of resistance. Furthermore, it is becoming increasingly important to explore feasible methods for accurate detection of different KPC enzymes.

Neonatal sepsis is a life-threatening condition with high mortality. Virulence determinants relevant in causing Gram-negative (GN) neonatal sepsis are still poorly characterized. A better understanding of virulence factors (VFs) associated with GN neonatal sepsis could offer new targets for therapeutic interventions. The aim of this review was to assess the role of GN VFs in neonatal sepsis. We primarily aimed to investigate the main VFs leading to adverse outcome and second to evaluate VFs associated with increased invasiveness/pathogenicity in neonates. MEDLINE, Embase, and Cochrane Library were systematically searched for studies reporting data on the role of virulome/VFs in bloodstream infections caused by Enterobacterales among neonates and infants aged 0–90 days. Twenty studies fulfilled the inclusion criteria. Only 4 studies reported data on the association between pathogen virulence determinants and neonatal mortality, whereas 16 studies were included in the secondary analyses. The quality of reporting was suboptimal in the great majority of the published studies. No consistent association between virulence determinants and GN strains causing neonatal sepsis was identified. Considerable heterogeneity was found in terms of VFs analysed and reported, included population and microbiological methods, with the included studies often showing conflicting data. This variability hampered the comparison of the results. In conclusions, pathogens responsible for neonatal sepsis are widely heterogenous and can use different pathways to develop invasive disease. The recent genome-wide approach needs to include multicentre studies with larger sample sizes, analyses of VF gene profiles instead of single VF genes, alongside a comprehensive collection of clinical information. A better understanding of the roles of virulence genes in neonatal GN bacteraemia may offer new vaccine targets and new markers of highly virulent strains. This information can potentially be used for screening and preventive interventions as well as for new targets for anti-virulence antibiotic-sparing therapies. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is the main cause of bacterial gastroenteritis and a public health problem worldwide. Little information is available on the genotypic characteristics of human C. jejuni in Spain. This study is based on an analysis of the resistome, virulome, and phylogenetic relationship, antibiogram prediction, and antimicrobial susceptibility of 114 human isolates of C. jejuni from a tertiary hospital in southern Spain from October 2020 to June 2023. The isolates were sequenced using Illumina technology, and a bioinformatic analysis was subsequently performed. The susceptibility of C. jejuni isolates to ciprofloxacin, tetracycline, and erythromycin was also tested. The resistance rates for each antibiotic were 90.3% for ciprofloxacin, 66.7% for tetracycline, and 0.88% for erythromycin. The fluoroquinolone resistance rate obtained is well above the European average (69.1%). CC-21 ( n = 23), ST-572 ( n = 13), and ST-6532 ( n = 13) were the most prevalent clonal complexes (CCs) and sequence types (STs). In the virulome, the cadF, ciaB , and cdtABC genes were detected in all the isolates. A prevalence of 20.1% was obtained for the genes wlaN and cstIII , which are related to the pathogenesis of Guillain-Barré syndrome (GBS). The prevalence of the main antimicrobial resistance markers detected were CmeABC (92.1%), RE-cmeABC (7.9%), the T86I substitution in gyrA (88.9%), bla OXA-61 (72.6%) , tet(O) (65.8%), and ant (6)-Ia (17.1%). High antibiogram prediction rates (>97%) were obtained, except for in the case of the erythromycin-resistant phenotype. This study contributes significantly to the knowledge of C. jejuni genomics for the prevention, treatment, and control of infections caused by this pathogen.IMPORTANCEDespite being the pathogen with the greatest number of gastroenteritis cases worldwide, Campylobacter jejuni remains a poorly studied microorganism. A sustained increase in fluoroquinolone resistance in human isolates is a problem when treating Campylobacter infections. The development of whole genome sequencing (WGS) techniques has allowed us to better understand the genotypic characteristics of this pathogen and relate them to antibiotic resistance phenotypes. These techniques complement the data obtained from the phenotypic analysis of C. jejuni isolates. The zoonotic transmission of C. jejuni through the consumption of contaminated poultry supports approaching the study of this pathogen through "One Health" approach. In addition, due to the limited information on the genomic characteristics of C. jejuni in Spain, this study provides important data and allows us to compare the results with those obtained in other countries., Competing Interests: The authors declare no conflict of interest.

This study presents a comprehensive analysis of samples from urban wastewater treatment plants using anoxic/oxic processes in Slovakia and Taiwan, focusing on microbiome, resistome, mobilome, and virulome, which were analyzed using a shotgun metagenomic approach. Distinct characteristics were observed; in Taiwan, a higher abundance and diversity of antibiotic resistance genes were found in both influent and effluent samples, while there was a higher prevalence of mobile genetic elements and virulence factor genes in Slovakia. Variations were noted in microbial community structures; influent samples in Taiwan were reflected from fecal and hospital sources, and those in Slovakia were derived from environmental elements. At the genus level, the samples from Taiwan's sewage treatment plants were dominated by Cloacibacterium and Bacteroides , while Acinetobacter was predominant in samples from Slovakia. Despite similar antibiotic usage patterns, distinct wastewater characteristics and operational disparities influenced microbiome, resistome, mobilome, and virulome compositions, with limited reduction of most resistance genes by the studied anoxic/oxic processes. These findings underscore the importance of region-specific insights into microbial communities for understanding the dynamics of antimicrobial resistance and pathogenicity in urban wastewater treatment systems. Such insights may lay the groundwork for optimizing treatment processes and reducing the dissemination of antibiotic resistance and pathogenicity genes for safeguarding public health., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Klebsiella pneumoniae is a ubiquitous nosocomial pathogen associated with various types of infections in hospitalized patients and different animal species. In the current study, 49  Klebsiella strains isolated from humans, dogs, and cats were investigated using NGS technology. MALDI-TOF failed to identify newly discovered K. variicola and K. quasipneumoniae isolates correctly. MLST analysis revealed different sequence types among K. pneumoniae isolates, and the most frequent STs were ST29, ST219, and ST37. Three ST23 that are generally known as hypervirulent type were identified but they lacked major discriminatory determinants for hypervirulent K. pneumoniae (hvKp). K. pneumoniae isolates showed high diversity, and several isolates from humans and animals were assigned to the same ST and were almost identical. Isolates from humans exhibited more pronounced resistance patterns compared to the animal isolates. High levels of resistance were observed for piperacillin, trimethoprim/sulfamethoxazole, and cephalosporins, and resistance to carbapenem compounds was only found in isolates of human origin. Three strains of human origin were extensively drug-resistant (XDR). A diverse range of resistance genes primarily confer resistance to beta-lactams., phenicol/quinolone, aminoglycoside, macrolide, sulfonamides, and fosfomycin were identified in silico . However, there were inconsistencies between the phenotypic characterization of isolates and the set of resistance genes detected in silico in this set of Klebsiella isolates. Further research using a larger number of isolates from various sources is necessary to fully comprehend the relationship between the presence of antimicrobial resistance determinants and phenotypic data. It is also necessary to monitor the spread of K. pneumoniae from a One Health perspective in Egypt., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Authors. Published by Elsevier B.V.)

Introduction. Staphylococcus aureus is a leading agent in community-acquired bacteraemia (CAB) and has been linked to elevated mortality rates and methicillin resistance in Costa Rica. Gap statement and aim. CAB cases in a tertiary hospital and delineated the sequence types (STs), virulome, and resistome of the implicated isolates. S. aureus CAB cases in a tertiary hospital and delineated the sequence types (STs), virulome, and resistome of the implicated isolates. Methodology. Clinical information was retrieved from patient files. Antibiotic susceptibility profiles were obtained with disc diffusion and automated phenotypic tests. Genomic data were exploited to type the isolates and for detection of resistance and virulence genes. Results. Primary infections predominantly manifested as bone and joint infections, followed by skin and soft tissue infections. Alarmingly, 70% of patients continued to exhibit positive haemocultures beyond 48 h of treatment modification, with nearly a quarter requiring mechanical ventilation or developing septic shock. The 30-day mortality rate reached an alarming 40%. More than 60% of the patients were found to have received suboptimal or inappropriate antibiotic treatment, and there was an alarming tendency towards the overuse of third-generation cephalosporins as empirical treatment. Laboratory tests indicated elevated creatinine levels, leukocytosis, and bandaemia within the first 24 h of hospitalization. However, most showed improvement after 48 h. The isolates were categorized into 13 STs, with a predominance of representatives from the clonal complexes CC72 (ST72), CC8 (ST8), CC5 (ST5, ST6), and CC1 (ST188). Twenty-four isolates tested positive for mecA , with ST72 strains accounting for 20. In addition, we detected genes conferring acquired resistance to aminoglycosides, MLS B antibiotics, trimethoprim/sulfamethoxazole, and mutations for fluoroquinolone resistance in the isolate collection. Genes associated with biofilm formation, capsule synthesis, and exotoxin production were prevalent, in contrast to the infrequent detection of enterotoxins or exfoliative toxin genes. Conclusions. Our findings broaden our understanding of S. aureus infections in a largely understudied region and can enhance patient management and treatment strategies.

We have previously studied carbapenem non-susceptible Pseudomonas aeruginosa (CNPA) strains from intensive care units (ICUs) in a referral hospital in Jakarta, Indonesia (Pelegrin et al., 2019). We documented that CNPA transmissions and acquisitions among patients were variable over time and that these were not significantly reduced by a set of infection control measures. Three high risk international CNPA clones (sequence type (ST)235, ST823, ST357) dominated, and carbapenem resistance was due to carbapenemase-encoding genes and mutations in the porin OprD. Pelegrin et al. (2019) reported core genome analysis of these strains. We present a more refined and detailed whole genome-based analysis of major clones represented in the same dataset. As per our knowledge, this is the first study reporting Single Nucleotide Polymorphisms (wgSNP) analysis of Pseudomonas strains. With whole genome-based Multi Locus Sequence Typing (wgMLST) of the 3 CNPA clones (ST235, ST357 and ST823), three to eleven subgroups with up to 200 allelic variants were observed for each of the CNPA clones. Furthermore, we analyzed these CNPA clone clusters for the presence of wgSNP to redefine CNPA transmission events during hospitalization. A maximum number 35350 SNPs (including non-informative wgSNPs) and 398 SNPs (ST-specific_informativewgSNPs) were found in ST235, 34,570 SNPs (including non-informative wgSNPs) and 111 SNPs (ST-specific_informative-wgSNPs) in ST357 and 26,443 SNPs (including non-informative SNPs) and 61 SNPs (ST-specific_informative-wgSNPs) in ST823. ST-specific_Informative-wgSNPs were commonly noticed in sensor-response regulator genes. However, the majority of non-informative wgSNPs was found in conserved hypothetical proteins or in uncharacterized proteins. Of note, antibiotic resistance and virulence genes segregated according to the wgSNP analyses. A total of 8 transmission chains for ST235 strains followed by 9 and 4 possible transmission chains for ST357 and ST823 were traceable on the basis of pairwise distances of informative-wgSNPs (0 to 4 SNPs) among the strains. The present study demonstrates the value of detailed whole genome sequence analysis for highly refined epidemiological analysis of P. aeruginosa. [ABSTRACT FROM AUTHOR]

ABSTRACT The Latin American Coalition for Escherichia coli Research (LACER) was created as a network of investigators using One Health approaches trying to understand infections caused by regional E. coli isolates and to sound the alarm due to the evolution of strains that are multiresistant to antibiotics (resistome) that also display different virulence profiles (virulome). After the COVID19 pandemic, a major concern by investigators has been the appearance of more virulent and resistant strains. Recently, a paper published in Microbiology Spectrum by Brazilian investigators (Fuga B., et al. Microbiol Spectr 10:e0125621, 2022, https://doi.org/10.1128/spectrum.01256-21) has used a genomic approach to demonstrate that during a period of 45 years, a wide resistome and virulome has converged, resulting in the appearance and persistence of high-risk clones affecting humans, animals and the environment, and its rapid dissemination is becoming an unattended international threat.

We have previously studied carbapenem non-susceptible Pseudomonas aeruginosa (CNPA) strains from intensive care units (ICUs) in a referral hospital in Jakarta, Indonesia (Pelegrin et al., 2019). We documented that CNPA transmissions and acquisitions among patients were variable over time and that these were not significantly reduced by a set of infection control measures. Three high risk international CNPA clones (sequence type (ST)235, ST823, ST357) dominated, and carbapenem resistance was due to carbapenemase-encoding genes and mutations in the porin OprD. Pelegrin et al. (2019) reported core genome analysis of these strains. We present a more refined and detailed whole genome-based analysis of major clones represented in the same dataset. As per our knowledge, this is the first study reporting Single Nucleotide Polymorphisms (wgSNP) analysis of Pseudomonas strains. With whole genome-based Multi Locus Sequence Typing (wgMLST) of the 3 CNPA clones (ST235, ST357 and ST823), three to eleven subgroups with up to 200 allelic variants were observed for each of the CNPA clones. Furthermore, we analyzed these CNPA clone clusters for the presence of wgSNP to redefine CNPA transmission events during hospitalization. A maximum number 35350 SNPs (including non-informative wgSNPs) and 398 SNPs (ST-specific_informative-wgSNPs) were found in ST235, 34,570 SNPs (including non-informative wgSNPs) and 111 SNPs (ST-specific_informative-wgSNPs) in ST357 and 26,443 SNPs (including non-informative SNPs) and 61 SNPs (ST-specific_informative-wgSNPs) in ST823. ST-specific_Informative-wgSNPs were commonly noticed in sensor-response regulator genes. However, the majority of non-informative wgSNPs was found in conserved hypothetical proteins or in uncharacterized proteins. Of note, antibiotic resistance and virulence genes segregated according to the wgSNP analyses. A total of 8 transmission chains for ST235 strains followed by 9 and 4 possible transmission chains for ST357 and ST823 were traceable on the basis of pairwise distances of informative-wgSNPs (0 to 4 SNPs) among the strains. The present study demonstrates the value of detailed whole genome sequence analysis for highly refined epidemiological analysis of P. aeruginosa.

American cranberry (Vaccinium macrocarpon) and lowbush/wild blueberry (V. angustifolium) pomace are polyphenol-rich products having potentially beneficial effects in broiler chickens. This study investigated the cecal microbiome of broiler-vaccinated or non-vaccinated birds against coccidiosis. Birds in each of the two groups (vaccinated or non-vaccinated) were fed a basal non-supplemented diet (NC), a basal diet supplemented with bacitracin (BAC), American cranberry (CP), and lowbush blueberry (BP) pomace alone or in combination (CP + BP). At 21 days of age, cecal DNA samples were extracted and analyzed using both whole-metagenome shotgun sequencing and targeted-resistome sequencing approaches. Ceca from vaccinated birds showed a lower abundance of Lactobacillus and a higher abundance of Escherichia coli than non-vaccinated birds (p < 0.05). The highest and lowest abundance of L. crispatus and E. coli, respectively, were observed in birds fed CP, BP, and CP + BP compared to those from NC or BAC treatments (p < 0.05). Coccidiosis vaccination affected the abundance of virulence genes (VGs) related to adherence, flagella, iron utilization, and secretion system. Toxin-related genes were observed in vaccinated birds (p < 0.05) in general, with less prevalence in birds fed CP, BP, and CP + BP than NC and BAC (p < 0.05). More than 75 antimicrobial resistance genes (ARGs) detected by the shotgun metagenomics sequencing were impacted by vaccination. Ceca from birds fed CP, BP, and CP + BP showed the lowest (p < 0.05) abundances of ARGs related to multi-drug efflux pumps, modifying/hydrolyzing enzyme and target-mediated mutation, when compared to ceca from birds fed BAC. Targeted metagenomics showed that resistome from BP treatment was distant to other groups for antimicrobials, such as aminoglycosides (p < 0.05). Significant differences in the richness were observed between the vaccinated and non-vaccinated groups for aminoglycosides, β-lactams, lincosamides, and trimethoprim resistance genes (p < 0.05). Overall, this study demonstrated that dietary berry pomaces and coccidiosis vaccination significantly impacted cecal microbiota, virulome, resistome, and metabolic pathways in broiler chickens.

Generic Escherichia coli is commonly used as an indicator of fecal contamination to assess water quality and human health risk. Where measured E. coli exceedances occur, the presence of other pathogenic microorganisms, such as Shiga toxin-producing E. coli (STEC), is assumed, but confirmatory data are lacking. Putative E. coli isolates (n = 709) were isolated from water, sediment, soil, periphyton, and feces samples (n = 189) from five sites representing native forest and agricultural environments. Ten E. coli isolates (1.41%) were stx2 positive, 19 (2.7%) were eae positive, and stx1-positive isolates were absent. At the sample level, stx2-positive E. coli (5 of 189, 2.6%) and eae-positive isolates (16 of 189, 8.5%) were rare. Using real-time PCR, these STEC-associated virulence factors were determined to be more prevalent in sample enrichments (stx1, 23.9%; stx2, 31.4%; eae, 53.7%) and positively correlated with generic E. coli isolate numbers (P, 0.05) determined using culture-based methods. Whole-genome sequencing (WGS) was undertaken on a subset of 238 isolates with assemblies representing seven E. coli phylogroups (A, B1, B2, C, D, E, and F), 22 Escherichia marmotae isolates, and 1 Escherichia ruysiae isolate. Virulence factors, including those from extraintestinal pathogenic E. coli, were extremely diverse in isolates from the different locations and were more common in phylogroup B2. Analysis of the virulome from WGS data permitted the identification of gene repertoires that may be involved in environmental fitness and broadly align with phylogroup. Although recovery of STEC isolates was low, our molecular data indicate that they are likely to be widely present in environmental samples containing diverse E. coli phylogroups. [ABSTRACT FROM AUTHOR]

It is increasingly being recognized that severe gastroduodenal diseases such as peptic ulcer and gastric cancer are not just the outcomes of Helicobacter pylori infection in the stomach. Rather, both diseases develop and progress due to the perfect storms created by a combination of multiple factors such as the expression of different H. pylori virulence proteins, consequent human immune responses, and dysbiosis in gastrointestinal microbiomes. In this mini review, we have discussed how the genomes of H. pylori and other gastrointestinal microbes as well as the genomes of different human populations encode complex and variable virulome–immunome interplay, which influences gastroduodenal health. The heterogeneities that are encrypted in the genomes of different human populations and in the genomes of their respective resident microbes partly explain the inconsistencies in clinical outcomes among the H. pylori -infected people. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is an opportunistic pathogen that can cause several infections, mainly in hospitalised or immunocompromised individuals. The spread of K. pneumoniae emerging virulent and multidrug-resistant clones is a worldwide concern and its identification is crucial to control these strains especially in hospitals. This article reports data related to multi-resistant K. pneumoniae strains, isolated from inpatients in the city of Manaus, Brazil, harbouring virulence and antimicrobial-resistance genes, including high-risk international clones belonging to clonal group (CG) 258. Twenty-one strains isolated from different patients admitted to four hospitals in the city of Manaus, located in the state of Amazonas, Northern Brazil (Amazon Rainforest region) were evaluated. The majority of strains (61.9% n = 13) were classified as multidrug-resistant (MDR), and five strains (23.8%) as extensively drug-resistant (XDR). Several virulence and antimicrobial-resistance genes were found among the strains and eight strains (38.1%) presented the hyper-mucoviscous phenotype. MLST analysis demonstrated a great diversity of STs among the strains, totaling 12 different STs (ST11, ST23, ST198, ST277, ST307, ST340, ST378, ST462, ST502, ST3991, ST3993 and ST5209). Three of these (ST11, ST23 and ST340) belong to CG258. [ABSTRACT FROM AUTHOR]

The emergence of Klebsiella pneumoniae (K. pneumoniae) in German healthcare is worrying. It is not well-investigated in the veterinary world and food chains. In the current study, antibiotic susceptibility profiles of 24 K. pneumoniae strains isolated from powdered milk samples produced in Germany were investigated by a microdilution test. Next-generation sequencing (NGS) was applied to identify genomic determinants for antimicrobial resistance (AMR), virulence-associated genes and plasmids replicons. All isolates were susceptible to the majority (14/18) of tested antibiotics. Resistance to colistin, fosfomycin, chloramphenicol and piperacillin was found. The ambler class A ß-lactamase, blaSHV variants were identified in all isolates, of which blaSHV-187 was most prevalent and found in 50% of isolates. Single-nucleotide-variants of oqxA and oqxB conferring resistance to phenicol/quinolone were found in all isolates, and the oqxB17 was the most prevalent found in 46% of isolates. 67% of isolates harbored fosA genes; however, only one was fosfomycin-resistant. Two isolates harbored genes conferring resistance to colistin, despite being susceptible. The majority of identified virulome genes were iron uptake siderophores. Two enterobactins (entB, fepC), six adherence-related genes belonging to E. coli common pilus (ECP) and one secretion system (ompA gene) were found in all isolates. In contrast, yersiniabactin was found in two isolates. One ST23 strain was susceptible to all tested antibiotics, and harbored determinants discriminatory for hypervirulent strains, e.g., aerobactin, salmochelin, yersiniabactin, enterobactin and regulator of mucoid phenotype A genes that are highly associated with hypervirulent K. pneumoniae. The IncF plasmid family was found in all strains, while almost half of the isolates harbored Col440I-type plasmids and nine isolates harbored various Inc-type plasmids. The presence of K. pneumoniae carrying different resistomes and major virulent specific virulomes in powdered milk samples is alarming. This could threaten public health, particularly of neonates and infants consuming dried milk. [ABSTRACT FROM AUTHOR]

It is increasingly being recognized that severe gastroduodenal diseases such as peptic ulcer and gastric cancer are not just the outcomes of Helicobacter pylori infection in the stomach. Rather, both diseases develop and progress due to the perfect storms created by a combination of multiple factors such as the expression of different H. pylori virulence proteins, consequent human immune responses, and dysbiosis in gastrointestinal microbiomes. In this mini review, we have discussed how the genomes of H. pylori and other gastrointestinal microbes as well as the genomes of different human populations encode complex and variable virulome–immunome interplay, which influences gastroduodenal health. The heterogeneities that are encrypted in the genomes of different human populations and in the genomes of their respective resident microbes partly explain the inconsistencies in clinical outcomes among the H. pylori-infected people.