Molecular epidemiology, antibiotic resistance profile and frequency of integron 1 and 2 in adherent-invasive Escherichia coli isolates of colorectal cancer patients.

This study explores the prevalence of adherent-invasive Escherichia coli (AIEC) in colorectal cancer (CRC) patients and investigates the potential of effective intracellular antibiotics as a therapeutic strategy for CRC patients with AIEC infections. Considering the pivotal role of integrons in bacterial antibiotic resistance, the frequency of class 1 and 2 integrons in AIEC isolated from CRC patients, in one of the referenced 3 gastroenterology clinics in Isfahan, Iran was examined. AIEC strains were isolated from the colorectal biopsies and their antimicrobial sensitivity was assessed using the disc diffusion method. Polymerase chain reaction (PCR) was employed to detect intl1 and intl2. The multilocus sequence typing (MLST) method was utilized to type 10 selected isolates. Of the 150 samples, 24 were identified as AIEC, with the highest number isolated from CRC2 (33.4%) and CRC1 (29.16%), and the least from the FH group (8.3%) and control group (12.5%). int1 in 79.2% and int2 in 45.8% of AIEC strains were found and 41.6% of strains had both integrons. AIEC isolates with int1 exhibited the highest sensitivity to trimethoprim-sulfamethoxazole (57.9%), while those with int2 showed the highest sensitivity to ciprofloxacin (63.6%). A significant association between resistance to rifampin and integron 2 presence in AIEC isolates was observed. Furthermore, a significant correlation between integron 1 presence, invasion, survival, and replication within macrophages in AIEC strains was identified. MLST analysis revealed ST131 from CC131 with integron 1 as the most common sequence type (ST). The emergence of such strains in CRC populations poses a serious public health threat. The distribution pattern of STs varied among studied groups, with pandemic STs highlighting the importance of examining and treating patients infected with these isolates. Comprehensive prospective clinical investigations are warranted to assess the prognostic value of detecting this pathovar in CRC and to evaluate therapeutic techniques targeting drug-resistant AIECs, such as phage therapy, bacteriocins, and anti-adhesion compounds, for CRC prevention and treatment.

Comparative Genomics of an Emerging Multidrug-Resistant blaNDM-Carrying ST182 Lineage in Enterobacter cloacae Complex.

BACKGROUND: Enterobacter cloacae, E. hormaechei and related subspecies remain the most clinically relevant among the Enterobacter cloacae complex (ECC). Carbapenemase-producing ECC strains are increasingly identified in hospital-acquired infections and usually belong to four main multilocus sequence types (MLST STs) named ST114, ST93, ST90 and ST78. Instead, ST182 has been sporadically reported among E. hormaechei strains, and recently, outbreaks of blaNDM-producing ST182 clonal strains have emerged. Herein, we aimed to investigate the presence of ST182 and explore its evolution and modes of blaNDM acquisition. METHODS: A phylogenetic analysis of 646 MLST STs identified among 4685 E. hormaechei whole-genome sequencing (WGS) assemblies deposited in public repositories was performed, as well as an in silico comparative and phylogenomic analyses for 55 WGS assemblies of ST182. blaNDM-harboring contigs were also compared to published plasmid sequences. RESULTS: ST182 E. hormaechei strains were recovered from patients on five continents during 2011-2021. They were divided into three major genomic clusters, comprising a separate clonal complex with six other STs. In 30 out of 55 ST182 WGS assemblies, blaNDM-harboring structures were identified that were similar to the plasmids predominant in Gram-negative bacteria, harboring resistance genes to multiple antibiotic classes and virulence genes. No associations between the genomic clusters and the country/continent of isolation or the presence and the plasmid types of the blaNDM-harboring contigs were observed. CONCLUSIONS: Our findings show that ST182 E. hormaechei strains have been identified in the past decade worldwide; 54.5% of them carried diverse blaNDM genetic structures, suggesting recent acquisition of the blaNDM alleles. Thus, blaNDM-harboring ST182 is an emerging multidrug-resistant and virulent lineage in ECC strains that requires close monitoring.

Whole-Genome Sequencing of Human and Porcine Escherichia coli Isolates on a Commercial Pig Farm in South Africa.

Escherichia coli is an indicator micro-organism in One Health antibiotic resistance surveillance programs. The purpose of the study was to describe and compare E. coli isolates obtained from pigs and human contacts from a commercial farm in South Africa using conventional methods and whole-genome sequencing (WGS). Porcine E. coli isolates were proportionally more resistant phenotypically and harbored a richer diversity of antibiotic resistance genes as compared to human E. coli isolates. Different pathovars, namely ExPEC (12.43%, 21/169), ETEC (4.14%, 7/169), EPEC (2.96%, 5/169), EAEC (2.96%, 5/169) and STEC (1.18%, 2/169), were detected at low frequencies. Sequence type complex (STc) 10 was the most prevalent (85.51%, 59/169) among human and porcine isolates. Six STcs (STc10, STc86, STc168, STc206, STc278 and STc469) were shared at the human-livestock interface according to multilocus sequence typing (MLST). Core-genome MLST and hierarchical clustering (HC) showed that human and porcine isolates were overall genetically diverse, but some clustering at HC2-HC200 was observed. In conclusion, even though the isolates shared a spatiotemporal relationship, there were still differences in the virulence potential, antibiotic resistance profiles and cgMLST and HC according to the source of isolation.

Molecular Characteristics of Virulence Genes in Carbapenem-Resistant and Carbapenem-Sensitive Klebsiella Pneumoniae in Relation to Different Capsule Serotypes in Ningbo, China.

Background: Klebsiella pneumoniae (KP) is a common nosocomial pathogen. Capsules are an important component of KP's virulence, among which the K1, K2, K5, K20, K54, and K57 serotypes are predominant and exhibit varying degrees of virulence. Methods: The capsule and virulence genes of 150 carbapenem-resistant Klebsiella pneumoniae (CRKP) and 213 carbapenem-sensitive Klebsiella pneumoniae (CSKP) isolates were examined by polymerase chain reaction (PCR). The isolates were tested for hypermucoviscosity by string tests. Phylogenetic relationships between KP isolates were analyzed using multilocus sequence typing (MLST) and a Galleria mellonella infection model confirmed the differences in virulence. Results: A total of 111 of 363 isolates of KP were detected, the highest detected serotypes were K1, K5, and K2, and CSKP was detected more frequently than CRKP. There was a greater prevalence of K1 and K2 serotypes in CSKP, while in CRKP, K5 serotypes were more prevalent. K1 isolates had the highest detection rates for hypermucoviscosity Klebsiella pneumoniae (hmKP) and hypervirulent Klebsiella pneumoniae (hvKP), and carried the most virulence genes. K54 isolates had the lowest detection rate of hmKP while K5 isolates had the lowest detection rate of hvKP and carried the fewest virulence genes. MLST results for serotypes K1, K20, and K57 showed significant homogeneity, while those for serotypes K2, K5, and K54 showed diversity. The Galleria mellonella infection model showed that the K1 serotype was the most virulent and the K54 serotype was the weakest. Conclusion: CSKP isolates were detected more frequently than CRKP isolates for capsular serotype detection. K1 isolates had the most virulence gene and strongest virulence, K5 isolates carried the fewest virulence genes, and K54 isolates had the weakest virulence. Furthermore, significant homogeneity was observed among K1, K20, and K57 isolates.

Rapid genotyping of Toxoplasma gondii isolates via Nanopore-based multi-locus sequencing.

Toxoplasma gondii is an obligate intracellular parasite associated with severe disease, especially in the immunosuppressed. It is also a cause of congenital malformation and abortion in both animals and humans and is considered one of the most important foodborne pathogens worldwide with different strains showing variable distribution and differing pathogenicity. Thus, strain-level differentiation of T. gondii isolates is an essential asset in the understanding of parasite's diversity, geographical distribution, epidemiology and health risk. Here, we designed and implemented an Oxford Nanopore MinION protocol to analyse genomic sequence variation including single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms (InDel's) of four different genomic loci, part of protein coding genes SAG2, SAG3, ROP17 and ROP21. This method provided results with the sequencing depth necessary for accurate differentiation of T. gondii strains and represents a rapid approach compared to conventional techniques which we further validated against environmental samples isolated from wild wood mice. In summary, multi-locus sequence typing (MLST) of both highly conserved and more polymorphic areas of the genome, provided robust data for strain classification in a platform ready for further adaption for other strains and pathogens.

Culture-independent Multilocus sequence typing screening for Haemophilus influenzae cross-infection in non-cystic fibrosis bronchiectasis.

Whether cross-infection of respiratory pathogens between patients with non-cystic fibrosis bronchiectasis occurs is debated. Investigation with traditional microbiological culture risks simplifying the lung microbiome. We demonstrate the use of culture-independent Multilocus sequence typing to screen for Haemophilus influenzae strain types in a cohort of twenty-eight patients with non-cystic fibrosis bronchiectasis.

Host specificity and virulence of Flavobacterium psychrophilum: a comparative study in ayu (Plecoglossus altivelis) and rainbow trout (Oncorhynchus mykiss) hosts.

Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease, is a devastating, worldwide distributed, fish pathogen causing significant economic loss in inland fish farms. Previous epidemiological studies showed that prevalent clonal complexes (CC) differ in fish species affected with disease such as rainbow trout, coho salmon and ayu, indicating significant associations between particular F. psychrophilum genotypes and host species. Yet, whether the population structure is driven by the trade of fish and eggs or by host-specific pathogenicity is uncertain. Notably, all F. psychrophilum isolates retrieved from ayu belong to Type-3 O antigen (O-Ag) whereas only very few strains retrieved from other fish species possess this O-Ag, suggesting a role in outbreaks affecting ayu. Thus, we investigated the links between genotype and pathogenicity by conducting comparative bath infection challenges in two fish hosts, ayu and rainbow trout, for a collection of isolates representing different MLST genotypes and O-Ag. Highly virulent strains in one host species exhibited low to no virulence in the other. F. psychrophilum strains associated with ayu and possessing Type-3 O-Ag demonstrated significant variability in pathogenicity in ayu, ranging from avirulent to highly virulent. Strikingly, F. psychrophilum strains retrieved from rainbow trout and possessing the Type-3 O-Ag were virulent for rainbow trout but not for ayu, indicating that Type-3 O-Ag alone is not sufficient for pathogenicity in ayu, nor does it prevent pathogenicity in rainbow trout. This study revealed that the association between a particular CC and host species partly depends on the pathogen's adaptation to specific host species.

Surveillance of vancomycin-resistant enterococci reveals shift in dominating clusters from vanA to vanB Enterococcus faecium clusters, Denmark, 2015 to 2022.

BackgroundVancomycin-resistant enterococci (VRE) are increasing in Denmark and Europe. Linezolid and vancomycin-resistant enterococci (LVRE) are of concern, as treatment options are limited. Vancomycin-variable enterococci (VVE) harbour the vanA gene complex but are phenotypically vancomycin-susceptible.AimThe aim was to describe clonal shifts for VRE and VVE in Denmark between 2015 and 2022 and to investigate genotypic linezolid resistance among the VRE and VVE.MethodsFrom 2015 to 2022, 4,090 Danish clinical VRE and VVE isolates were whole genome sequenced. We extracted vancomycin resistance genes and sequence types (STs) from the sequencing data and performed core genome multilocus sequence typing (cgMLST) analysis for Enterococcus faecium. All isolates were tested for the presence of mutations or genes encoding linezolid resistance.ResultsIn total 99% of the VRE and VVE isolates were E. faecium. From 2015 through 2019, 91.1% of the VRE and VVE were vanA E. faecium. During 2020, to the number of vanB E. faecium increased to 254 of 509 VRE and VVE isolates. Between 2015 and 2022, seven E. faecium clusters dominated: ST80-CT14 vanA, ST117-CT24 vanA, ST203-CT859 vanA, ST1421-CT1134 vanA (VVE cluster), ST80-CT1064 vanA/vanB, ST117-CT36 vanB and ST80-CT2406 vanB. We detected 35 linezolid vancomycin-resistant E. faecium and eight linezolid-resistant VVEfm.ConclusionFrom 2015 to 2022, the numbers of VRE and VVE increased. The spread of the VVE cluster ST1421-CT1134 vanA E. faecium in Denmark is a concern, especially since VVE diagnostics are challenging. The finding of LVRE, although in small numbers, ia also a concern, as treatment options are limited.

In Silico Typing and Identification Confirmation of Isolates.

Streptococcus suis is an important zoonotic pathogen causing severe infections in pigs and humans. Serotyping of S. suis strains is crucial for epidemiological surveillance, outbreak investigations, and understanding the pathogenesis of this bacterium. Here, we describe a step-by-step approach that enhances a previously developed pipeline by utilizing a computational script for efficient and accurate typing of S. suis strains. The pipeline is implemented in Perl programming language and leverages the Short Read Sequence Typing for Bacterial Pathogens (SRST2) tool. It integrates various bioinformatics techniques and utilizes multiple databases, including a serotype database, cpsH confirmation database, multi-locus sequence typing (MLST) database, recN species-specific gene database, and virulence gene database. These databases contain comprehensive information on S. suis serotypes, genetic markers, and virulence factors. The script can utilize paired-end or single-end fastq files as input and first confirms the species by sequence read data aligning to the recN gene, ensuring the accurate identification of S. suis strains. The pipeline next performs MLST typing and virulence factor identification using SRST2 while in a parallel processes it performs in silico serotyping of the strains. The pipeline offers a streamlined and semiautomated approach to serotyping S. suis strains, facilitating large-scale studies and reducing the manual effort required for data analysis.

Prevalence, Genotypic Characteristics, and Antibiotic Resistance of Listeria monocytogenes From Retail Foods in Huzhou, China.

Listeria monocytogenes are considered to be the major foodborne pathogen worldwide. To understand the prevalence and potential risk of L. monocytogenes in retail foods, a total of 1243 retail foods in 12 food categories were sampled and screened for L. monocytogenes from 2020 to 2022 in Huzhou, China. A total of 46 out of 1234 samples were confirmed to be L. monocytogenes positive with a total rate of 3.7%. The contamination rate of seasoned raw meat (15.2%) was the highest, followed by raw poultry meat and raw livestock meat (9.9%) and salmon sashimi (9.5%). The L. monocytogenes isolates belonged to four serotypes, 1/2a,1/2b, 1/2c, and 4b, with the most prevalent serotype being 1/2a (47.9%). All isolates were grouped into 15 sequence types (STs) belonging to 14 clonal complexes (CCs) via multilocus sequence typing (MLST). The most prevalent ST was ST9/CC9 (23.9%), followed by ST3/CC3 (19.6%) and ST121/CC121 (17.4%). Notably, 11 STs were detected from ready-to-eat (RTE) foods, some of them have been verified to be strongly associated with clinical origin listeriosis cases, such as ST3, ST2, ST5, ST8, and ST87. Listeria pathogenicity islands 1 (LIPI-1) and LIPI-2 were detected in approximately all L. monocytogenes isolates, whereas the distribution of both LIPI-3 genes and LIPI-4 genes exhibited association with specific ST, with LIPI-3 in ST3 and ST288, and LIPI-4 in ST87. The strains carrying LIPI-3 and LIPI-4 virulence genes in this study were all isolated from RTE foods. Antimicrobial susceptibility tests showed that >90% of isolates were susceptible to PEN, AMP, ERY, CIP, SXT, VAN, CHL, and GEN, indicating the antibiotic treatment might be still efficient for most of the L. monocytogenes strains. However, for the three clinical first-line antibiotics (PEN, AMP, and GEN), we also observed three and four strains showing MIC values greater than the susceptibility standards for PEN and AMP, respectively, and one strain showing resistance to GEN.

Puerperal mastitis caused by limited community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) clones.

Objective: To outline the epidemiology of puerperal mastitis caused by methicillin-resistant Staphylococcus aureus (MRSA) and evaluate the effect of an infection control bundle on its incidence. Methods: A surge in MRSA puerperal mastitis was noted in a community hospital in September 2009. MRSA samples from mastitis cases and the environment underwent typing using multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec), gene encoding surface protein A (spa), accessory gene regulator (agr), and pulsed-field gel electrophoresis (PFGE). The phenotypic characteristics, including superantigen toxin profiles, gene encoding Panton-Valentine leucocidin (pvl), and minimal inhibitory concentration (MIC) against vancomycin, were ascertained. Subsequently, an infection control bundle emphasizing contact precautions was introduced, and mastitis incidence rates pre- and post-intervention were compared. Results: The majority of cases occurred within 6 weeks post-delivery in first-time mothers. Of the 42 S. aureus isolates (27 from mastitis and 15 from colonized staff and environmental sources), 25 (92.6%) clinical and 3 (20%) colonized MRSA were identified as ST59-SCCmecVT-spa t437-agr group I with a vancomycin MIC of 1 mg/L, pvl-positive, and predominantly with a consistent toxin profile (seb-selk-selr). PFGE revealed 13 patterns; pulsotype B exhibited clonal relatedness between two clinical and three colonized MRSA samples. Post-intervention, the incidence of both mastitis and MRSA mastitis notably decreased from 13.01 to 1.78 and from 3.70 to 0.99 episodes per 100 deliveries, respectively. Conclusion: Distinct community-associated MRSA (CA-MRSA) clones were detected among puerperal mastitis patients and colonized staff. The outbreak was effectively controlled following the implementation of a targeted infection control bundle.

Molecular epidemiology and molecular typing methods of Acinetobacter baumannii: An updated review.

The aim of this study was to go through the molecular methods used for typing of carbapenem-resistant Acientobacter baumannii (CRAB) isolates for investigating the molecular epidemiology all over the world. Multiple typing techniques are required to understand the source and nature of outbreaks caused by Acientobacter baumannii (A. baumannii) and acquired resistance to antimicrobials. Nowadays, there is gradual shift from traditional typing methods to modern molecular methods to study molecular epidemiology and infection control. Molecular typing of A. baumannii strains has been revolutionized significantly in the last 2 decades. A few sequencing-based techniques have been proven as a breakthrough and opened new prospects, which have not been achieved by the traditional methods. In this review, discussed different pre-existing and recently used typing methods to explore the molecular epidemiology of A. baumannii pertaining in context with human infections.

Multi-locus sequence typing, antimicrobials resistance and virulence profiles of Salmonella enterica isolated from bovine carcasses in Minas Gerais state, Brazil.

The aim of this study was to identify virulence and antimicrobial resistance profiles and determine the sequence type (ST) by multilocus sequence typing (MLST) of Salmonella enterica isolates from bovine carcasses from slaughterhouse located in Minas Gerais state, Brazil, and its relationship with bovine isolates obtained on the American continent based on sequence type profile. The MLST results were compared with all Salmonella STs associated with cattle on American continent, and a multi-locus sequence tree (MS tree) was built. Among the 17 S. enterica isolates, five ST profiles identified, and ST10 were the most frequent, grouping seven (41.2%) isolates. The isolates presented 11 different profiles of virulence genes, and six different antibiotics resistance profiles. The survey on Enterobase platform showed 333 Salmonella STs from American continent, grouped into four different clusters. Most of the isolates in the present study (13/17), were concentrated in a single cluster (L4) composed by 74 STs. As a conclusion, five different STs were identified, with ST10 being the most common. The isolates showed great diversity of virulence genes and antibiotics resistance profiles. Most of the isolates of this study were grouped into a single cluster composed by 74 STs formed by bovine isolates obtained on the American continent.

Genomic insights into the diversity, virulence, and antimicrobial resistance of group B Streptococcus clinical isolates from Saudi Arabia.

Introduction: Detailed assessment of the population structure of group B Streptococcus (GBS) among adults is still lacking in Saudi Arabia. Here we characterized a representative collection of isolates from colonized and infected adults. Methods: GBS isolates (n=89) were sequenced by Illumina and screened for virulence and antimicrobial resistance determinants. Genetic diversity was assessed by single nucleotide polymorphisms and core-genome MLST analyses. Results: Genome sequences revealed 28 sequence types (STs) and nine distinct serotypes, including uncommon serotypes VII and VIII. Majority of these STs (n=76) belonged to the human-associated clonal complexes (CCs) CC1 (33.71%), CC19 (25.84%), CC17 (11.24%), CC10/CC12 (7.87%), and CC452 (6.74%). Major CCs exhibited intra-lineage serotype diversity, except for the hypervirulent CC17, which exclusively expressed serotype III. Virulence profiling revealed that nearly all isolates (94.38%) carried at least one of the four alpha family protein genes (i.e., alphaC, alp1, alp2/3, and rib), and 92.13% expressed one of the two serine-rich repeat surface proteins Srr1 or Srr2. In addition, most isolates harbored the pilus island (PI)-2a alone (15.73%) or in combination with PI-1 (62.92%), and those carrying PI-2b alone (10.11%) belonged to CC17. Phylogenetic analysis grouped the sequenced isolates according to CCs and further subdivided them along with their serotypes. Overall, isolates across all CC1 phylogenetic clusters expressed Srr1 and carried the PI-1 and PI-2a loci, but differed in genes encoding the alpha-like proteins. CC19 clusters were dominated by the III/rib/srr1/PI-1+PI-2a (43.48%, 10/23) and V/alp1/srr1/PI-1+PI-2a (34.78%, 8/23) lineages, whereas most CC17 isolates (90%, 9/10) had the same III/rib/srr2/P1-2b genetic background. Interestingly, genes encoding the CC17-specific adhesins HvgA and Srr2 were detected in phylogenetically distant isolates belonging to ST1212, suggesting that other highly virulent strains might be circulating within the species. Resistance to macrolides and/or lincosamides across all major CCs (n=48) was associated with the acquisition of erm(B) (62.5%, 30/48), erm(A) (27.1%, 13/48), lsa(C) (8.3%, 4/48), and mef(A) (2.1%, 1/48) genes, whereas resistance to tetracycline was mainly mediated by presence of tet(M) (64.18%, 43/67) and tet(O) (20.9%, 14/67) alone or in combination (13.43%, 9/67). Discussion: These findings underscore the necessity for more rigorous characterization of GBS isolates causing infections.

Hybrid Illumina-Nanopore assembly improves identification of multilocus sequence types and antimicrobial resistance genes of Staphylococcus aureus isolated from Vermont dairy farms: comparison to Illumina-only and R9.4.1 nanopore-only assemblies.

Antimicrobial resistance (AMR) in Staphylococcus aureus is a pressing public health challenge with significant implications for the dairy industry, encompassing bovine mastitis concerns and potential zoonotic threats. To delve deeper into the resistance mechanisms of S. aureus, this study employed a hybrid whole genome assembly approach that synergized the precision of Illumina with the continuity of Oxford Nanopore. A total of 62 isolates, collected from multiple sources from Vermont dairy farms, were sequenced using the GridION Oxford Nanopore R9.4.1 platform and the Illumina platform, and subsequently processed through our long-read first bioinformatics pipeline. Our analyses showcased the hybrid-assembled genome's superior completeness compared to Oxford Nanopore (R9.4.1)-only or Illumina-only assembled genomes. Furthermore, the hybrid assembly accurately determined multilocus sequence typing (MLST) strain types across all isolates. The comprehensive probe for antibiotic resistance genes (ARGs) using databases like CARD, Resfinder, and MEGARES 2.0 characterized AMR in S. aureus isolates from Vermont dairy farms, and revealed the presence of notable resistance genes, including beta-lactam genes blaZ, blaI, and blaR. In conclusion, the hybrid assembly approach emerged as a tool for uncovering the genomic nuances of S. aureus isolates collected from multiple sources on dairy farms. Our findings offer a pathway for detecting AMR gene prevalence and shaping AMR management strategies crucial for safeguarding human and animal health.

Multi-locus sequence typing of Escherichia coli isolated from clinical samples in Jordan.

INTRODUCTION: Escherichia coli (E. coli) is the major cause of extraintestinal infections in the urinary tracts and bloodstream in humans in the community and health care institutions. Several studies on the genetic characterization of E. coli among clinical and environmental isolates were performed and revealed a wide diversity of sequence types (STs). In Jordan, phenotypic and genetic features of E. coli were extensively studied but there is still a need to identify the STs that inhabit the community. METHODOLOGY: In this study, multi-locus sequence typing (MLST) was performed on archived clinical E. coli isolates collected from different hospitals in Jordan and the identified STs were extensively analyzed. RESULTS: Genotyping of 92 E. coli isolates revealed 34 STs and 9 clonal complexes. The frequencies of STs ranged between 1 to 23 observations. The most frequent STs among E. coli isolates were ST131 (n = 23), ST69 (n = 19), ST998 (n = 7), ST2083 (n = 5), and ST540 (n = 4). These five ST accounted for up to 60% of the 92 E. coli isolates. Based on the MLST database, the STs reported in this work were world widely recognized in humans, animals, and in the environment. CONCLUSIONS: This study has elaborated more knowledge about the genotypes of E. coli in Jordan, with recommendations for future studies to correlate its genotypes with virulence and resistance genes.

Fecal Shedding of Clostridioides difficile in Calves in Sao Paulo State, Brazil.

OBJECTIVE: This study aimed to evaluate the fecal shedding of C. difficile in calves on farms in Sao Paulo State, Brazil. MATERIALS AND METHODS: Fecal samples (n=300) were collected from diarrheic (n=78) and nondiarrheic (n=222) calves less than 60 days of age from 20 farms. Fecal samples were inoculated into enrichment broth supplemented with taurocholate and cultured under anaerobic conditions. Colonies suspected to be C. difficile were harvested for DNA extraction and then multiplex PCR for the detection of genes encoding toxins A and B and binary toxins. All toxigenic isolates were ribotyped and tested for antimicrobial susceptibility, and five selected strains were subjected to whole-genome sequencing to determine their sequence type. RESULTS AND DISCUSSION: C. difficile was isolated from 29.3% (88/300) of the samples. All toxigenic isolates (17/88, 19.3%) were classified as ribotypes RT046 (13/17 -79.47%, A+B+ CDT-) and RT126 (4/17=20.53%, A+B+ CDT+). The sequenced strains from RT046 were classified as ST35 (Clade 1), while those from RT126 were classified as ST11 (Clade 5). No associations between the epidemiological factors in any of the groups and C. difficile isolation were observed. Most of the toxigenic isolates (16/17=94.41%) were classified as multidrug-resistant. Calves can be an important source of toxigenic C. difficile strains, including multidrug-resistant isolates from ribotypes commonly observed in humans.

Antimicrobial resistance, virulence potential and genomic epidemiology of global genomes of the rare Salmonella enterica serovar Orion.

AIMS: Our aim is to characterize through whole-genome sequencing (WGS) the antimicrobial resistance (AMR) and heavy metal tolerance (HMT) genes content, plasmid presence, virulence potential and genomic diversity of the rare non-typhoid Salmonella enterica serovar Orion (S. Orion) from 19 countries of the African, American, Eastern Mediterranean, European, Southeastern Asia and Western Pacific regions. METHODS AND RESULTS: Totally 324 S. Orion genomes were screened for AMR, HMT and virulence genes, plasmids and Salmonella Pathogenicity Islands (SPIs). Genomic diversity was investigated using Multi-Locus Sequence Typing (MLST) and core-genome MLST (cgMLST). Efflux pump encoding genes mdsA and mdsB were present in all genomes analysed, while quinolone chromosomal point mutations and aminoglycoside, beta-lactam, colistin, lincosamide, macrolide, phenicol, sulphonamide, trimethoprim, tetracycline and disinfectant resistance genes were found in 0.3%-5.9%. A total of 17 genomes (5.2%) from Canada, the United Kingdom, the USA and Tanzania showed a potential multi-drug resistance profile. Gold tolerance genes golS and golT were detected in all genomes analysed, while arsenic, copper, mercury, silver and tellurium tolerance genes were found in 0.3%-35.5%. Col(MGD2) was the most frequently detected plasmid, in 15.4% of the genomes. Virulence genes related to adherence, macrophage induction, magnesium uptake, regulation, serum resistance, stress adaptation, type III secretion systems and six SPIs (1, 2, 3, 4, 5, 9, 12, 13, 14 and C63PI) were detected. ST639 was assigned to 89.2% of the S. Orion genomes, while cgMLST showed core-genome STs and clusters of strains specific by countries. CONCLUSION: The high virulence factor frequencies, the genomic similarity among some non-clinical and clinical strains circulating worldwide and the presence of a strain carrying a resistance gene against a last resource antimicrobial like colistin, highlight the potential risk of S. Orion strains for public health and food safety and reinforce the importance to not underestimate the potential hazard of rare non-typhoid Salmonella serovars.

Molecular Characterization of Klebsiella pneumoniae Clinical Isolates Through Whole-Genome Sequencing: A Comprehensive Analysis of Serotypes, Sequence Types, and Antimicrobial and Virulence Genes.

Introduction Antimicrobial resistance (AMR) has become a menace, spreading among bacterial species globally. AMR is now recognized as a silent pandemic responsible for treatment failures. Therefore, an effective surveillance mechanism is warranted to understand the bacterial species isolated from human clinical specimens. The present study employed next-generation sequencing (NGS) or whole-genome sequencing (WGS) to identify the resistance and virulence genes, sequence type, and serotypes. Methods This study included 18 multidrug-resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) isolates obtained from patients suffering from different infections attending the Prathima Institute of Medical Sciences, Karimnagar, India. All isolates were identified, and antimicrobial susceptibility profiles were determined through conventional microbiological techniques and confirmed by automated systems. All the isolates were investigated using NGS or WGS to identify the genes coding for resistance, such as extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases, and virulence genes. Multilocus sequence typing (MLST) was conducted to identify the sequence types, and Kleborate analysis was performed to confirm the species, genes for AMR, and virulence and evaluate the capsular polysaccharide (KL) and cell wall/lipopolysaccharide (O) serotypes carried by the isolates. Results The mean age of the patients was 46.11±20.35 years. Among the patients included, 12 (66.66%) were males and 6 (33.33%) were females. A high percentage (>50%) of hypervirulent K. pneumoniae (hvKp) strains that had genes coding for AMR and plasmids having the potential to carry blaNDM and resistance genes were observed. Among the isolates, 16 (88.88%) revealed the presence of multiple antibiotic-resistant genes with evidence of at least one gene coding for beta-lactamase resistance. There was a high prevalence of blaSHV (17/18; 94.44%) and blaCTX-M-15 (16/18; 88.88%) AMR genes. Other AMR genes identified included blaTEM (83.33%; 15/18) and blaOXA (14/18; 77.77%). Two (11.11%) strains each showed the presence of blaNDM-1 and blaNDM-5 genes. The virulence genes identified included gapA, infB, mdh, pgi, phoE, rpoB, tonB, and ybt. The most frequent K. pneumoniae serotypes found were KL51:O1v2 (3/18, 16.66%), KL17:O1v1 (3/18, 16.66%), and KL64:O2v1 (3/18, 16.66%). KL64 (4/18; 22.22%) was the most common capsular serotype identified among the isolates. The most frequent MLST-based sequence type (ST) identified included ST-147 (5/18, 27.77%), followed by ST-231 (3/18, 16.66%) and ST-101 (2/18, 11.11%). Conclusions The molecular analysis of K. pneumoniae isolates revealed multiple AMR, plasmid, and virulence genes. Additionally, many global STs were noticed by MLST. The results noted a high prevalence of hvKp strains. Molecular characterization of bacterial strains using NGS/WGS is important to understand the epidemiology of bacterial strains and the antibiotic resistance and virulence genes they are potentially carrying. The data obtained from this study may be utilized to devise careful antibiotic-prescribing approaches and improve patient management practices.

Phylogenomic characterization of Flavobacterium psychrophilum isolates retrieved from Turkish rainbow trout farms.

Flavobacterium psychrophilum, a devastating fish pathogen, is responsible for bacterial cold-water disease (BCWD), also known as rainbow trout fry syndrome. F. psychrophilum is the main causative agent of outbreaks in rainbow trout farms, especially at early live stages. In the present study, we aimed to characterize F. psychrophilum Turkish isolates. Eighteen isolates were retrieved from BCWD outbreaks between 2014 and 2021. In vitro phenotypic characterization showed gelatin and casein hydrolysis capacities and in vitro adhesion for all isolates, whereas elastinolytic activity was present for 16 of 18 isolates. We used complete genome sequencing to infer MLST-type, serotype and phylogenetic reconstruction. Strikingly, one strain isolated from Coruh trout (FP-369) belongs to ST393, a previously undescribed ST, and is phylogenetically distant from the other isolates. However, all strains retrieved from rainbow trout belong to the well-characterized clonal complex CC-ST10, 12 of 17 were tightly connected in a single cluster. Several serotypes (Types -1, -2 and -3) were represented among isolates, but no correlation was observed with geographic origins. This analysis suggests a regional dissemination of an epidemic, disease-producing bacterial population. This study provides a basis for epidemiological surveillance of isolates circulating in Turkey and phenotypic data for future molecular studies of virulence traits of this important fish pathogen.