Toxin Genes Research Articles

Whole genome sequencing revealed high proportions of ST152 MRSA among clinical Staphylococcus aureus isolates from ten hospitals in Ghana.

Previous studies in Ghana indicated low prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and predominance of ST152 methicillin-susceptible S. aureus (MSSA) among clinical isolates. ST152 MRSA clones are associated with severe infections and epidemics. Using whole genome sequencing (WGS), 159 S. aureus isolated from clinical sources (wound, blood, urine, ear, abscess, umbilical cord, eye, vaginal samples, and others) from 10 hospitals across Ghana were investigated. mecA (gene for methicillin resistance) was detected in 38% of the isolates. Panton-Valentine leucocidin toxin (PVL) gene occurred in 65% isolates, with 84% of the MRSA's harboring the PVL gene. ST152 was the major clone, with 74% harboring the mecA gene. Other MRSA clones detected were ST5, ST5204, ST852, and ST1. MSSA clones included ST3249, ST152, ST5, ST1, and ST8. Twenty-three genes encoding resistance to 12 antimicrobial classes were observed with blaZ (97%) being the most prevalent. Other predominant resistance genes included tetK (46%), cat (42%), and dfrG (36%) encoding resistance for tetracyclines, phenicols, and diaminopyrimidine, respectively. Virulence genes for enterotoxins, biofilms, toxic-shock-syndrome toxins, hemolysins, and leukotoxins were also detected. Phylogenetic analysis revealed a shift in the dominant clone from MSSA ST152 to MRSA ST152 over the past decade. The study provides valuable insights into the genomic content of S. aureus from clinical sources in Ghana. The finding of ST152 MRSA in high numbers suggests a shifting epidemiological landscape of these pathogens and continuous surveillance using robust tools like WGS is needed to monitor the rise and spread of these epidemic clones in the country.IMPORTANCESince its emergence in 1959, MRSA has been a significant public health concern, causing infections in both clinical and community settings. Patients with MRSA-related infections experience higher mortality rates due to its ability to evade antimicrobials and immune defenses. In Ghana, understanding the molecular epidemiology of MRSA has been hindered by the lack of appropriate laboratory infrastructure and the limited capacity for molecular data analysis. This study, the largest genomic study of S. aureus in Ghana, addresses this gap by utilizing whole genome sequencing to examine the diversity of circulating S. aureus strains from 10 hospitals. Our findings highlight the predominance of pandemic clones, particularly ST152, and the notable transition of ST152 MSSA to ST152 MRSA over the past decade. The findings from this study supports AMR surveillance efforts in Ghana and emphasize the importance of implementing genomic surveillance using WGS to comprehensively monitor the rise and spread of multi-drug-resitant organisms such as MRSA in the country.

Prevalence of Exfoliative Toxin Genes (eta and etb) in Staphylococcus aureus Isolates from Tonsillitis, Wound, and Urinary Tract Infections in Iraq

Prevalence of Exfoliative Toxin Genes (eta and etb) in Staphylococcus aureus Isolates from Tonsillitis, Wound, and Urinary Tract Infections in Iraq

Analyses of Xenorhabdus griffiniae genomes reveal two distinct sub-species that display intra-species variation due to prophages.

Nematodes of the genus Steinernema and their Xenorhabdus bacterial symbionts are lethal entomopathogens that are useful in the biocontrol of insect pests, as sources of diverse natural products, and as research models for mutualism and parasitism. Xenorhabdus play a central role in all aspects of the Steinernema lifecycle, and a deeper understanding of their genomes therefore has the potential to spur advances in each of these applications. Here, we report a comparative genomics analysis of Xenorhabdus griffiniae, including the symbiont of Steinernema hermaphroditum nematodes, for which genetic and genomic tools are being developed. We sequenced and assembled circularized genomes for three Xenorhabdus strains: HGB2511, ID10 and TH1. We then determined their relationships to other Xenorhabdus and delineated their species via phylogenomic analyses, concluding that HGB2511 and ID10 are Xenorhabdus griffiniae while TH1 is a novel species. These additions to the existing X. griffiniae landscape further allowed for the identification of two subspecies within the clade. Consistent with other Xenorhabdus, the analysed X. griffiniae genomes each encode a wide array of antimicrobials and virulence-related proteins. Comparative genomic analyses, including the creation of a pangenome, revealed that a large amount of the intraspecies variation in X. griffiniae is contained within the mobilome and attributable to prophage loci. In addition, CRISPR arrays, secondary metabolite potential and toxin genes all varied among strains within the X. griffiniae species. Our findings suggest that phage-related genes drive the genomic diversity in closely related Xenorhabdus symbionts, and that these may underlie some of the traits most associated with the lifestyle and survival of entomopathogenic nematodes and their bacteria: virulence and competition. This study establishes a broad knowledge base for further exploration of not only the relationships between X. griffiniae species and their nematode hosts but also the molecular mechanisms that underlie their entomopathogenic lifestyle.

Isolation and Characterization of a Novel Escherichia Bacteriophage with Potential to Control Multidrug-Resistant Avian Pathogenic Escherichia coli and Biofilms

Background/Objectives: Avian pathogenic Escherichia coli (APEC) infection is a significant problem for the global chicken industry, as it decreases animal welfare and is associated with substantial economic losses. Traditionally, APEC infections have been controlled through the use of antibiotics, which has led to an increased prevalence of antibiotic-resistant E. coli. Therefore, developing alternative treatments for APEC infection is crucial. Methods: In this study, an Escherichia phage specific to multidrug-resistant (MDR) APEC, designated as phage vB_EcoP_PW8 (phage vECPW8), was isolated. The morphology, phage adsorption to host cells, one-step growth curve, thermal stability, pH stability, whole-genome sequencing, antibacterial ability, and antibiofilm efficacy of phage vECPW8 were evaluated. Results: The results demonstrated that phage vECPW8 has a Podoviridae morphology and is effective at lysing bacteria. Phage vECPW8 exhibited a high absorption rate to bacterial cells (more than 85% within 10 min) and had a latent period of 20 min, with a burst size of 143 plaque-forming units per cell. Additionally, phage vECPW8 showed good temperature and pH stability. The phage displayed strong antibacterial activity in vitro, and its efficacy in controlling bacteria was confirmed through scanning electron microscopy. Whole-genome sequencing revealed that the phage has a linear genome with 69,579 base pairs. The genome analysis supported the safety of the phage, as no toxin, virulence, or resistance-related genes were detected. Phage vECPW8 was identified as a novel lytic phage in the Gamaleyavirus genus and Schitoviridae family. The phage also demonstrated antibiofilm efficacy by reducing and preventing biofilm formation, as evidenced by biofilm biomass and bacterial cell viability measurements. Conclusions: These results indicate that phage vECPW8 is a promising candidate for the effective treatment of MDR APEC infections in poultry.

Clostridioides difficile infections caused by hypervirulent strains: a single-centre real-life study.

Clostridioides difficile infection (CDI) is a leading cause of healthcare-associated infections worldwide, with hypervirulent strains linked to severe disease and higher mortality. This study aims to analyze the epidemiology of CDI at a tertiary-care hospital in Italy and compare clinical outcomes between patients infected with hypervirulent and non-hypervirulent strains. A retrospective comparative study was conducted on patients diagnosed with CDI at ASST Spedali Civili di Brescia, Italy, from January 2015 to June 2023. Hypervirulent strains were identified using the GeneXpert assay as positive for cytotoxin gene (tcdB), binary toxin genes (tcdA and tcdB) and a single nucleotide deletion at position 117 in the tcdC gene and compared to a randomized matched control group with non-hypervirulent CDI. Clinical data were collected and analyzed, with multivariate logistic regression employed to identify risk factors for hypervirulent CDI. Of 1,059 positive C. difficile specimens, a statistically significant trend between January 2015 to June 2023 was found in the increasing incidence of CDI cases per 1,000 hospital admissions and 10,000 bed-days. Notably, a remarkable increase of hypervirulent strains was recorded in 2021 and 2022 when compared to previous years. A total of 130 patients were analyzed: 62 (47.7%) with hypervirulent CDI and 68 (52.3%) controls. Hypervirulent CDI was associated with higher 30-day mortality (18% vs. 5.8%, p = 0.03). Multivariate analysis showed that hypervirulent CDI significantly increased 30-day mortality risk (OR = 9.915, CI = 2.37-61.05, p = 0.005) and that prior antibiotic therapy was a significant risk factor (OR = 5.49, CI = 1.19-39.96, p = 0.047). Our epidemiological data, while suggesting a potential resurgence in CDI transmission during COVID-19 pandemic, are derived from a single-center experience with limited generalizability to the broader population. Nonetheless, they highlight the need for strengthened antimicrobial stewardship and national surveillance systems to effectively monitor and manage these strains.

Characterization of two lytic bacteriophages infecting carbapenem-resistant clinical Klebsiella pneumoniae in Dhaka, Bangladesh

Bacteriophages or bacteria infecting viruses are genetically diverse. Due to the emergence of antimicrobial-resistant bacteria, lytic bacteriophages are gaining enormous attention for treating superbug infections. Klebsiella pneumoniae is one of the eight most significant nosocomial pathogens and is addressed as a critical priority pathogen by WHO, requiring alternative treatment options. We reported two highly lytic bacteriophages, Klebsiella phage Kpn BM7 and the novel Klebsiella phage Kpn BU9, isolated from hospital wastewater and exhibiting lytic activity against different clinical isolates. Whole-genome analysis revealed that phages BM7 and BU9 belong to class Caudoviricetes. Phage BM7, with a genome length of 170,558 bp, is a member of the genus Marfavirus and the species Marfavirus F48. While phage BU9, with a genome length of 60,450 bp, remains unclassified. Neither phage harbors any lysogenic, toxin, or antimicrobial resistance genes. Both phages can steadily survive up to 40 °C and at pH 5–7. The optimal MOI was 0.1 for BM7 and 1 for BU9, with short latent periods of 10 and 25 min and burst sizes of 85 PFU/cell and 12 PFU/cell, respectively. This is the first carbapenem-resistant K. pneumoniae targeting lytic phages to be reported from Bangladesh. This study suggests that BM7 and BU9 are potential candidates for targeting carbapenem-resistant K. pneumoniae.

Etiological characteristics and whole genome sequence analysis of Clostridium perfringens causing a food poisoning outbreak

This study investigated the etiological characteristics and whole genome sequencing of clostridium perfringens (CP) from a food poisoning outbreak. Multiplex real-time PCR was employed to screen pathogens in collected samples. Based on the preliminary screening results, the isolation, cultivation, and identification of suspected pathogenic CP bacteria were performed. The nucleic acid tests were conducted for CP-related virulence genes on CP isolates, anal swab specimens and food samples. The molecular typing of CP isolates was analyzed by using pulsed-field gel electrophoresis (PFGE). A phylogenetic tree was established to analyze nucleotide and amino acid homology after sequencing the PLC gene. The whole genome sequencing and gene annotation on the representative strain QD2022FB4-CP4 were performed to explore its drug resistance, toxin genes and biological characteristics. The analysis revealed that the food poisoning was triggered by F-type Clostridium perfringens, which infected the consumers by contaminating'roujiamo' sandwiches. The whole genome sequencing of the strain QD2022FB4-CP found that it had active metabolic processes, multiple virulence genes and multidrug resistance characteristics.

Clinicopathological findings and etiological characterization in caprine gangrenous mastitis

Gangrenous mastitis ranks among the most economically significant diseases affecting small ruminants. In the present study, along with clinicopathological studies, the etiological characterisation and the contribution of various virulence factors of caprine gangrenous mastitis was studied. Staphylococcus aureus with colony count ranging from 106 to 108 CFU/mL has been identified as the etiological agent in 15 gangrenous mastitis affected goats based on cultural isolation, biochemical characteristics and molecular confirmation. Haematological and serum biochemical analysis was carried out in the affected goats which revealed leucocytosis with thrombocytopenia and hypoproteinemia. Phenotypic identification of antibiotic resistance revealed significantly high resistance to enrofloxacin, sulpha-trimethoprim, tetracycline, and gentamicin whereas the antibiotics cefoperazone, ceftriaxone- tazobactam and amoxicillin clavulanate showed least resistance. Toxin genes lukMF, and hla were detected in 66.7 per cent and 60 per cent of the isolates respectively. Antibiotic resistant genes blaZ (13.3 %), tetM (20 %), sul1 (13.3 %), mecA (80 %) and biofilm forming genes like icaA (26.6 %) icaD (40 %) were also detected in the S. aureus isolates. Majority of the isolates revealed significantly higher antibiotic resistance along with toxin genes and all the isolates showed biofilm formation. Biofilm forming ability of S. aureus plays a crucial role in its virulence whereas, antimicrobial resistance has a significant role in potentiating the virulence of pathogen.

Existence of Thermotolerant and Salt-Loving Diarrheagenic Vibrio alginolyticus in Non-Saline Potable Water System: A Novel Finding from India.

We present a novel report of abundance of halophilic Vibrio alginolyticus with thermotolerant and enterotoxigenic characteristics from community water system of an inland-focus of India causing diarrheal outbreak as an index pathogen. Though, Vibrio alginolyticus causing diarrhea after exposure to marine water and consumption of seafood was reported globally, its existence in non-saline drinking-water sources with pathogenic viability was unknown. A 'matched-pair-case-control' study identified the primary source of V. alginolyticus infection as 'tap-water' distributed by the municipality, used for drinking (MOR: 8.33; 95% CI 2.51-27.6) and household chores (MOR: 3.75; 95% CI 1.24-11.3). Cardinal toxin gene 'tdh' and other pathogenicity markers viz.tlh, vppC, toxR, VPI, T3SS1 and sxt were detected in V. alginolyticus isolates. Expression potential of the hemolytic genes are demonstrated by hemolysis assay and transcriptome analysis. Altogether 30.55% of isolates exhibited strong hemolytic potential in vitro. RT-PCR revealed uninterrupted virulence gene expression in outbreak strains under heat stress. Surprisingly, ~ 100% of V. alginolyticus from the outbreak focus were sensitive/partially sensitive to all group of antibiotics except β-lactums, carbapenem and quinolones. High drug-sensitivity suggested lack of previous human gut exposure and indicated a fresh dissemination from the environmental niche to the community domain. The maximum likelihood phylogeny depicted multiple clades in V. alginolyticus strains from Pan India sources. Isolated outbreak strains shared common ancestry with the strains from nearby riverine system, a source of 'drinking water' supplied to the affected community, confirming its environmental origin. V. alginolyticus, traditionally a fish-pathogen, is steadily gaining an emerging epidemiological relevance alongside other waterborne diarrheagenic bacteria and its 'thermotolerant' attribute poses additional threat under the canvas of climate change.

Elevated levels of butyric acid in the jejunum of an animal model of broiler chickens: from early onset of Clostridium perfringens infection to clinical disease of necrotic enteritis

BackgroundNecrotic enteritis (NE) is an economically important disease of broiler chickens caused by Clostridium perfringens (CP). The pathogenesis, or disease process, of NE is still not clear. This study aimed to identify the alterations of metabolites and metabolic pathways associated with subclinical or clinical NE in CP infected birds and to investigate the possible variations in the metabolic profile of birds infected with different isolates of CP.MethodologyUsing a well-established NE model, the protein content of feed was changed abruptly before exposing birds to CP isolates with different toxin genes combinations (cpa, cpb2, netB, tpeL; cpa, cpb2, netB; or cpa, cpb2). Metabolomics analysis of jejunal contents was performed by a targeted, fully quantitative LC-MS/MS based assay.ResultsThis study detected statistically significant differential expression of 34 metabolites including organic acids, amino acids, fatty acids, and biogenic amines, including elevation of butyric acid at onset of NE in broiler chickens. Subsequent analysis of broilers infected with CP isolates with different toxin gene combinations confirmed an elevation of butyric acid consistently among 21 differentially expressed metabolites including organic acids, amino acids, and biogenic amines, underscoring its potential role during the development of NE. Furthermore, protein-metabolite network analysis revealed significant alterations in butyric acid and arginine-proline metabolisms.ConclusionThis study indicates a significant metabolic difference between CP-infected and non-infected broiler chickens. Among all the metabolites, butyric acid increased significantly in CP-infected birds compared to non-infected healthy broilers. Logistic regression analysis revealed a positive association between butyric acid (coefficient: 1.23, P < 0.01) and CP infection, while showing a negative association with amino acid metabolism. These findings suggest that butyric acid could be a crucial metabolite linked to the occurrence of NE in broiler chickens and may serve as an early indicator of the disease at the farm level. Further metabolomic experiments using different NE animal models and field studies are needed to determine the specificity and to validate metabolites associated with NE, regardless of predisposing factors.

Assessing the Risk of Spreading Clostridioides difficile and Its Toxins Within the Dairy Farm.

Clostridioides difficile currently constitutes a major pathogen of the gastrointestinal tract, which poses a significant growing burden on medicine and veterinary medicine in many regions. A farm was assessed (feed table, silage pit, and feces (healthy animals, emaciated animals, and animals with mastitis)) for the presence of C. difficile toxins using the PCR method and for the microbiome in cow feed and feces using NGS technology, one month apart. C. difficile toxin A and binary toxin were detected in feed samples. C. difficile toxin genes were found in the feces of sick animals two to three times more often than in healthy animals. Analysis of the microbial community of cow feces revealed that, during the month, the animals experienced major changes in the community structure associated with the accumulation of pathogenic bacteria, in particular Paeniclostridium sp., as well as with the development of methanogenic archaea of the Methanobacteriaceae and associated microorganisms (Lachnospiraceae and Anaerovoracaceae), which may speak of a decrease in feed efficiency and, subsequently, animal productivity. Thus, it seems likely that C. difficile enters the gastrointestinal tract of animals through feed, while animals weakened by diseases are more sensitive to the reproduction of pathogens in the GIT due to a weakened organism.

Genotypic analysis of Shiga toxin-producing Escherichia coli clonal complex 17 in England and Wales, 2014-2022.

Introduction. Shiga toxin-producing Escherichia coli (STEC) are zoonotic, gastrointestinal pathogens characterized by the presence of the Shiga toxin (stx) gene. Historically, STEC O157:H7 clonal complex (CC) 11 has been the most clinically significant serotype; however, recently there has been an increase in non-O157 STEC serotypes, including STEC O103:H2 belonging to CC17.Gap statement. STEC O103:H2 is an STEC serotype frequently isolated in England, although little is known about the epidemiology, clinical significance, associated public health burden or evolutionary context of this strain.Aim. Surveillance data and whole-genome sequencing data were analysed to determine the microbiological characteristics and public health burden of CC17, including the clinically significant serotype O103:H2, in England and Wales.Methodology. Isolates of E. coli belonging to CC17 (n=425) submitted to the Gastrointestinal Bacteria Reference Unit from 2014 to 2022 were whole genome sequenced, integrated with enhanced surveillance questionnaire data and analysed retrospectively.Results. Overall, diagnoses of CC17 infection increased every year since 2014. Most cases were female (58.5%), with the highest proportion of cases belonging to the 0-4 age group (n=83/424, 19.6%). Clinical presentation data identified diarrhoea (92.1%), abdominal pain (72.4%) and blood in stool (55.3%) as the most frequent symptoms, while 20.4% cases were admitted to hospital and 1.3% developed haemolytic uraemic syndrome. The five most common established serotypes were O103:H2 (64.5%), O123:H2 (11.1%), O151:H2 (6.6%), O71:H2 (3.3%) and O4:H2 (2.6%). The majority of CC17 isolates (78.6%) had the stx1a/eae virulence gene combination. Nine outbreak clusters of STEC infections that were mainly geographically dispersed and temporally related were identified and associated with foodborne transmission.Conclusions. Nationwide implementation of PCR to detect non-O157 STEC and improvements to algorithms for the follow-up of PCR-positive faecal specimens is recommended. Enhanced surveillance is necessary to assess the incidence of CC17 infection and overall burden of this CC within the UK population.

Sample Type and Processing Plant Differences in the Proportion of Enterohemorrhagic Escherichia coli O157 and Non-O157 Serogroups in Feces and on Hides of Cull Dairy Cattle at Slaughter.

The study was conducted to determine the proportion and concentration of enterohemorrhagic Escherichia coli (EHEC) O157 and six non-O157 (O26, O45, O103, O111, O121, and O145) serogroups and identify seasonal and processing plant differences in feces and on hides of cull dairy cattle processed in commercial slaughterhouses in the United States. Approximately 60 rectal and 60 hide-on samples from matched carcasses were collected in each of three processing plants, in two periods; summer of 2017 and spring of 2018. Samples before enrichment were spiral plated to quantify EHEC, and postenriched samples underwent culture methods that included immuno-magnetic separation, plating on selective media, and PCR assays for identification and serogroup confirmation of putative isolates. An isolate was considered EHEC O157 positive if it harbored serogroup-specific (rfbE), Shiga toxin (stx1 and/or stx2), and intimin (eae) genes and EHEC non-O157 positive if at least one of the non-O157 serogroup-specific, stx1 and/or stx2, and eae genes was identified. Generalized linear mixed models were fitted to estimate overall proportion of positives for EHEC O157 and non-O157 EHEC serogroups, as well as seasonal and processing plant differences in fecal and hide-on proportion of positives. The fecal EHEC proportion at the sample level was 1.8% (95% CI = 0.0-92.2%) and 4.2% (95% CI = 0.0-100.0%) for EHEC O157 and EHEC non-O157, respectively. Hide sample level proportion of positives was 3.0% (95% CI = 0.0-99.9%) for EHEC O157 and 1.6% (95% CI = 0.0-100.0%) for EHEC non-O157. The proportion of EHEC O157 and non-O157 significantly differed by processing plant and sample type (hide vs. feces), but not by season. The association between proportion of EHEC serogroups in feces with the proportion on hides collected from matched cattle was 7.8% (95% CI = 0.6-53.3%) and 3.8% (95% CI = 0.3-30.8%) for EHEC O157 and non-O157, respectively. Taken together, our findings provide evidence of a low proportion of EHEC serogroups in the feces and on hides of cull dairy cattle and that their proportion varies across processing plants.

Clinical Snapshot of Group A Streptococcal Isolates from an Australian Tertiary Hospital

Streptococcus pyogenes (Group A Streptococcus, GAS) is a human-restricted pathogen that causes a wide range of diseases from pharyngitis and scarlet fever to more severe, invasive infections such as necrotising fasciitis and streptococcal toxic shock syndrome. There has been a global increase in both scarlet fever and invasive infections during the COVID-19 post-pandemic period. The aim of this study was the molecular characterisation of 17 invasive and non-invasive clinical non-emm1 GAS isolates from an Australian tertiary hospital collected between 2021 and 2022. Whole genome sequencing revealed a total of nine different GAS emm types with the most prevalent being emm22, emm12 and emm3 (each 3/17, 18%). Most isolates (14/17, 82%) carried at least one superantigen gene associated with contemporary scarlet fever outbreaks, and the carriage of these toxin genes was non-emm type specific. Several mutations within key regulatory genes were identified across the different GAS isolates, which may be linked to an increased expression of several virulence factors. This study from a single Australian centre provides a snapshot of non-emm1 GAS clinical isolates that are multiclonal and linked with distinct epidemiological markers commonly observed in high-income settings. These findings highlight the need for continual surveillance to monitor genetic markers that may drive future outbreaks.

Clinical Outcomes and Molecular Characteristics of Bacteroides fragilis Infections.

Bacteroides fragilis is the most common opportunistic anaerobic pathogen. In the absence of appropriate antimicrobial therapy, mortality rates associated with B. fragilis group infections can reach as high as 50%. Therefore, we aimed to elucidate the clinical characteristics and outcomes of B. fragilis infections and the molecular genetic characteristics of B. fragilis isolates. Forty B. fragilis clinical isolates were collected at Hanyang University Hospital between January 2022 and December 2023. Antimicrobial susceptibility was tested using the agar dilution method. Whole-genome sequencing was conducted using the Illumina platform (Illumina, San Diego, CA, USA). Various multilocus sequence types of B. fragilis were identified, including ST149 (N=4), ST11 (N=4), ST1 (N=3), ST21 (N=2), and ST157 (N=1). The insertion sequence (IS) IS1187, located upstream of cfiA, was associated with high-level carbapenem resistance in the ST157 isolate. B. fragilis toxin genes (bft ) were identified in 30% of isolates. The most common comorbidities were diabetes mellitus (26.5%) and non-metastatic cancer (23.5%). Five patients (14.7%) died within 30 days, and two (5.9%) deaths were directly attributable to B. fragilis infection. The emergence of high-level MIC carbapenem-resistant B. fragilis ST157 has led to caution in the presence of B. fragilis infections.

Epidemiology of Clostridioides difficile PCR ribotype 181 after the COVID-19 pandemic in Northern Greece.

Clostridioides difficile infection (CDI) is one of the most significant causes of diarrhea in hospitalized patients. The aim of this study was to investigate the incidence and epidemiology of CDI after the COVID-19 pandemic in hospitalized patients in a rehabilitation center in Thessaloniki, Greece. Αretrospective observational cohort study was performed in inpatients diagnosed with diarrhea of all ages (January 2023 - December 2023) who were initially screened for CDI. From the total cohort of patients with proven CDI, some patients were randomly selected based on their monthly isolation incidence throughout the study period, to investigate their epidemiological data and clinical characteristics. Laboratory diagnosis of CDI was performed by enzyme immunoassay, followed by specific anaerobic culture and molecular testing for detection of toxigenic C. difficile. The isolated C. difficile strains were further characterized by PCR ribotyping. The annual incidence of CDI during the studyperiod was 27.1% (130/480). The linear trend of CDI incidence decreased from 32.5% to 18.2% (P= 0.024). The all-cause mortality rate was 5.0% (3/60). A positive correlation was observed between the length of hospital stay and the number of recurrences (r = 0.546, P < 0.001), while 28 patients (46.7%) experienced recurrence of the infection. Seven different PCR ribotypes were identified in thisstudy. C. difficile tcdA+, tcdB+, cdtA+, cdtB+ PCR ribotype 181 (RT181) was the predominant (76.6%, 46/60), followed by toxin A-negative PCR RT017 (11.6%, 7/60). The annual incidence of CDIdecreased after the COVID-19 pandemic. Our study demonstrates predominance of C. difficile RT181 with tcdA+, tcdB+, cdtA+, cdtB+ toxin gene profile after COVID-19 pandemic in Northern Greece.

First detection of multidrug-resistant and toxigenic Pasteurella aerogenes in sow vaginal discharge: a novel threat to swine health in Thailand

Pasteurella aerogenes has been implicated in reproductive disorders in sows, yet its prevalence and characteristics in vaginal discharge are not well understood. This study aimed to detect P. aerogenes in sow vaginal discharge samples and investigate its antibiotic resistance profile, toxin genes, and toxicity. P. aerogenes was isolated from 40% (8/20) of samples. Antimicrobial susceptibility testing revealed universal resistance to amoxicillin-clavulanate (4:1), with 87.5% of isolates also resistant to oxytetracycline, amoxicillin, ceftriaxone, and enrofloxacin. The colistin resistance gene mcr-2 was detected in 75% of isolates, while class 1 integron (int1) was found in 12.5%. The pax toxin gene cluster was present in 75% of isolates. Toxicity assays using Panagrellus redivivus demonstrated dose-dependent effects of P. aerogenes supernatant containing pax toxins. This study represents the first report of P. aerogenes isolation from sow vaginal discharge in Thailand. The high prevalence of antibiotic resistance, presence of the mcr-2 gene, and toxicity of pax toxin-positive isolates suggest that P. aerogenes may be an underestimated factor in swine reproductive health. These findings highlight the need for further investigation into the role of P. aerogenes in sow reproductive disorders and its potential impact on swine production.

Genomic adaptation of Clostridium perfringens to human intestine

AbstractClostridium perfringens is often associated with foodborne diseases, posing significant public health risks. However, genomic investigation of C. perfringens isolates from the human population has been lacking. This study aims to fill this knowledge gap by examining the genomic characteristics of C. perfringens isolates from 699 individuals at a provincial hospital in China. We further conducted evolutionary and pan‐genomic analyses, incorporating isolates from humans and animals worldwide. The results reveal potential regional and transregional transmission of C. perfringens among individuals, along with the common transfer of small gene clusters during this process. Notably, the food poisoning‐associated toxin gene cpe was identified in a fusion plasmid for the first time in an isolate, indicating fusion of pCP13‐like and pCW3‐like plasmids and the potential for transfer of cpe across genetic backgrounds. Moreover, we observed that the genomic characteristics of C. perfringens correlate with host species, with specific toxin genes, such as pfoA and colA, potentially influencing host selectivity. Through this comprehensive genomic analysis, we provide novel insights into the fusion of pCW3‐like and pCP13‐like plasmids, the genetic location of cpe, the transmission dynamics of C. perfringens strains, and the relationship between toxin genes and host relevance. These findings expand our understanding of C. perfringens and its implications for public health.

Influence of diverse pH and temperatures on the gene expression of toxin-antitoxin systems in Klebsiella pneumoniae

Background: The type II toxin-antitoxin system consists of tightly linked genetic units that produce a toxin and its specific antitoxin. Under typical circumstances, the antitoxin neutralizes the toxic effects caused by the toxin. However, the toxin-antitoxin systems during stress regulate bacterial growth and persistence by affecting translation or DNA replication. This study aimed to assess how variations in temperature and pH stress influence the regulation of type II toxin-antitoxin gene expression within Klebsiella pneumoniae isolates.Methods: Sixty-five Klebsiella pneumoniae isolates were identified by the VITEK®2 compact system and confirmed identity by Polymerase Chain Reaction assay by amplified rpoB gene (Housekeeping gene). The isolates were subjected to Reverse Transcriptase-polymerase chain Reaction assay to determine the transcriptional variations of the type II toxin-antitoxin system genes at diverse temperature and pH values.Results: The results of the current study demonstrated higher expression levels of the antitoxin hipB gene at (37℃, 30℃, 44℃, pH 7, pH 9), mqsR toxin gene at (37℃, 30℃, pH 7, pH 5, pH 11) and relE toxin gene at (pH 5, pH 11).Conclusion: These findings suggest the possible influence of pH and temperature on toxin-antitoxin systems, which might enhance bacterial antibiotic tolerance and persistence.Keywords: Toxin-antitoxin systems; pH stress; Temperature stress; Klebsiella pneumoniae; Gene expression; Reverse Transcriptase PCR

Prevalence and virulence profiles of ESBL-producing Escherichia coli in urinary and blood infections in South Korea.

Escherichia coli is a significant pathogen in extraintestinal infections, and ESBL-producing E. coli poses a major clinical challenge due to its antibiotic resistance. This study comprehensively analyzed E. coli isolates from urine and blood samples of patients with urinary tract and bloodstream infections at three major tertiary hospitals in South Korea. The goal was to provide insights into the distribution, antibiotic resistance, and virulence factors of these strains. Our analysis identified CTX-M and TEM as the dominant ESBL types, found in 71.7% and 61.7% of isolates, respectively, with 46.7% showing co-occurrence. Multilocus sequence typing (MLST) revealed the predominance of high-risk clones such as ST131, ST69, ST73, and ST95, with rare sequence types like ST410 and ST405 also identified. The high prevalence of virulence factors, including iutA (80.8%) and kpsMII (74.2%), further highlights the complexity of these strains. In addition, 38.3% of clinical isolates contained a combination of siderophore, adhesin, protectin, and toxin-related genes. There was no significant difference between urinary tract and bloodstream infections or regional differentiation in Korea. This study highlights the importance of controlling ESBL-producing E. coli infections, especially given the increasing incidence among patients with underlying medical conditions and older adults who are more susceptible to urinary tract infections. These findings serve as valuable indicators for pathogen analysis, especially those harboring antibiotic resistance and toxin genes. The insights gained are expected to contribute significantly to the development of infectious disease prevention and control strategies.