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Enterococcus faecalis is a gram-positive bacterium and a common cause of hospital-associated infections. Three major CRISPR loci have been discovered in this species, namely, CRISPR1-cas, CRISPR2 and CRISPR3-cas. We developed novel primers which target the CRISPR1-cas loci in E. faecalis and tested these primers on 26 E. faecalis isolates isolated from diverse settings from Segamat, Malaysia. Half of the isolates were found to carry the CRISPR1-cas9 locus, and the CRISPR1 array was successfully amplified in 12 out of 13 isolates that contained the cas9 gene. Characterization of the CRISPR array shows that CRISPR1-cas shares similar array length and typical repeat sequences with CRISPR2 but differs significantly in terms of spacer identities and terminal repeat (TR) sequences. Most CRISPR spacers encode for chromosomal DNA sequences. Genotype characterization based on ancestral spacer (AS) and TR sequences indicates that E. faecalis with the same CRISPR1-AS genotype do not always harbour the same CRISPR2-AS genotypes and vice versa. A combined CRISPR1-cas and CRISPR2 typing offers comparable discriminatory power to MLST, suggesting its potential to be used in short-term strain identification and epidemiological surveillance at a lower sequencing cost. Our study provides a genetic reference for future studies in Southeast Asia.

Assessment of the pathogenic potential (virulence and toxicity) in non-pathogenic bacterial species is a challenge as it relies on methods developed for assessment of species known to be pathogenic. Here, we have applied and evaluated some of these methods on industrially relevant bacteria to differentiate between 'true' virulence factors applying only to pathogens and niche factors being defined as promoting colonization and survival rather than pathogenicity and as being present also in non-pathogenic bacteria. We examined the pathogenicity of 49 strains from 9 industrially relevant bacterial species (Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus delbrueckii, Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum, Latilactobacillus curvatus, Ligilactobacillus salivarius, Staphylococcus carnosus and Staphylococcus xylosus), including 14 clinical isolates of the same species, through genomic screening and phenotypically through assays established for pathogenic bacteria. The genomes were screened against the Virulence Factor Database (VFDB), and thresholds (>80% nucleotide or protein identity, >70% coverage) provided by the European Food Safety Authority (EFSA) were adopted to differentiate between genes of potential concern and genes of no concern. Core genome analysis was performed to determine whether the clinical isolates were phylogenetically related to the industrial isolates. The genotypic assessment did not reveal the presence of true virulence factors in the examined strains, and in the core genome analysis, the clinical isolates could not be distinguished from the industrial strains. Furthermore, cytotoxicity toward Vero cells, negative impact on Caco-2 cell viability and haemolytic activity on blood agar plates were examined, and none of the tested strains exhibited any activity in these assays. Overall, the results suggest that VFDB screening with the EFSA thresholds can be used to differentiate between true virulence factors and niche factors. Furthermore, the use of phenotypic assays supports the genotypic assessment, albeit expert knowledge is required to interpret the results.

Here, we report the draft sequence of a rapid-growing nontuberculous Mycobacterium isolated from a urine sample at the Scottish Mycobacteria Reference Laboratory, Royal Infirmary of Edinburgh, UK. The reported genome has a length of 6,749,454 bp, a G+C content of 67.2 mol% and 6,336 protein CDSs. Average nucleotide identity (ANI) analysis identified Mycobacterium vanbaalenii PYR-1 as the closest relative (83.32% ANI), indicating that this isolate likely represents a novel species within the genus. Notably, phenotypic characterization revealed a distinct antimicrobial resistance (AMR) profile. This assembly provides a valuable resource for studying the evolution of AMR mechanisms in nontuberculous mycobacteria and offers insight into resistance phenotypes observed in clinical isolates.

Tuberculosis (TB) is an infectious disease that affects humans and animals. The pathogens that cause TB belong to the Mycobacterium tuberculosis complex (MTBC), with M. tuberculosis and Mycobacterium bovis as the main representatives of human- and animal-adapted strains, respectively. One key genetic regulator of the MTBC members is the PhoPR system, which controls many processes, including the stress response, lipid metabolism and pathogenesis, among others. Previous studies identified a key G71I substitution in the M. bovis PhoR orthologue relative to M. tuberculosis PhoR and suggested that PhoPR might be non-functional in animal-adapted strains, but recent work has highlighted the functionality of PhoPR in M. bovis despite the G71I substitution. Here, we compare the transcriptional effects of the PhoPR system of M. tuberculosis H37Rv and M. bovis AF2122/97 on an M. bovis AF2122/97 ΔphoPR knockout background. Our results show common patterns of gene expression between the two orthologues, but also clear differences in the expression of rubredoxin genes and lipid biosynthetic loci. This work adds to the evidence that the PhoPR system is indeed functional in M. bovis and suggests that PhoPR controls differential transcriptional programmes that are important in the adaptation to human or animal hosts.

Background. Carbapenem and third-generation cephalosporin (3GC) resistance among Gram-negative bacteria poses a serious threat to human and animal health. This study aimed to identify and characterize carbapenem- and 3GC-resistant Enterobacterales isolated from poultry in Lusaka Province, Zambia. Methods. Ninety pooled cloacal samples were collected from market-ready broiler chickens in the Chongwe and Chilanga districts of Lusaka Province. The isolates were screened for 3GC and carbapenem resistance using the disc diffusion and broth microdilution methods. PCR and Sanger sequencing were performed for species identification and detection of β-lactamase-encoding (bla) genes, including bla _CTX-M, bla _TEM, bla _OXA-1 and bla _SHV. Hierarchical clustering was used to assess phenotypic and genotypic relationships. Results. A total of 83 3GC-resistant Gram-negative isolates were recovered, of which 12% were also carbapenem resistant. Escherichia coli was the most prevalent species, followed by Klebsiella pneumoniae and Enterobacter spp., then Pseudomonas aeruginosa, other Pseudomonas spp., Acinetobacter baumannii, Citrobacter freundii and Aeromonas caviae. Multidrug resistance occurred in 84.3% of the isolates, with the highest resistance to ampicillin, tetracycline and co-trimoxazole. Overall, 80.7% of the isolates harboured at least one of the four tested bla genes, with bla _CTX-M and bla _TEM being the most common. Hierarchical clustering revealed that isolates from both districts shared similar phenotypic and genotypic resistance patterns. Conclusions. The presence of multidrug- and carbapenem-resistant Enterobacterales from poultry highlights the emergence of carbapenem resistance in Zambia's food production sector. The detection of imipenem-resistant isolates indicates the potential for transmission of resistance genes between animals and humans. These findings underscore the need for prudent antimicrobial use, strengthened stewardship and a One Health surveillance approach to contain the spread of carbapenem resistance genes.

Capnocytophaga canimorsus is a fastidious Gram-negative bacterium found in the mouths of dogs and cats. It is a rare cause of infective endocarditis, when it is often associated with dog bites. We present a case of C. canimorsus infective endocarditis complicated by aortic regurgitation and root abscess in a patient with a history of previous infective endocarditis. The patient underwent redo aortic valve surgery with aortic valve replacement. Blood cultures and 16S ribosomal ribonucleic acid gene amplification and sequencing from the excised valve tissue confirmed C. canimorsus as the cause. The patient was treated with beta-lactam antibiotics and discharged home. Rather than secondary to a dog bite, infection most likely occurred due to a dog licking an open wound. It is important to remember that dog contact, often perceived as innocuous, such as being licked, can be a source of serious infection, particularly in the context of an open wound. Over a third of households in the UK own a dog as a pet. With C. canimorsus infections thought to be on the rise, in part due to increased pet ownership, there is a need to ensure pet owners, particularly those at risk of infections and chronic skin wounds, are educated on such risks and the appropriate preventative steps.

The intracellular autophagy receptor p62 (also known as Sequestosome-1) plays a dual role in autophagic flux and downstream Toll-like receptor signalling and has been implicated in modulating immune responses. However, its specific function in controlling intracellular bacterial survival, particularly in macrophages, remains less well characterized. Salmonella enterica serovar Typhimurium (S. Tm) is a major global pathogen and a leading cause of gastroenteritis-associated morbidity. We have previously demonstrated that host restriction of S. Tm in macrophages involves the GTPase Rab32 and the BLOC-3 complex. In the present study, we identify a novel interaction between p62 and Rab32. Notably, p62 restricts Salmonella survival independently of the Rab32/BLOC-3 pathway. Indeed, p62-knockdown in macrophages resulted in significantly increased intracellular bacterial survival, an effect that did not correlate with altered recruitment of canonical autophagy-related proteins, as assessed by fluorescence microscopy. Through real-time polymerase chain reaction (RT-qPCR) and infection assays, we further show that p62-depleted macrophages exhibit a dampened pro-inflammatory response, which corresponds with the increased bacterial burden. These findings provide new mechanistic insight into the role of p62 in modulating the macrophage inflammatory response during Salmonella infection, highlighting its contribution to host defence beyond its canonical functions in autophagy.

Lactococcus garvieae is a facultatively anaerobic Gram-positive coccus which causes lactococcosis, a septicaemic illness in fish of major aquacultural significance. This pathogen has emerged in recent years as a cause of zoonotic infections including infective endocarditis, primary bacteraemia, peritonitis and orthopaedic infections, with putative risk factors such as raw seafood ingestion and underlying gastrointestinal disease. We report a case of L. garvieae bioprosthetic aortic and mitral valve endocarditis in a 75-year-old man, complicated by multiple cerebral septic emboli, lumbar discitis/osteomyelitis and bilateral pulmonary nodules. Despite initial stabilization with antibiotic therapy and medical heart failure management, the patient deteriorated approximately a year after his index admission with worsening valvulopathy and ultimately died of complications of his infection. This case highlights the emergence of L. garvieae as an opportunistic pathogen in humans, with increasing cases identified due to improved recognition and enhanced diagnostics.

Background. World Health Organization (WHO) priority bacterial pathogens and ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) organisms in household wastewater pose critical community transmission risks, yet surveillance data from Sub-Saharan Africa remain limited. This study provides the first comprehensive priority pathogen detection and antimicrobial resistance assessment in household wastewater from Gombe State, Nigeria, focusing on organisms of highest clinical concern. Methods. We conducted targeted surveillance for WHO priority pathogens in 320 household wastewater samples across seven districts in Gombe using multi-stage sampling. Some priority Gram-negative pathogens (Escherichia coli, K. pneumoniae, P. aeruginosa and Enterobacter spp.) were specifically isolated and characterized. Antimicrobial susceptibility testing followed Clinical and Laboratory Standards Institute (CLSI) 2024 guidelines across 12 antibiotics targeting critical resistance patterns. Extended-spectrum beta-lactamase (ESBL) and carbapenemase detection focused on priority pathogen isolates, with PCR confirmation of key resistance genes. Results. Priority pathogen detection revealed E. coli (131 isolates, 32.6%) as the dominant WHO priority pathogen, followed by K. pneumoniae (77 isolates, 19.2%) and P. aeruginosa (45 isolates, 11.2%). The ESKAPE pathogen P. aeruginosa showed 73.3% multidrug resistance (MDR), with carbapenemase gene detection [Verona integron-encoded metallo-beta-lactamase gene (blaVIM), 60%; K. pneumoniae carbapenemase gene (blaKPC), 20%]. The critical priority pathogen K. pneumoniae demonstrated a 79.2% MDR prevalence, with universal beta-lactamase CTX-M gene (blaCTX-M) gene presence (100%) among ESBL producers. Priority pathogen E. coli exhibited an alarming 84.7% MDR rate, with widespread ESBL production (51.5%). Carbapenem resistance among priority pathogens reached 41.3%, indicating last-resort antibiotic failure in critical organisms. Conclusions. Household wastewater in Gombe harbours WHO priority pathogens and ESKAPE organisms with high antimicrobial resistance prevalence. These preliminary findings suggest substantial environmental circulation of resistant bacteria and highlight the need for enhanced surveillance, further investigation of community transmission risks and strengthened antimicrobial stewardship programmes.

The environmental spread of antibiotic-resistant bacteria is a growing global health concern, particularly in low- and middle-income countries where limited wastewater treatment infrastructure may facilitate the dissemination of multidrug-resistant (MDR) organisms. Escherichia coli and Klebsiella spp. are clinically significant MDR pathogens commonly associated with healthcare-associated infections and known to carry diverse antimicrobial resistance genes (ARGs). In this study, we conducted genomic and phenotypic analyses of E. coli and Klebsiella spp. isolated from hospital wastewater and paediatric patient samples at a tertiary hospital in Lima, Peru, between 2017 and 2019. A total of 157 isolates were collected (E. coli, n=113; Klebsiella spp., n=44). Whole-genome sequencing was performed to identify ARGs and assess sequence types (STs). MDR phenotypes were more prevalent among wastewater isolates (73.5%) compared to clinical isolates (56.8%, P=0.014), while extended-spectrum β-lactamase production was more frequent in clinical isolates (52.9 % vs. 13.9 %, P<0.001). Carbapenemase-producing isolates were found only in wastewater, whereas colistin resistance was restricted to a subset of clinical E. coli isolates from urine. Genomic analysis revealed greater sequence type diversity among wastewater isolates, including high-risk STs such as ST10, ST131 and ST405. The Shannon diversity index was higher for wastewater-derived isolates (H=3.45) compared to clinical isolates (H=2.95), indicating a more heterogeneous resistance reservoir. In total, 1,302 resistance gene hits were identified, with clinical isolates carrying significantly more ARGs per genome than wastewater isolates. A small number of shared STs were detected in both sources, suggesting possible overlap in bacterial populations. Our findings highlight the potential role of hospital wastewater as a reservoir of antimicrobial resistance and support the value of integrating environmental and clinical genomic surveillance. Wastewater-based monitoring may inform infection control efforts and guide interventions to curb the spread of AMR within healthcare settings and their surrounding environments.