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.

UNASSIGNED: This study was to identify and analyze the pathogen responsible for food poisoning in a tourist group traveling from Macao to Zhuhai.
UNASSIGNED: Samples were obtained from 27 patients of 96 cases, as well as samples of contaminated food in Macau. The collected samples were subjected to serological identification, drug sensitivity analysis, drug resistance gene identification, virulence factor analysis, and tracing.
UNASSIGNED: Twenty-six isolates and the salad isolate were S. enteritidis ST11. Isolates from patients were exhibited significant resistance to Penicillin AMP (Ampicillin) and quinolones NAL (Nalidixic acid). Among these isolates, 21 strains were resistant to two or more antibiotics, indicating the multi-drug resistance (MDR). Genomic characteristics and phylogenetic analysis were performed on 9 of the isolates using whole genome sequencing (WGS). The analysis revealed that the resistance to AMP and NAL was primarily caused by a gryA mutation D87Y (9/9, 100%), and the presence of beta-lactam resistance genes blaOXA-1 (1/9, 11.11%), blaTEM-141 (1/9, 11.11%), and blaTEM-1B (8/9, 88.89%). It was also found a strains isolated from patients had two resistance genes to quinolones or beta-lactam drugs (1/8, 12.5%), respectively. The strains were found to possess 165 virulence genes, one adherence class virulence factor, one invasion class virulence factor and various pathogenicity islands, including SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-9, SPI-10, SPI-13, SPI-14, SPI-15, SGI 1, CS54_island, and C63PI-1. Additionally, the virulence plasmids were detected, including IncFIB(s)-IncFII(s)-IncX1 (55.56%), IncFIB(s)-IncFII(s) (33.33%), and IncFIB(s)-IncFII(s)-IncHI2-IncHI2A (11.11%). PFGE (Pulsed Field Gel Electrophoresis) and phylogenetic tree analysis revealed a high degree of similarity between Salmonella isolates from patients and food samples from Macao.
UNASSIGNED: This study identified Salmonella enterica ST11 as the cause of the food poisoning outbreak. The findings highlight the importance of phenotypic characterization and next-generation sequencing (NGS) tools in epidemiological studies and emphasize the potential risk of a new emerging multi-antibiotic ST11 clone for S. enteritidis.

We studied 34 isolates of Tigecycline-Non-Susceptible A. baumannii (TNAB) obtained from clinical specimens at a large tertiary care hospital in Chongqing, China. These 34 strains belonged to 8 different clones including ST195 (35.3%) and ST208 (17.7%). EBURST analysis found that these 8 ST types belonged to the Clonal Complex 92. Tigecycline resistance-associated genes adeR, adeS, adeL, adeN, rrf, rpsJ, and trm were detected in most strains. The expression level of the resistance-nodulation-cell division (RND) efflux pumps in TNAB strains was higher than the reference strain ATCC19606. 58.8% of strains had a decrease in the tigecycline minimum inhibitory concentration (MIC) after the addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The TNAB strains in our hospital have a high degree of affinity and antibiotic resistance. Regular surveillance should be conducted to prevent outbreaks of TNAB epidemics.

Streptococcus uberis is a globally endemic and poorly controlled cause of bovine mastitis impacting the sustainability of the modern dairy industry. A core genome was derived from 579 newly sequenced S. uberis isolates, along with 305 publicly available genome sequences of S. uberis isolated from 11 countries around the world and used to develop a core genome multi-locus sequence typing (cgMLST) scheme. The S. uberis core genome comprised 1475 genes, and these were used to identify 1447 curated loci that were indexed into the cgMLST scheme. This was able to type 1012 of 1037 (>97  %) isolates used and differentiated the associated sequences into 932 discrete core genome sequence types (cgSTs). Analysis of the phylogenetic relationships of cgSTs revealed no clear clustering of isolates based on metadata such as disease status or year of isolation. Geographical clustering of cgSTs was limited to identification of a UK-centric clade, but cgSTs from UK isolates were also dispersed with those originating from other geographical regions across the entire phylogenetic topology. The cgMLST scheme offers a new tool for the detailed analysis of this globally important pathogen of dairy cattle. Initial analysis has re-emphasized and exemplified the genetically diverse nature of the global population of this opportunistic pathogen.

Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a systemic mycosis that infects a wide range of species. Recent molecular biological investigations have allowed for the genotyping of these species, providing more detailed information on their pathogenicity and infection routes. Koalas (Phascolarctos cinereus) are frequently colonized by Cryptococcus spp., but molecular epidemiological studies have yet to be conducted in Japan. Here, we conducted multi-locus sequence typing (MLST) analysis on Cryptococcus spp. colonization isolates obtained from all koalas kept in seven parks across Japan. Out of 46 koalas examined, 10 (22%) were positive for C. gattii and 3 (6.5%) were positive for C. neoformans. All C. gattii isolates belonged to molecular type VGI and were either sequence type (ST) 51 or a novel ST, and all C. neoformans isolates belonged to molecular type VNI and ST23. Despite the frequent movement of koalas between parks, the STs were relatively park-specific, suggesting that the floor of the rearing barns is a source of infection and may act as a reservoir. MLST analysis confirmed that C. gattii was transported, established, and spread by koalas in areas where C. gattii was not originally present. MLST analysis is considered useful in assessing the pathogenicity and tracing the transmission routes of Cryptococcus spp. carried by koalas.IMPORTANCEThis is the first study to conduct a multi-locus sequence typing analysis on Cryptococcus spp. carried by captive koalas in Japan. Cryptococcosis remains a globally high-fatality fungal infection in humans, and captive koalas are known to carry a high percentage of Cryptococcus spp. Through this research, the molecular types and transmission routes of Cryptococcus spp. carried by koalas have been elucidated, revealing the potential role of enclosure flooring as a reservoir. It has been confirmed that Cryptococcus gattii, which is not endemic in Japan, has become established through koalas and is spreading to new individuals in Japan. This study is believed to provide valuable insights into koala conservation and contribute to the One Health approach for Cryptococcosis, a zoonotic infection.

This study aimed to investigate the genetic diversity of 108 geographically and temporally diverse strains of Mycoplasma hominis using a multi-locus sequence typing scheme (MLST). We extracted MLST data of 87 strains from PubMLST database and retrieved MLST gene sequences from 21 complete genomes of M. hominis available in GenBank database. MLST scheme identified 65 Sequence types (STs), which were grouped into five clonal complexes (CC) and 47 singletons. Phylogenetic analysis revealed that the majority of M. hominis isolates were clustered according to their country of origin, showing some significant specificity trends for the nation. Although recombination was detected, it was not significant enough to alter the clonal population structure of M. hominis. In sum, MLST scheme provides insightful data on the phylogenetics of international strains of M. hominis, arguing for the existence of genetically differentiable STs according to their origin of isolation.

OBJECTIVE: Clostridium perfringens (C. perfringens) is a significant opportunistic pathogen. This study aims to examine the occurrence of C. perfringens in patients with diarrhoea and food poisoning and compare the genetic similarities with strains found in poultry retail markets and poultry farms in the same city (Tai\'an, China).
METHODS: Clostridium perfringens was isolated from 30 human faecal samples and genotyped using multiplex PCR. The antimicrobial susceptibility test was conducted using the Kirby-Bauer disk diffusion method. Genetic relationships were analysed through Multi-locus sequence typing (MLST) and Phylogenetic analysis.
RESULTS: The positive rate of C. perfringens was found to be 96.67%. Among the positive samples, 91.67% of the faecal samples from patients with food poisoning contained type F strains of C. perfringens, while only 16.67% of the samples from diarrhoea cases contained type F. The drug susceptibility test revealed that the majority of isolates displayed broad-spectrum antimicrobial resistance. Out of the 57 isolates tested for drug susceptibility, 89.47% demonstrated resistance to at least three antibiotics. The MLST results indicated that strains originating from the same host and environment tended to be more closely related. However, certain strains associated with food poisoning and diarrhoea in patients shared the same ST and CC as some strains found in the retail market. These strains were also found to be phylogenetically similar to some retail market strains, suggesting potential risks to human health.
CONCLUSIONS: Therefore, it is crucial to enhance the management of poultry retail markets in order to mitigate these associated risks.

Listeria monocytogenes is the etiologic agent of listeriosis, a foodborne disease that poses a threat to public health globally. Chicken meat exhibits heightened susceptibility to L. monocytogenes contamination during butchery. The persistence of this pathogen in the slaughterhouse environment enables recurring contamination of meat products. This study aimed at identifying the sources and transmission routes of L. monocytogenes contamination within an abattoir where it was consistently detected for three consecutive years (2019-2021). Furthermore, the environmental factors aiding contamination along chicken processing lines were determined by surveying the microbiome within the facility. Samples collected in 2019 to 2021 were subjected to culture-dependent analysis to assess the prevalence, serotypes, and multi-locus sequence typing (MLST) of L. monocytogenes. Additionally, the specimens collected in 2021 underwent culture-independent analysis via real-time quantitative polymerase chain reaction (qPCR) and 16S rRNA gene amplicon sequencing to identify the contamination sources and characterize the entire microbial community within the slaughterhouse. L. monocytogenes was isolated only from the clean zone, where the final slaughtering stage occurs. Most strains isolated from the final carcasses showed the same genetic cluster as the isolate in the chilling water and were assigned to MLST profile ST3. Culture-independent qPCR confirmed L. monocytogenes contamination in all samples, excluding post-scalding carcasses, prewashed post-evisceration carcasses, and the bleeding areas. Consequently, qPCR enabled more comprehensive identification of L. monocytogenes contamination points than culture-dependent approaches. Moreover, 16S rRNA gene amplicon sequencing demonstrated that psychro-tolerant and spoilage-related bacteria with L. monocytogenes-like attributes exhibited enhanced viability in the clean zone and immersion-chilling water. Metagenomics-based source tracking analysis further revealed that the shackles and chilling waters represent predominant sources of cross-contamination between different slaughterhouse zones, whereas the grading and packaging workstations and chilling water in the clean zone were deemed crucial sources affecting final carcass contamination. Collectively, these findings demonstrate through culture-dependent and -independent methods that L. monocytogenes spreads along the slaughter line, contaminating the slaughterhouse. Moreover, by investigating changes in microbial community and bacterial flow along the slaughter line within the facility, the sources influencing carcass contamination can be effectively traced.

BACKGROUND: Staphylococcus aureus, one of the most prevalent opportunistic pathogens, mainly colonizes the nasal cavity and is a risk factor for severe infections. Virulence factors and accessory gene regulator (agr) are key to the severity and diversity of staphylococcal infection. In this study, we aimed to characterise S. aureus agr-types and virulence genes and correlated them with genetic background and antibiotic-resistant phenotypes.
RESULTS: Agr types were identified in 704 isolates (98.5%), with only 11 isolates were negative for agr type. Most of our isolates were classified as agr type I, followed by types III, II and IV. The enterotoxin c gene (sec) was detected in 48.6% of isolates, showing the highest prevalence among the five enterotoxin genes detected. The positivity rates for the lukS/F-PV and tsst genes were 4% and 2.2%, respectively, while neither sed nor SasX were detected. ST45, ST59, ST338, ST188, ST6, ST7, ST22, ST25, ST398, and ST944 belonged to agr I group, while ST5 and ST15 belonged to agr II group. ST30 and ST1 were classified into agr III group, and ST121 was assigned into agr IV group. The tsst gene was found exclusively within agr I and III types belonging to ST7 and ST30 isolates, while the lukS/F-PV was predominantly carried by agr I type isolates primarily within CC59 and CC22 clones. Among the methicillin-resistant S. aureus (MRSA) isolates, 89.7% belonged to agr I group, and 97.8% of rifampicin-resistant or intermediate isolates were assigned to agr I group. MRSA isolates harboured more tested virulence genes compared to methicillin-susceptible S. aureus isolates.
CONCLUSIONS: We characterized the distributions of agr types and eight major virulence genes of 715 S. aureus isolates, and our findings revealed clear associations between agr types and STs, as well as virulence genes, and drug resistant phenotypes.

Clostridioides difficile is the most important pathogen causing antimicrobial-associated diarrhea and has recently been recognized as a cause of community-associated C. difficile infection (CA-CDI). This study aimed to characterize virulence factors, antimicrobial resistance (AMR), ribotype (RT) distribution and genetic relationship of C. difficile isolates from diverse fecally contaminated environmental sources. C. difficile isolates were recovered from different environmental samples in Northern Germany. Antimicrobial susceptibility testing was determined by E-test or disk diffusion method. Toxin genes (tcdA and tcdB), genes coding for binary toxins (cdtAB) and ribotyping were determined by PCR. Furthermore, 166 isolates were subjected to whole genome sequencing (WGS) for core genome multi-locus sequence typing (cgMLST) and extraction of AMR and virulence-encoding genes. Eighty-nine percent (148/166) of isolates were toxigenic, and 51% (76/148) were positive for cdtAB. Eighteen isolates (11%) were non-toxigenic. Thirty distinct RTs were identified. The most common RTs were RT127, RT126, RT001, RT078, and RT014. MLST identified 32 different sequence types (ST). The dominant STs were ST11, followed by ST2, ST3, and ST109. All isolates were susceptible to vancomycin and metronidazole and displayed a variable rate of resistance to moxifloxacin (14%), clarithromycin (26%) and rifampicin (2%). AMR genes, such as gyrA/B, blaCDD-1/2, aph(3\')-llla-sat-4-ant(6)-la cassette, ermB, tet(M), tet(40), and tetA/B(P), conferring resistance toward fluoroquinolone, beta-lactam, aminoglycoside, macrolide and tetracycline antimicrobials, were found in 166, 137, 29, 32, 21, 72, 17, and 9 isolates, respectively. Eleven \"hypervirulent\" RT078 strains were detected, and several isolates belonged to RTs (i.e., RT127, RT126, RT023, RT017, RT001, RT014, RT020, and RT106) associated with CA-CDI, indicating possible transmission between humans and environmental sources pointing out to a zoonotic potential.