Rapid detection of the source of a Listeria monocytogenes outbreak in Switzerland through routine interviewing of patients and whole-genome sequencing.

AIMS OF THE STUDY: Listeriosis is a notifiable disease in Switzerland. In summer 2022, the Swiss Federal Office of Public Health noticed an increase in reports of listeriosis cases, indicating a possible ongoing outbreak. Here we present the approaches applied for rapidly confirming the outbreak, detecting the underlying source of infection and the measures put in place to eliminate it and contain the outbreak. METHODS: For close surveillance and early detection of outbreak situations with their possible sources, listeriosis patients in Switzerland are systematically interviewed about risk behaviours and foods consumed prior to the infection. Listeria monocytogenes isolates derived from patients in medical laboratories are sent to the National Reference Laboratory for Enteropathogenic Bacteria and Listeria, where they routinely undergo whole-genome sequencing. Interview and whole-genome sequencing data are continuously linked for comparison and analysis. RESULTS: In summer 2022, 20 patient-derived L. monocytogenes serotype 4b sequence type 388 strains were found to belong to an outbreak cluster (≤10 different alleles between neighbouring isolates) based on core genome multilocus sequence typing analysis. Geographically, 18 of 20 outbreak cases occurred in northeastern Switzerland. The median age of patients was 77.4 years (range: 58.1-89.7), with both sexes equally affected. Rolling analysis of the interview data revealed smoked trout from a local producer as a suspected infection source, triggering an on-site investigation of the production facility and sampling of the suspected products by the responsible cantonal food inspection team on 15 July 2022. Seven of ten samples tested positive for L. monocytogenes and the respective cantonal authority ordered a ban on production and distribution as well as a product recall. The Federal Food Safety and Veterinary Office released a nationwide public alert covering the smoked fish products concerned. Whole-genome sequencing analysis confirmed the interrelatedness of the L. monocytogenes smoked trout product isolates and the patient-derived isolates. Following the ban on production and distribution and the product recall, reporting of new outbreak-related cases rapidly dropped to zero. CONCLUSIONS: This listeriosis outbreak could be contained within a relatively short time thanks to identification of the source of contamination through the established combined approach of timely interviewing of every listeriosis patient or a representative and continuous molecular analysis of the patient- and food-derived L. monocytogenes isolates. These findings highlight the effectiveness of this well-established, joint approach involving the federal and cantonal authorities and the research institutions mandated to contain listeriosis outbreaks in Switzerland.

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.

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.

High clonal diversity of Staphylococcus aureus isolates from children's playgrounds in Hungary.

Staphylococcus aureus is one of the most important human pathogenic bacteria and environmental surfaces play an important role in the spread of the bacterium. Presence of S. aureus on children's playgrounds and on toys was described in international studies, however, little is known about the prevalence and characteristics of S. aureus at playgrounds in Europe. In this study, 355 samples were collected from playgrounds from 16 cities in Hungary. Antibiotic susceptibility of the isolates was tested for nine antibiotics. Presence of virulence factors was detected by PCR. Clonal diversity of the isolates was tested by PFGE and MLST. The overall prevalence of S. aureus was 2.81% (10/355) and no MRSA isolates were found. Presence of spa (10), fnbA (10), fnbB (5), icaA (8), cna (7), sea (2), hla (10), hlb (2) and hlg (6) virulence genes were detected. The isolates had diverse PFGE pulsotypes. With MLST, we have detected isolates belonging to ST8 (CC8), ST22 (CC22), ST944 and ST182 (CC182), ST398 (CC398), ST6609 (CC45), ST3029 and ST2816. We have identified a new sequence type, ST6609 of CC45. S. aureus isolates are present on Hungarian playgrounds, especially on plastic surfaces. The isolates were clonally diverse and showed resistance to commonly used antibiotics. These data reinforce the importance of the outdoor environment in the spread for S. aureus in the community.

Posibilities for use of whole genome sequencing (WGS) for the analysis of Streptococcus pneumoniae isolates.

Streptococcus pneumoniae (pneumococcus) is a Gram-positive coccus causing both non-invasive and invasive infectious diseases. Pneumococcal diseases are vaccine preventable. Invasive pneumococcal diseases (IPD) meeting the international case definition are reported nationally and internationally and are subject to surveillance programmes in many countries, including the Czech Republic. An important part of IPD surveillance is the monitoring of causative serotypes and their frequency over time and in relation to ongoing vaccination programmes. In the world and in the Czech Republic, whole genome sequencing (WGS) is increasingly used for pneumococci, which allows for serotyping from sequencing data, precise analysis of their genetic relationships, and the study of genes present in their genome. Whole-genome sequencing enables the generation of reliable and internationally comparable data that can be easily shared. Sequencing data are analysed using bioinformatics tools that require knowledge in the field of natural sciences with an emphasis on genetics and expertise in bioinformatics. This publication presents some options for pneumococcal analysis, i.e., serotyping, multilocus sequence typing (MLST), ribosomal MLST (rMLST), core genome MLST (cgMLST), whole genome MLST (wgMLST), single nucleotide polymorphism (SNP) analysis, assignment to Global Pneumococcal Sequence Cluster (GPSC), and identification of virulence genes and antibiotic resistance genes. The WGS strategies and applications for Europe and WGS implementation in practice are presented. WGS analysis of pneumococci allows for improved IPD surveillance, thanks to molecular serotyping, more detailed typing, generation of internationally comparable data, and improved evaluation of the effectiveness of vaccination programmes.

Whole-genome analysis of Escherichia coli isolated from wild Amur tiger (Panthera tigris altaica) and North China leopard (Panthera pardus japonensis).

Background: Escherichia coli is an important intestinal flora, of which pathogenic E. coli is capable of causing many enteric and extra-intestinal diseases. Antibiotics are essential for the treatment of bacterial infections caused by pathogenic E. coli; however, with the widespread use of antibiotics, drug resistance in E. coli has become particularly serious, posing a global threat to human, animal, and environmental health. While the drug resistance and pathogenicity of E. coli carried by tigers and leopards in captivity have been studied intensively in recent years, there is an extreme lack of information on E. coli in these top predators in the wild environment. Methods: Whole genome sequencing data of 32 E. coli strains collected from the feces of wild Amur tiger (Panthera tigris altaica, n = 24) and North China leopard (Panthera pardus japonensis, n = 8) were analyzed in this article. The multi-locus sequence types, serotypes, virulence and resistance genotypes, plasmid replicon types, and core genomic SNPs phylogeny of these isolates were studied. Additionally, antimicrobial susceptibility testing (AST) was performed on these E. coli isolates. Results: Among the E. coli isolates studied, 18 different sequence types were identified, with ST939 (21.9%), ST10 (15.6%), and ST3246 (9.4%) being the most prevalent. A total of 111 virulence genes were detected, averaging about 54 virulence genes per sample. They contribute to invasion, adherence, immune evasion, efflux pump, toxin, motility, stress adaption, and other virulence-related functions of E. coli. Sixty-eight AMR genes and point mutations were identified. Among the detected resistance genes, those belonging to the efflux pump family were the most abundant. Thirty-two E. coli isolates showed the highest rate of resistance to tetracycline (14/32; 43.8%), followed by imipenem (4/32; 12.5%), ciprofloxacin (3/32; 9.4%), doxycycline (2/32; 6.3%), and norfloxacin (1/32; 3.1%). Conclusions: Our results suggest that E. coli isolates carried by wild Amur tigers and North China leopards have potential pathogenicity and drug resistance.

Characterization of MLST-99 Salmonella Typhimurium and the monophasic variant I:4,[5],12:i:- isolated from Canadian Atlantic coast shellfish.

Salmonella enterica subsp. enterica Typhimurium and its monophasic variant I 1;4,[5],12:i:- (MVST) are responsible for thousands of reported cases of salmonellosis each year in Canada, and countries worldwide. We investigated S. Typhimurium and MVST isolates recovered from raw shellfish harvested in Atlantic Canada by the Canadian Food Inspection Agency (CFIA) over the past decade, to assess the potential impact of these isolates on human illness and to explore possible routes of shellfish contamination. Whole-genome sequence analysis was performed on 210 isolates of S. Typhimurium and MVST recovered from various food sources, including shellfish. The objective was to identify genetic markers linked to ST-99, a sequence type specifically associated with shellfish, which could explain their high prevalence in shellfish. We also investigated the genetic similarity amongst CFIA ST-99 isolates recovered in different years and geographical locations. Finally, the study aimed to enhance the molecular serotyping of ST-99 isolates, as they are serologically classified as MVST but are frequently misidentified as S. Typhimurium through sequence analysis. To ensure recovery of ST-99 from shellfish was not due to favourable growth kinetics, we measured the growth rates of these isolates relative to other Salmonella and determined that ST-99 did not have a faster growth rate and/or shorter lag phase than other Salmonella evaluated. The CFIA ST-99 isolates from shellfish were highly clonal, with up to 81 high-quality single nucleotide variants amongst isolates. ST-99 isolates both within the CFIA collection and those isolated globally carried numerous unique deletions, insertions and mutations in genes, including some considered important for virulence, such as gene deletions in the type VI secretion system. Interestingly, several of these genetic characteristics appear to be unique to North America. Most notably was a large genomic region showing a high prevalence in genomes from Canadian isolates compared to those from the USA. Although the functions of the majority of the proteins encoded within this region remain unknown, the genes umuC and umuD, known to be protective against UV light damage, were present. While this study did not specifically examine the effects of mutations and insertions, results indicate that these isolates may be adapted to survive in specific environments, such as ocean water, where wild birds and/or animals serve as the natural hosts. Our hypothesis is reinforced by a global phylogenetic analysis, which indicates that isolates obtained from North American shellfish and wild birds are infrequently connected to isolates from human sources. These findings suggest a distinct ecological niche for ST-99, potentially indicating their specialization and adaptation to non-human hosts and environments, such as oceanic habitats.

Characterization of linezolid- and methicillin-resistant coagulase-negative staphylococci in a tertiary hospital in China.

BACKGROUND: Recently, linezolid-resistant staphylococci have become an emerging problem worldwide. Understanding the mechanisms of resistance, molecular epidemiology and transmission of linezolid-resistant CoNS in hospitals is very important. METHODS: The antimicrobial susceptibilities of all isolates were determined by the microdilution method. The resistance mechanisms and molecular characteristics of the strains were determined using whole-genome sequencing and PCR. RESULTS: All the strains were resistant to oxacillin and carried the mecA gene; 13 patients (36.1%) had prior linezolid exposure. Most S. epidermidis and S. hominis isolates were ST22 and ST1, respectively. MLST typing and evolutionary analysis indicated most linezolid-resistant CoNS strains were genetically related. In this study, we revealed that distinct CoNS strains have different mechanisms of linezolid resistance. Among ST22-type S. epidermidis, acquisition of the T2504A and C2534T mutations in the V domain of the 23 S rRNA gene, as well as mutations in the ribosomal proteins L3 (L101V, G152D, and D159Y) and L4 (N158S), were linked to the development of linezolid resistance. In S. cohnii isolates, cfr, S158Y and D159Y mutations in the ribosomal protein L3 were detected. Additionally, emergence of the G2576T mutation and the cfr gene were major causes of linezolid resistance in S. hominis isolates. The cfr gene, G2576T and C2104T mutations, M156T change in L3 protein, and I188S change in L4 protein were found in S. capitis isolates. CONCLUSION: The emergence of linezolid-resistant CoNS in the environment is concerning because it involves clonal dissemination and frequently coexists with various drug resistance mechanisms.

Phenotypic and genotypic analysis of antimicrobial resistance and population structure of gastroenteritis-related Aeromonas isolates.

BACKGROUND: The population structure and the correlation between antimicrobial resistance (AMR) phenotypes and genotypes in Aeromonas species isolated from patients with gastroenteritis are not well understood. The aims of the study were to: (1) investigate the antimicrobial susceptibility profiles of Aeromonas species isolated from patients with gastroenteritis; (2) explore the relationship between AMR genes and resistance phenotypes; and (3) describe the population structure of these isolates and provide evidence of transmission events among them. METHODS: This microbiological survey was performed at the Microbiology Laboratory of the Emek Medical Center in Afula, Israel. Cultivation of Aeromonas was attempted from stool samples that tested positive by PCR. Antimicrobial susceptibility testing (AST) was performed using the Sensititre GN3F microdilution panel. Whole genome sequencing (WGS) was done using the Illumina NextSeq500/550 system. Phylogenetic studies involved multi-locus sequence typing (MLST) and core genome (cg) MLST. Resistance mechanisms were identified using the Comprehensive Antibiotic Resistance Database and compared with the AST results. RESULTS: The study included 67 patient-unique isolates. The species that were identified included A. caviae (n = 58), A. dhakensis (n = 3), A. media (n = 2), A. veronii (n = 2) and A. hydrophila (n = 2). Isolates were almost uniformly susceptible to amikacin, gentamicin, aztreonam, cefepime, ceftazidime, ciprofloxacin and meropenem. All isolates with the exception of 1-2 isolates were resistant to ampicillin, cefazolin and ampicillin-sulbactam which was compatible with the presence of the blaOXA genes. Variable resistance rates were observed to cefuroxime, cefoxitin, ceftriaxone, piperacillin-tazobactam that were not correlated with the presence of other ß-lactamase genes. Resistance to tetracycline and trimethoprim-sulfamethoxazole correlated with the presence of tetA and sul1, respectively. The population structure of A. caviae was highly diverse with the minority of the isolates (16/57) clustering into six defined sequence types. A cgMLST-based distance of four genes was found in one pair of isolates, suggesting common source transmission. CONCLUSIONS: A. caviae is the dominant species related to gastroenteritis and is characterized by a diverse population structure, with almost no evidence for common-source transmission. Resistance rates to most antimicrobial agents were low and partially matched with the presence of resistance genes.

Comparative genomics reveals high genetic similarity among strains of Salmonella enterica serovar Infantis isolated from multiple sources in Brazil.

Background: Salmonella enterica serovar Infantis (Salmonella Infantis) is a zoonotic, ubiquitous and foodborne pathogen of worldwide distribution. Despite Brazil's relevance as a major meat exporter, few studies were conducted to characterize strains of this serovar by genomic analyses in this country. Therefore, this study aimed to assess the diversity of 80 Salmonella Infantis strains isolated from veterinary, food and human sources in Brazil between 2013 and 2018 by comparative genomic analyses. Additional genomes of non-Brazilian countries (n = 18) were included for comparison purposes in some analyses. Methods: Analyses of whole-genome multi-locus sequence typing (wgMLST), using PGAdb-builder, and of fragmented genomes, using Gegenees, were conducted to compare the 80 Brazilian strains to the 18 non-Brazilian genomes. Pangenome analyses and calculations were performed for all Salmonella Infantis genomes analyzed. The presence of prophages was determined using PHASTER for the 80 Brazilian strains. The genome plasticity using BLAST Ring Image Generator (BRIG) and gene synteny using Mauve were evaluated for 20 selected Salmonella Infantis genomes from Brazil and ten from non-Brazilian countries. Unique orthologous protein clusters were searched in ten selected Salmonella Infantis genomes from Brazil and ten from non-Brazilian countries. Results: wgMLST and Gegenees showed a high genomic similarity among some Brazilian Salmonella Infantis genomes, and also the correlation of some clusters with non-Brazilian genomes. Gegenees also showed an overall similarity >91% among all Salmonella Infantis genomes. Pangenome calculations revealed an open pangenome for all Salmonella Infantis subsets analyzed and a high gene content in the core genomes. Fifteen types of prophages were detected among 97.5% of the Brazilian strains. BRIG and Mauve demonstrated a high structural similarity among the Brazilian and non-Brazilian isolates. Unique orthologous protein clusters related to biological processes, molecular functions, and cellular components were detected among Brazilian and non-Brazilian genomes. Conclusion: The results presented using different genomic approaches emphasized the significant genomic similarity among Brazilian Salmonella Infantis genomes analyzed, suggesting wide distribution of closely related genotypes among diverse sources in Brazil. The data generated contributed to novel information regarding the genomic diversity of Brazilian and non-Brazilian Salmonella Infantis in comparison. The different genetically related subtypes of Salmonella Infantis from Brazil can either occur exclusively within the country, or also in other countries, suggesting that some exportation of the Brazilian genotypes may have already occurred.

FosA8-producing E. coli ST131: clinical cases in Italy, February 2023.

Fosfomycin-resistant FosA8-producing Enterobacterales are uncommon strains with extremely low incidence in Europe, based on only three reports in the literature. We detected FosA8-producing Escherichia coli ST131 in clinical isolates from two patients admitted in February 2023 to a rehabilitation unit in Italy. The occurrence of rare fosA-like genes in the high-risk clone ST131 is of clinical relevance. The dissemination of FosA-producing E. coli, although still at low levels, should be continuously monitored.

Multi-locus sequence analysis of Anaplasma bovis in goats and ticks from Thailand, with the initial identification of an uncultured Anaplasma species closely related to Anaplasma phagocytophilum-like 1.

Ticks and tick-borne pathogens (TTBP) pose a serious threat to animal and human health globally. Anaplasma bovis, an obligatory intracellular bacterium, is one of the more recent species of the Family Anaplasmaceae to be formally described. Owing to its diminutive size, microscopic detection presents a formidable challenge, leading to it being overlooked in laboratory settings lacking advanced equipment or resources, as observed in various regions, including Thailand. This study aimed to undertake a genetic analysis of A. bovis and determine its prevalence in goats and ticks utilizing three genetic markers (16S rRNA, gltA, groEL). A total of 601 goat blood and 118 tick samples were collected from 12 sampling sites throughout Thailand. Two tick species, Haemaphysalis bispinosa (n = 109), and Rhipicephalus microplus (n = 9) were identified. The results herein showed that 13.8 % (83/601) of goats at several farms and 5 % (1/20) of ticks were infected with A. bovis. Among infected ticks, A. bovis and an uncultured Anaplasma sp. which are closely related to A. phagocytophilum-like 1, were detected in each of H. bispinosa ticks. The remaining R. microplus ticks tested positive for the Anaplasma genus. A nucleotide sequence type network showed that A. bovis originated from Nan and Narathiwat were positioned within the same cluster and closely related to China isolates. This observation suggests the potential dispersal of A. bovis over considerable distances, likely facilitated by activities such as live animal trade or the transportation of infected ticks via migratory birds. The authors believe that the findings from this study will provide valuable information about TTBP in animals.

Temporal variations in the serogroup distribution of invasive meningococcal disease in Quebec, Canada, due to emerging unique clade of serogroup Y strain belonging to the Sequence Type-23 clonal complex.

OBJECTIVE: To identify recent trends in invasive meningococcal diseases (IMD) in Quebec, Canada, with a focus on MenY cases and MenY strains. METHODS: IMD cases and MenY strains from January 1, 2015 to August 11, 2023 were analyzed for clonal analysis and prediction of susceptibility to MenB vaccines. MenY strains of ST-23 CC from Quebec were analyzed with global MenY strains by core-genomic multi-locus sequence typing (cg-MLST). RESULTS: Since 2015 the serogroup distribution of IMD in Quebec has shifted from predominantly MenB to mainly MenY, with most (80.9 %) of the latter belonging to ST-23 CC. The median age of MenY cases due to ST-23 CC were statistically younger than MenY cases due to non-ST-23 CC. MenY of ST-23 CC showed genetic diversity and the major genetic cluster were similar to the Swedish Y1 strain. The increase in invasive MenY disease in Quebec was due to a sub-clade of Lineage 23.1 which caused an elevated proportion of severe disease in young adults. CONCLUSION: The increase in invasive MenY disease in Quebec, Canada was driven by the expansion of a sub-clade of Lineage 23.1 in young adults. Currently available quadrivalent A,C,W,Y-conjugate meningococcal vaccines were predicted to provide protection against these strains.

Chromosomal integration and plasmid fusion occurring in ST20 carbapenem-resistant Klebsiella pneumoniae isolates coharboring blaNDM-1 and blaIMP-4 induce resistance transmission and fitness variation.

To investigate the epidemiology of ST20 carbapenem-resistant Klebsiella pneumoniae (CRKP) in China, and further explore the genomic characteristics of blaIMP-4 and blaNDM-1 coharboring isolates and plasmid contributions to resistance and fitness. Seven ST20 CRKP isolates were collected nationwide, and antimicrobial susceptibility testing was performed. Antimicrobial resistance genes, virulence genes, and plasmid replicons were identified via whole-genome sequencing, and clonality assessed via core-genome multilocus sequence typing. Furthermore, we found four dual-metallo-ß-lactamases (MBL)-harbouring isolates, the gene location was detected by Southern blotting, and plasmid location analysis showed that blaIMP-4 was located on a separate plasmid, a self-conjugative fusion plasmid, or the bacterial chromosome. These isolates were subjected to long-read sequencing, the presence of blaIMP-4 in different locations was identified by genomic comparison, and transposon units were detected via inverse PCR. We subsequently found that blaIMP-4 on the fusion plasmid and bacterial chromosome was formed via intact plasmid recombination by the IS26 and ltrA, respectively, and the circular transposon unit was related to cointegration, however, blaIMP-4 in different locations did not affect the gene stability. The blaNDM-1-harbouring plasmid contributed to the increased resistance to ß-lactams and shortened survival lag time which was revealed in plasmid cured isolates. In summary, the K. pneumoniae ST20 clone is a high-risk resistant clone. With the use of ceftazidime/avibactam, MBL-positive isolates, especially dual-MBL-harbouring isolates, should be given additional attention.

Microbiome and mitogenomics of the chigger mite Pentidionis agamae: potential role as an Orientia vector and associations with divergent clades of Wolbachia and Borrelia.

BACKGROUND: Trombiculid mites are globally distributed, highly diverse arachnids that largely lack molecular resources such as whole mitogenomes for the elucidation of taxonomic relationships. Trombiculid larvae (chiggers) parasitise vertebrates and can transmit bacteria (Orientia spp.) responsible for scrub typhus, a zoonotic febrile illness. Orientia tsutsugamushi causes most cases of scrub typhus and is endemic to the Asia-Pacific Region, where it is transmitted by Leptotrombidium spp. chiggers. However, in Dubai, Candidatus Orientia chuto was isolated from a case of scrub typhus and is also known to circulate among rodents in Saudi Arabia and Kenya, although its vectors remain poorly defined. In addition to Orientia, chiggers are often infected with other potential pathogens or arthropod-specific endosymbionts, but their significance for trombiculid biology and public health is unclear. RESULTS: Ten chigger species were collected from rodents in southwestern Saudi Arabia. Chiggers were pooled according to species and screened for Orientia DNA by PCR. Two species (Microtrombicula muhaylensis and Pentidionis agamae) produced positive results for the htrA gene, although Ca. Orientia chuto DNA was confirmed by Sanger sequencing only in P. agamae. Metagenomic sequencing of three pools of P. agamae provided evidence for two other bacterial associates: a spirochaete and a Wolbachia symbiont. Phylogenetic analysis of 16S rRNA and multi-locus sequence typing genes placed the spirochaete in a clade of micromammal-associated Borrelia spp. that are widely-distributed globally with no known vector. For the Wolbachia symbiont, a genome assembly was obtained that allowed phylogenetic localisation in a novel, divergent clade. Cytochrome c oxidase I (COI) barcodes for Saudi Arabian chiggers enabled comparisons with global chigger diversity, revealing several cases of discordance with classical taxonomy. Complete mitogenome assemblies were obtained for the three P. agamae pools and almost 50 SNPs were identified, despite a common geographic origin. CONCLUSIONS: P. agamae was identified as a potential vector of Ca. Orientia chuto on the Arabian Peninsula. The detection of an unusual Borrelia sp. and a divergent Wolbachia symbiont in P. agamae indicated links with chigger microbiomes in other parts of the world, while COI barcoding and mitogenomic analyses greatly extended our understanding of inter- and intraspecific relationships in trombiculid mites.

Emergence of eravacycline heteroresistance in carbapenem-resistant Acinetobacter baumannii isolates in China.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is resistant to almost all antibiotics. Eravacycline, a newer treatment option, has the potential to treat CRAB infections, however, the mechanism by which CRAB isolates develop resistance to eravacycline has yet to be clarified. This study sought to investigate the features and mechanisms of eravacycline heteroresistance among CRAB clinical isolates. A total of 287 isolates were collected in China from 2020 to 2022. The minimum inhibitory concentration (MIC) of eravacycline and other clinically available agents against A. baumannii were determined using broth microdilution. The frequency of eravacycline heteroresistance was determined by population analysis profiling (PAP). Mutations and expression levels of resistance genes in heteroresistant isolates were determined by polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR), respectively. Antisense RNA silencing was used to validate the function of eravacycline heteroresistant candidate genes. Twenty-five eravacycline heteroresistant isolates (17.36%) were detected among 144 CRAB isolates with eravacycline MIC values ≤4 mg/L while no eravacycline heteroresistant strains were detected in carbapenem-susceptible A. baumannii (CSAB) isolates. All eravacycline heteroresistant strains contained OXA-23 carbapenemase and the predominant multilocus sequence typing (MLST) was ST208 (72%). Cross-resistance was observed between eravacycline, tigecycline, and levofloxacin in the resistant subpopulations. The addition of efflux pump inhibitors significantly reduced the eravacycline MIC in resistant subpopulations and weakened the formation of eravacycline heteroresistance in CRAB isolates. The expression levels of adeABC and adeRS were significantly higher in resistant subpopulations than in eravacycline heteroresistant parental strains (P < 0.05). An ISAba1 insertion in the adeS gene was identified in 40% (10/25) of the resistant subpopulations. Decreasing the expression of adeABC or adeRS by antisense RNA silencing significantly inhibited eravacycline heteroresistance. In conclusion, this study identified the emergence of eravacycline heteroresistance in CRAB isolates in China, which is associated with high expression of AdeABC and AdeRS.

Analysis of virulence profiles in clinical isolates of Klebsiella pneumoniae from renal abscesses: clinical significance of hypervirulent isolates.

Introduction: Klebsiella pneumoniae can cause a wide range of infections. Hypervirulent K. pneumoniae (hvKp), particularly associated with the K1 and K2 capsular types, is an increasingly significant microorganism with the potential to cause invasive infections, including renal abscesses. Despite the rising prevalence of hvKp infections, information on renal abscesses caused by K. pneumoniae is limited, and the clinical significance of hvKp associated with specific virulence genes remains elusive. Methods: This study performed at a 1200-bed tertiary hospital sought to identify the clinical and microbiological characteristics of renal abscesses caused by K. pneumoniae, focusing on various virulence genes, including capsular serotypes and multilocus sequence typing (MLST). Results: Over an 8-year period, 64 patients with suspected renal abscesses were reviewed. Ten patients diagnosed with K. pneumoniae-related renal abscesses were ultimately enrolled in the study. Among the isolates from the 10 patients, capsular serotype K2 was predominant (40.0%), followed by K1 (30.0%). The most common sequence type by MLST was 23 (40.0%). In particular, six patients (60.0%) harbored specific genes indicative of hvKp: iucA, peg-344, rmpA, and rmpA2. Conclusions: Our findings highlight the importance of hvKp as a pathogen in renal abscesses. Although the nature of hvKp is relatively unknown, it is widely recognized as a highly virulent pathogen that can infect relatively healthy individuals of various ages and simultaneously cause infections at multiple anatomical sites. Therefore, when treating patients with K. pneumoniae-related renal abscesses, caution is necessary when considering the characteristics of hvKp, such as potential bacteremia, multi-organ abscess formation, and metastatic spread.

Genetic characterization of the Entamoeba moshkovskii population based on different potential genetic markers.

Entamoeba moshkovskii, according to recent studies, appears to exert a more significant impact on diarrhoeal infections than previously believed. The efficient identification and genetic characterization of E. moshkovskii isolates from endemic areas worldwide are crucial for understanding the impact of parasite genomes on amoebic infections. In this study, we employed a multilocus sequence typing system to characterize E. moshkovskii isolates, with the aim of assessing the role of genetic variation in the pathogenic potential of E. moshkovskii. We incorporated 3 potential genetic markers: KERP1, a protein rich in lysine and glutamic acid; amoebapore C (apc) and chitinase. Sequencing was attempted for all target loci in 68 positive E. moshkovskii samples, and successfully sequenced a total of 33 samples for all 3 loci. The analysis revealed 17 distinct genotypes, labelled M1­M17, across the tested samples when combining all loci. Notably, genotype M1 demonstrated a statistically significant association with diarrhoeal incidence within E. moshkovskii infection (P = 0.0394). This suggests that M1 may represent a pathogenic strain with the highest potential for causing diarrhoeal symptoms. Additionally, we have identified a few single-nucleotide polymorphisms in the studied loci that can be utilized as genetic markers for recognizing the most potentially pathogenic E. moshkovskii isolates. In our genetic diversity study, the apc locus demonstrated the highest Hd value and π value, indicating its pivotal role in reflecting the evolutionary history and adaptation of the E. moshkovskii population. Furthermore, analyses of linkage disequilibrium and recombination within the E. moshkovskii population suggested that the apc locus could play a crucial role in determining the virulence of E. moshkovskii.

[Comparison of Three Different Methods in Investigating the Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates]. / Karbapeneme Dirençli Acinetobacter baumannii Klinik Izolatlarinin Moleküler Epidemiyoloji Açisindan Arastirilmasinda Üç Farkli Yöntemin Karsilastirilmasi.

The aim of the study was to evaluate the relationship between carbapenem-resistant Acinetobacter baumannii isolates carrying oxacillinase-type carbapenemase genes with "international high-risk clones" (IC I, II, and III) by different molecular epidemiological methods and to statistically compare the concordance and discrimination power of the methods. Carbapenem-resistant and moderately susceptible A.baumannii isolates from non-repeating blood cultures of 72 patients were included in the study. The presence of "blaOXA-23 , blaOXA-24 , blaOXA-51 ve blaOXA-58 " genes within OXA-type carbapenemases was detected by polymerase chain reaction (PCR) method and confirmed by DNA sequence analysis. Pulsed f ield gel electrophoresis (PFGE), multilocus sequence typing (MLST) and matrix-assisted laser desorption/ ionization time- of-flight mass spectrometry (MALDI-TOF MS) analyses were performed to evaluate the clonal relations of IC I, II and III clones together with clinical isolates. In the statistical comparison of the methods, discrimination power was evaluated by Simpson index of diversity (SID) and concordance by "Wallace coefficient". All of the isolates were found to carry blaOXA-23 and blaOXA-51 genes. As a result of the bioinformatic analysis of the four isolates selected for sequence analysis; blaOXA-23 and blaOXA-51 genes were detected in the selected isolates, and the analysis of two isolates carrying blaOXA-51 gene showed 99% similarity with blaOXA-92 gene. The isolates were clustered into five pulsotypes (A, B, C, D and E) according to ≥ 85% similarity coefficient by PFGE. The isolates and RUH 875, RUH 134, LUH 5875 strains belonging to high-risk clones ICI, ICII and ICIII, respectively, were divided into five main groups [A (n= 58), B (n= 8), C (n= 4), D (n= 4) and E (n= 1)] and 10 subgroups (A1, A2, A4, A5, A6, A9, B1, B4, C3, D1) by PFGE. IC clone III (E1) and seven strains showed singleton PFGE profiles (A3, A7, A8, B2, B3, C1, C2). ICII was found in A5 subtype, ICI in C1 subtype and ICIII in E1 subtype. By PFGE subtype groups, 18 pulsotypes were determined and ST1, ST2, ST81, ST157 and ST604 sequence types were found in 20 isolates randomly selected from pulsotypes according to MLST Pasteur scheme (cpn60, fusA, gltA, pyrG, recA, rplB, rpoB). Principal component analysis (PCA) of the spectra of 72 A. baumannii isolates and ICI, ICII and ICIII clones was performed by MALDI-TOF MS. In PCA analysis, the cluster distance level was defined as 1.5 and the isolates were divided into three clusters. IC clone I, II and III together with 70 clinical isolates were grouped in one cluster, while two clinical isolates (AB083 and AB0115) formed singleton clusters. There was no significant agreement between MALDI-TOF MS; MLST and PFGE data according to Wallace coefficient. It was found that PFGE method gave significant results in terms of discrimination power with SID coefficient, MALDI-TOF MS PCA analysis had the lowest discrimination power value, and the Wallace coefficient result of PFGE and MLST was concordant. In conclusion, MALDI-TOF MS may not function as a gold standard method like PFGE and MLST for epidemiological analysis in A.baumannii species and the epidemiological typing protocols used for MALDI-TOF MS need to be improved and developed.

[Etiological characterization of invasive non-typhoid Salmonella strains in Guangdong Province from 2018 to 2022].

Objective: To understand the serotype distribution, drug resistance and molecular characterization of invasive non-typhoid Salmonella (iNTS) in Guangdong Province from 2018 to 2022 and provide scientific evidence for the prevention and treatment of blood flow infection caused by Salmonella. Methods: Serological identification, antimicrobial susceptibility testing, multilocus sequence typing (MLST), and whole genome sequencing were performed on Salmonella isolated from blood and stool samples in Guangdong from 2018 to 2022. Simultaneously, annotated the sequencing results for drug resistance genes and virulence factors by a microbial gene annotation system. Results: The 136 iNTS strains were divided into 25 serotypes, and Salmonella enteritidis accounted for 38.24% (52/136). The OR of other iNTS serotypes were calculated with Salmonella typhimurium as the control. The OR values of Oreninburg, Rysson, and Pomona serotypes were the highest, which were 423.50, 352.92, and 211.75, respectively. The drug resistance rate of iNTS was 0.74%-66.91%, which was lower than that of non-iNTS (3.90%-77.21%). The main iNTS of drug resistance were ampicillin and tetracycline, with resistance rates of 66.91% (91/136) and 50.00% (68/136), respectively, while the resistance rates to ciprofloxacin (5.88%,8/136), ceftazidime (5.88%,8/136), gentamicin (5.13%,7/136) and cefoxitin (0.74%, 1/136) were relatively low. iNTS carried a variety of drug-resistance genes and virulence factors, but no standard virulence factor distribution has been found. MLST cluster analysis showed that iNTS was divided into 26 sequence types, and ST11 accounted for 38.24% (52/136). Conclusions: The iNTS strains in Guangdong were dominated by Salmonella enteritidis, of which three serotypes, Oreninburg, Rison, and Pomona, may be associated with a higher risk of invasive infection during 2018 to 2022. iNTS was sensitive to clinical first-line therapeutic drugs (cephalosporins and fluoroquinolones), with highly diverse sequences and clear phylogenetic branches. ST11 was the local dominant clone group.