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Purpose: In the Zhejiang region, research on Helicobacter pylori is lacking. The purpose of this study was to assess the extent of antibiotic resistance in H. pylori in this region, explore alternative methods for predicting the resistance patterns of H. pylori, and investigate the colonization of native gastric mucosa by other clades of H. pylori in the structure population of this bacterium. Methods: Strains were cultured under microaerobic conditions, and antimicrobial susceptibility testing (AST) was performed via agar dilution. Whole-genome sequencing (WGS) was performed via next-generation sequencing (NGS) technology. Epidemiological data including data from this study and reported articles from Zhejiang, China, were included. Further analyses based on AST, WGS, and epidemiological date include virulence genes, antibiotic resistance-related mutations, and phylogenetic trees based on 7 housekeeping genes and core-genome single nucleotide polymorphisms (SNPs). Results: The bacterial isolates in this study presented higher antibiotic resistance rates than previously reported, especially against levofloxacin and clarithromycin. The point mutation A2147G in 23 S rRNA is specific to clarithromycin resistance. Mutations at position/s 87 and/or 91 of the gyrA gene amino acid sequence are highly consistent with levofloxacin resistance highly. The point mutations C1707T in 23 S rRNA and E463K in the gyrB gene have not been previously documented in China. All the bacterial isolates belong to Asian branches in the structure population. The resistance rate to clarithromycin of isolates from hosts born after January 1, 1977 is statistically higher than that of hosts born before 1977. Conclusion: Eradication therapy based on AST results is urgently needed in Zhejiang. The point mutation A2147G in 23 S rRNA and point mutations in the gyrA gene at amino acid/s 87 and/or 91 are sufficient for predicting resistance to clarithromycin and levofloxacin, respectively. The isolate with the mutation E463K in the gyrB gene represents a significant contribution to the field. Mutations in 23 S rRNA may offer valuable insights into the dynamics of H. pylori transmission among hosts. [ABSTRACT FROM AUTHOR]

Background: Characteristics of non-clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) especially from fishery environment are poorly understood. This research, in addition to comprehensive characterisation, sought to delineate the genetic relatedness between the MRSA strains originating from clinical as well as non-clinical settings. Out of 39 methicillin-resistant staphylococcal isolates from 197 fish samples, 6 (Three each of methicillin-resistant S. haemolyticus (MRSH) and MRSA) with distinct resistance profiles were selected for whole-genome sequencing. Using respective bioinformatics tools, MRSA genomes were comprehensively characterized for resistome, virulomes, molecular epidemiology and phylogenetic analysis. Simultaneously, MRSH genomes were specifically examined to characterize antimicrobial resistance genes (ARGs), owing to the fact that MRSH is often recognized as a reservoir for resistance determinants. Results: Three MRSA clones identified in this study include ST672-IVd/t13599 (sequence type-SCCmec type/spa type), ST88-V/t2526, and ST672-IVa/t1309. Though, the isolates were phenotypically vancomycin-sensitive, five of the six genomes carried vancomycin resistance genes including the VanT (VanG cluster) or VanY (VanM cluster). Among the three MRSA, only one harbored the gene encoding Panton-Valentine Leukocidin (PVL) toxin, while staphylococcal enterotoxin (SEs) genes such as sea and seb, associated with staphylococcal food poisoning were identified in two other MRSA. Genomes of MRSH carried a composite of type V staphylococcal cassette chromosome mec (SCCmec) elements (5C2 & 5). This finding may be explained by the inversion and recombination events that may facilitate the integration of type V elements to the SCC elements of S. aureus with a methicillin-susceptible phenotype. Phylogenetically, MRSA from a non-clinical setting displayed a considerable relatedness to that from clinical settings. Conclusion: This study highlights the genetic diversity and resistance profiles of MRSA and MRSH, with non-clinical MRSA showing notable relatedness to clinical strains. Future research should explore resistance gene transfer mechanisms and environmental reservoirs to better manage MRSA spread. [ABSTRACT FROM AUTHOR]

Uropathogenic Escherichia coli (UPEC) is a typical cystitis-causing organism that can migrate from the vagina to the bladder and cause recurrent cystitis (RC). Few reports have compared the characteristics of urinary and vaginal UPEC in patients with RC. We carried out molecular biological analyses of Escherichia coli (E. coli) strains and their antimicrobial susceptibility to assess the association between urinary and vaginally UPEC. We included E. coli isolated from urinary and vaginal samples at the onset of cystitis in postmenopausal women with RC between 2014 and 2019 in our hospital. Pulsed-field gel electrophoresis (PFGE) was performed using a restriction enzyme (Xba I). These sequences were compared with 17 antimicrobial susceptibilities determined by a micro-liquid dilution method. Multilocus sequence typing (MLST) and classification of extended-spectrum β-lactamase (ESBL) genotypes by multiplex polymerase chain reaction (PCR) were performed on ESBL-producing E. coli. We analyzed 14 specimens (each seven urine and vaginal) from seven patients in total. On PFGE, the similarity of urinary and vaginal E. coli per patient ranged from 89.5 to 100 %, including four patients with 100 % matches. MLST demonstrated that 29 % (4/14 specimens) were strain sequence type 131. Two specimens contained ESBL-producing strains and identified the CTX-M-27 genotype for each specimen. For each patient, antimicrobial susceptibilities between urinary and vaginal E. coli were mostly identical. Thus, urinary- and vaginally-derived E. coli were identical in postmenopausal women with RC. Management targeting both urinary and vaginal UPEC is essential for RC, indicating the importance of a vagina-targeted approach. [ABSTRACT FROM AUTHOR]

Background: Acinetobacter baumannii (A. baumannii) is a life-threatening and challenging pathogen. In addition, it accounts for numerous serious infections, particularly among immunocompromised patients. Resistance to nearly all clinically used antibiotics and their ability to spread this resistance is one of the most important concerns related to this bacterium. Objectives: This study describes different molecular mechanisms of two multidrug-resistant A. baumannii isolates obtained from endotracheal aspirates collected from the neonatal intensive care unit (NICU), Ain Shams University Hospital, Egypt. Methods: Following the identification of two isolates, they were examined for susceptibility to antimicrobial agents. This was followed by multilocus sequence typing as well as whole-genome sequence (WGS). Additionally, a Pathosystems Resources Integration Center (PATRIC) analysis was performed. Results: Two isolates, Ab119 and Ab123, exhibited resistance to all tested antibiotics except for tigecycline and colistin. The WGS analysis of antimicrobial resistance genes (AMR) indicated that both isolates shared beta-lactam, aminoglycoside, macrolides, and sulfonamide resistance genes. Furthermore, each strain revealed different resistance genes such as blaNDM-1, blaNDM-10, OXA-64, aph (3')-VI, Tet-B in Ab119 strain and blaOXA-68, blaPER-1, blaPER-7, Tet-39 in Ab123 strain. Multiple efflux pump genes were detected. Multilocus sequence typing indicated that both isolates belong to the same sequence type (ST931), which belongs to international clone (IC3). Both isolates exhibited the presence of multiple mobile genetic elements (MGEs), but no plasmid was detected in either of them. Conclusions: A low prevalence of the IC3 sequence type was identified among two A. baumannii isolates obtained from the NICU in Egypt, exhibiting a high resistance level. Healthcare workers must have knowledge regarding the prevalence of A. baumannii among different populations in order to administer suitable treatment, improve patient outcomes, and apply effective infection control practices. [ABSTRACT FROM AUTHOR]

The worldwide spread of Pseudomonas aeruginosa, especially carbapenem-resistant P. aeruginosa (CRPA), poses a serious threat to global public health. In this research, we collected and studied the clinical prevalence, molecular epidemiology, and resistance mechanisms of CRPA in Fujian, China. Among 167 non-duplicated P. aeruginosa isolates collected during 2019-2021, strains from respiratory specimens and wound secretions of older males in the intensive care unit dominated. Ninety-eight isolates (58.7%) were resistant to at least one tested antibiotic, among which 70 strains were carbapenem-resistant. Moleclar typing of the CRPA isolates revealed they were highly divergent, belonging to 46 different sequence types. It is noteworthy that two previously reported high risk clones, ST1971 specific to China and the globally prevalent ST357, were found. Several carbapenemresistance-related characteristics were also explored in 70 CRPA isolates. Firstly, carbapenemase was phenotypically positive in 22.9% of CRPA, genetically predominant by metallo-b-lactamase (MBL) and co-carrige of different carbapenemase genes. Then, mutations of the carbapenem-specific porins oprD and opdP were commonly observed, with frequencies of 97.1% and 100.0%, respectively. Furthermore, the biofilm formation and relative transcription levels of 8 multidrug efflux pump genes were also found to be increased in 48.6% and 72.9% of CRPA isolates compared to the reference strain PAO1. These findings will help fill the data gaps in molecular characteristics of CRPA on the southeastern coast of China and emphasize the urgent need for data-based specific stewardship for antipseudomonal practices to prevent the dissemination of CRPA. [ABSTRACT FROM AUTHOR]

Background: Streptococcus mutans is studied for its acidogenic and aciduric characteristics, notably its biofilm formation in the presence of sucrose, toward its role in the caries process. Variations in both genotype and phenotype have been reported among clinical isolates of S. mutans. This study aimed to examine genotypic and phenotypic characteristics of S. mutans obtained from Thai children with varying caries statuses. Methods: We determined the presence of S. mutans and caries status in 395 children aged 3–4 years. From 325 children carrying S. mutans, we selected 90 with different caries statuses—caries-free (CF; n = 30), low severity of caries (LC; n = 30), or high severity of caries (HC; n = 30). Three isolates of S. mutans were taken from each child, thus, a total of 270 isolates were obtained. Multilocus sequence typing (MLST) was used to genotype the isolates and assess their clonal relationships. The properties, including biofilm formation, collagen binding, and acid production and tolerance were also evaluated. Results: Children with carious lesions showed a higher detection rate and number of S. mutans in saliva than those without caries. S. mutans from individuals with HC status showed the lowest biofilm formation ability, while this group had the highest detection rate of collagen-binding isolates. There was no difference in acid production or tolerance by caries status. Genotyping by MLST did not reveal any clone of S. mutans specific to CF status. This result remained even when we included MLST data from the open-access PubMLST database. MLST did identify clones containing only strains from caries-affected hosts, but tests of their phenotypic properties did not reveal any differences between S. mutans from these clones and clones that were from both caries-free and caries-affected children. Conclusions: The clonal relationships of S. mutans indicated by MLST were not associated with the status of dental caries in the host. [ABSTRACT FROM AUTHOR]

Vancomycin variable Enterococcus (VVE) bacteria are phenotypically susceptible to vancomycin, but they harbor the vanA gene. We aimed to ascertain the prevalence of VVE among clinically isolated vancomycin-susceptible Enterococcus faecium (VSE) isolates, as well as elucidate the molecular characteristics of the vanA gene cluster within these isolates. Notably, we investigated the prevalence and structure of the vanA gene cluster of VVE. Between June 2021 and May 2022, we collected consecutive, non-duplicated vancomycin-susceptible Enterococcus faecium (VSE) samples. Real-time PCR was performed to detect the presence of vanA, vanB, and vanC. Overlapping PCR with sequencing and whole -genome sequencing were performed for structural analysis. Sequence types (STs) were determined by multilocus sequence typing. Exposure testing was performed to assess the ability of the isolates to acquire vancomycin resistance. Among 282 VSE isolates tested, 20 isolates (7.1%) were VVE. Among them, 17 isolates had partial deletions in the IS1216 or IS1542 sequences in vanS (N=10), vanR (N=5), or vanH (N=2). All VVE isolates belonged to the CC17 complex and comprised five STs, namely ST17 (40.0%), ST1421 (25.0%), ST80 (25.0%), ST787 (5.0%), and ST981 (5.0%). Most isolates were related to three hospital-associated clones (ST17, ST1421, and ST80). After vancomycin exposure, 18 of the 20 VVEs acquired vancomycin resistance. Considering the high reversion rate, detecting VVE by screening VSE for vanA is critical for appropriate treatment and infection control. [ABSTRACT FROM AUTHOR]

We used whole genome sequencing (WGS) as an epidemiologic surveillance tool to elucidate the transmission dynamics of Shiga toxin-producing Escherichia coli (STEC) strains along the beef production chain in South Africa. Isolates were obtained from a cattle farm, abattoirs and retail outlets. Isolates were analysed using WGS on a MiSeq platform (Illumina, San Diego, CA, USA) and phylogenetic analysis was carried out. Of the 85 isolates, 39% (33) carried the stx gene and 61% (52) had lost the stx gene. The prevalence of stx subtypes was as follows; stx1a 55% (18/33), stx1b 52% (17/33), stx2a 55% (18/33), stx2b 27% (9/33), stx2dB 30% (10/33) and stx2d1A 15% (5/33). Thirty-five different serogenotypes were detected, of which 65% (56) were flagellar H-antigens and 34% (29) were both O-antigens and flagellar H-antigens. We identified 50 different sequence types (STs), and only nine of the isolates were assigned to three different clonal complexes. Core genome phylogenetic analysis revealed genetic relatedness, and isolates clustered mainly according to their STs and serogenotypes regardless of stx subtypes. This study provides evidence of horizontal transmission and recirculation of STEC strains in Gauteng province and demonstrates that every stage of the beef production chain plays a significant role in STEC entry into the food chain. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is a zoonotic parasite that can cause severe morbidity and mortality in warm-blooded animals, including marine mammals such as sea otters. Free-ranging cats can shed environmentally resistant T. gondii oocysts in their feces, which are transported through rain-driven runoff from land to sea. Despite their large population sizes and ability to contribute to environmental oocyst contamination, there are limited studies on T. gondii oocyst shedding by free-ranging cats. We aimed to determine the frequency and genotypes of T. gondii oocysts shed by free-ranging domestic cats in central coastal California and evaluate whether genotypes present in feces are similar to those identified in sea otters that died from fatal toxoplasmosis. We utilized a longitudinal field study of four free-ranging cat colonies to assess oocyst shedding prevalence using microscopy and molecular testing with polymerase chain reaction (PCR). T. gondii DNA was confirmed with primers targeting the ITS1 locus and positive samples were genotyped at the B1 locus. While oocysts were not visualized using microscopy (0/404), we detected T. gondii DNA in 25.9% (94/362) of fecal samples. We genotyped 27 samples at the B1 locus and characterized 13 of these samples at one to three additional loci using multi locus sequence typing (MLST). Parasite DNA detection was significantly higher during the wet season (16.3%, 59/362) compared to the dry season (9.7%; 35/362), suggesting seasonal variation in T. gondii DNA presence in feces. High diversity of T. gondii strains was characterized at the B1 locus, including non-archetypal strains previously associated with sea otter mortalities. Free-ranging cats may thus play an important role in the transmission of virulent T. gondii genotypes that cause morbidity and mortality in marine wildlife. Management of free-ranging cat colonies could reduce environmental contamination with oocysts and subsequent T. gondii infection in endangered marine mammals and people.

Abstract Purpose In the Zhejiang region, research on Helicobacter pylori is lacking. The purpose of this study was to assess the extent of antibiotic resistance in H. pylori in this region, explore alternative methods for predicting the resistance patterns of H. pylori, and investigate the colonization of native gastric mucosa by other clades of H. pylori in the structure population of this bacterium. Methods Strains were cultured under microaerobic conditions, and antimicrobial susceptibility testing (AST) was performed via agar dilution. Whole-genome sequencing (WGS) was performed via next-generation sequencing (NGS) technology. Epidemiological data including data from this study and reported articles from Zhejiang, China, were included. Further analyses based on AST, WGS, and epidemiological date include virulence genes, antibiotic resistance-related mutations, and phylogenetic trees based on 7 housekeeping genes and core-genome single nucleotide polymorphisms (SNPs). Results The bacterial isolates in this study presented higher antibiotic resistance rates than previously reported, especially against levofloxacin and clarithromycin. The point mutation A2147G in 23 S rRNA is specific to clarithromycin resistance. Mutations at position/s 87 and/or 91 of the gyrA gene amino acid sequence are highly consistent with levofloxacin resistance highly. The point mutations C1707T in 23 S rRNA and E463K in the gyrB gene have not been previously documented in China. All the bacterial isolates belong to Asian branches in the structure population. The resistance rate to clarithromycin of isolates from hosts born after January 1, 1977 is statistically higher than that of hosts born before 1977. Conclusion Eradication therapy based on AST results is urgently needed in Zhejiang. The point mutation A2147G in 23 S rRNA and point mutations in the gyrA gene at amino acid/s 87 and/or 91 are sufficient for predicting resistance to clarithromycin and levofloxacin, respectively. The isolate with the mutation E463K in the gyrB gene represents a significant contribution to the field. Mutations in 23 S rRNA may offer valuable insights into the dynamics of H. pylori transmission among hosts.

Abstract Worldwide, more than 90% of contemporary syphilis strains belong to SS14-like clade. This study aimed to describe the molecular profile of circulating Treponema pallidum subsp. pallidum (TPA) strains in Barcelona, Spain, from 2021 to 2023 building upon our report in 2015 which showed that 94.8% of typed strains belonged to the SS14 clade. Multilocus sequence typing (MLST) was conducted on TPA-positive samples obtained from swab samples by sequencing the tp0136, tp0548, and tp0705 loci. Strains were classified as Nichols-like or SS14-like clade. Macrolide and tetracycline resistance‑associated mutations were determined through analysis of 23S rDNA and 16S rRNA gene sequences. Of the 96 typeable samples, 47.9% belonged to SS14-like and 52.1% to the Nichols-like. Fourteen haplotypes were identified, with ST26 representing 43.8% of the samples, distributed across 11 haplotypes in the SS14-like and 3 haplotypes in the Nichols-like. All the samples showed macrolide resistance-associated mutations, while none exhibited tetracycline-associated mutations. Our findings revealed a substantial shift in the proportion of TPA clades within the Barcelona population from 2021 to 2023, characterized by a higher proportion of Nichols-like strains compared to 2015 and international trends. The varying temporal and geographical trends underscore the need for regular surveillance to understand regional variations in syphilis and strengthen control programs.

Several Chlamydia trachomatis lineages identified through outer membrane protein A genotyping or multilocus sequence typing have been circulating worldwide among men who have sex with men. In a study in Tokyo, Japan, we demonstrate that such lineages commonly belong to a specific polymorphic membrane protein E clade across genotypes.

Abstract Background Acinetobacter baumannii (A. baumannii) is a life-threatening and challenging pathogen. In addition, it accounts for numerous serious infections, particularly among immunocompromised patients. Resistance to nearly all clinically used antibiotics and their ability to spread this resistance is one of the most important concerns related to this bacterium. Objectives This study describes different molecular mechanisms of two multidrug-resistant A. baumannii isolates obtained from endotracheal aspirates collected from the neonatal intensive care unit (NICU), Ain Shams University Hospital, Egypt. Methods Following the identification of two isolates, they were examined for susceptibility to antimicrobial agents. This was followed by multilocus sequence typing as well as whole-genome sequence (WGS). Additionally, a Pathosystems Resources Integration Center (PATRIC) analysis was performed. Results Two isolates, Ab119 and Ab123, exhibited resistance to all tested antibiotics except for tigecycline and colistin. The WGS analysis of antimicrobial resistance genes (AMR) indicated that both isolates shared beta-lactam, aminoglycoside, macrolides, and sulfonamide resistance genes. Furthermore, each strain revealed different resistance genes such as blaNDM-1, blaNDM-10, OXA-64, aph (3')-VI, Tet-B in Ab119 strain and blaOXA-68, blaPER-1, blaPER-7, Tet-39 in Ab123 strain. Multiple efflux pump genes were detected. Multilocus sequence typing indicated that both isolates belong to the same sequence type (ST931), which belongs to international clone (IC3). Both isolates exhibited the presence of multiple mobile genetic elements (MGEs), but no plasmid was detected in either of them. Conclusions A low prevalence of the IC3 sequence type was identified among two A. baumannii isolates obtained from the NICU in Egypt, exhibiting a high resistance level. Healthcare workers must have knowledge regarding the prevalence of A. baumannii among different populations in order to administer suitable treatment, improve patient outcomes, and apply effective infection control practices.

Abstract Background The emergence of Streptococcus agalactiae infections in patients with bacteremia is increasing. It is crucial to investigate the epidemiology, molecular characteristics, biofilm status, and virulence analysis of Streptococcus agalactiae in these patients. Methods In this cross-sectional study, 61 S. agalactiae isolated from blood infection were subjected to characterization through antimicrobial susceptibility tests, biofilm formation, multilocus sequence typing (MLST), and PCR analysis for detecting resistance (tet and erm family) and virulence genes (alp2/3, alp4, bca, bac, eps, rib, lmb, cylE, and pilus island genes). Results Overall, 32.7% of the isolates demonstrated an inducible clindamycin resistance phenotype. The results showed that 49.2, 24.6, and 8.2% of confirmed Streptococcus agalactiae strains were classified as strong, intermediate, and weak biofilm-forming strains, respectively. tet(M) (57.1%) was recovered most, followed by tet(M) + tet(L) (14.3%), tet(S) + tet(K) (10.7%), tet(M) + tet(K) (8.9%), tet(M) + tet(K) + tet(O) (5.4%), and tet(S) + tet(L) + tet(O) (3.6%). Three virulence gene profiles of cylE, lmb, bca, rib (24.6%; 15/61), cylE, lmb, rib, alp3 (19.7%; 12/61), and cylE, lmb, bac, rib (16.4%; 10/61) were detected in approximately two-thirds of the isolates. MLST revealed that the 61 isolates belonged to six clonal complexes, including CC1 (49.2%), followed by CC12 (18%), CC19 (13.1%), CC22 (9.8%), CC17 (6.6%), and CC283 (3.3%), and 11 different sequence types (STs), including ST1 (24.6%), ST7 (14.8%), ST918 (13.1%), ST2118 (9.8%), ST19 (9.8%), ST48 (6.6%), ST1372 (4.9%), ST22 (4.9%), ST40 (4.9%), ST734 (3.3%), and ST283 (3.3%). Remarkably, all CC1 and CC12 isolates, three-fourths of CC19, and half of CC22 were confirmed as biofilm producers. Conversely, CC17 and CC28 isolates were found to be nonproducers. The occurrence of strong biofilm formation was limited to specific CCs, namely CC1 (34.4%), CC12 (8.2%), CC19 (3.3%), and CC22 (3.3%). Conclusion The high prevalence of CC1 and CC12 clones among S. agalactiae strains reflects the emergence of these lineages as successful clones in Iran, which is a serious concern and poses a potential threat to patients.

Abstract Background Characteristics of non-clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) especially from fishery environment are poorly understood. This research, in addition to comprehensive characterisation, sought to delineate the genetic relatedness between the MRSA strains originating from clinical as well as non-clinical settings. Out of 39 methicillin-resistant staphylococcal isolates from 197 fish samples, 6 (Three each of methicillin-resistant S. haemolyticus (MRSH) and MRSA) with distinct resistance profiles were selected for whole-genome sequencing. Using respective bioinformatics tools, MRSA genomes were comprehensively characterized for resistome, virulomes, molecular epidemiology and phylogenetic analysis. Simultaneously, MRSH genomes were specifically examined to characterize antimicrobial resistance genes (ARGs), owing to the fact that MRSH is often recognized as a reservoir for resistance determinants. Results Three MRSA clones identified in this study include ST672-IVd/t13599 (sequence type-SCCmec type/spa type), ST88-V/t2526, and ST672-IVa/t1309. Though, the isolates were phenotypically vancomycin-sensitive, five of the six genomes carried vancomycin resistance genes including the VanT (VanG cluster) or VanY (VanM cluster). Among the three MRSA, only one harbored the gene encoding Panton-Valentine Leukocidin (PVL) toxin, while staphylococcal enterotoxin (SEs) genes such as sea and seb, associated with staphylococcal food poisoning were identified in two other MRSA. Genomes of MRSH carried a composite of type V staphylococcal cassette chromosome mec (SCCmec) elements (5C2 & 5). This finding may be explained by the inversion and recombination events that may facilitate the integration of type V elements to the SCC elements of S. aureus with a methicillin-susceptible phenotype. Phylogenetically, MRSA from a non-clinical setting displayed a considerable relatedness to that from clinical settings. Conclusion This study highlights the genetic diversity and resistance profiles of MRSA and MRSH, with non-clinical MRSA showing notable relatedness to clinical strains. Future research should explore resistance gene transfer mechanisms and environmental reservoirs to better manage MRSA spread.

Abstract Background Streptococcus mutans is studied for its acidogenic and aciduric characteristics, notably its biofilm formation in the presence of sucrose, toward its role in the caries process. Variations in both genotype and phenotype have been reported among clinical isolates of S. mutans. This study aimed to examine genotypic and phenotypic characteristics of S. mutans obtained from Thai children with varying caries statuses. Methods We determined the presence of S. mutans and caries status in 395 children aged 3–4 years. From 325 children carrying S. mutans, we selected 90 with different caries statuses—caries-free (CF; n = 30), low severity of caries (LC; n = 30), or high severity of caries (HC; n = 30). Three isolates of S. mutans were taken from each child, thus, a total of 270 isolates were obtained. Multilocus sequence typing (MLST) was used to genotype the isolates and assess their clonal relationships. The properties, including biofilm formation, collagen binding, and acid production and tolerance were also evaluated. Results Children with carious lesions showed a higher detection rate and number of S. mutans in saliva than those without caries. S. mutans from individuals with HC status showed the lowest biofilm formation ability, while this group had the highest detection rate of collagen-binding isolates. There was no difference in acid production or tolerance by caries status. Genotyping by MLST did not reveal any clone of S. mutans specific to CF status. This result remained even when we included MLST data from the open-access PubMLST database. MLST did identify clones containing only strains from caries-affected hosts, but tests of their phenotypic properties did not reveal any differences between S. mutans from these clones and clones that were from both caries-free and caries-affected children. Conclusions The clonal relationships of S. mutans indicated by MLST were not associated with the status of dental caries in the host.

Abstract Background Commonly used approaches for genomic investigation of bacterial outbreaks, including SNP and gene-by-gene approaches, are limited by the requirement for background genomes and curated allele schemes, respectively. As a result, they only work on a select subset of known organisms, and fail on novel or less studied pathogens. We introduce refMLST, a gene-by-gene approach using the reference genome of a bacterium to form a scalable, reproducible and robust method to perform outbreak investigation. Results When applied to multiple outbreak causing bacteria including 1263 Salmonella enterica, 331 Yersinia enterocolitica and 6526 Campylobacter jejuni genomes, refMLST enabled consistent clustering, improved resolution, and faster processing in comparison to commonly used tools like chewieSnake. Conclusions refMLST is a novel multilocus sequence typing approach that is applicable to any bacterial species with a public reference genome, does not require a curated scheme, and automatically accounts for genetic recombination. Availability and implementation: refMLST is freely available for academic use at https://bugseq.com/academic .

Abstract Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a major public health problem, requiring the use of last-resort antibiotics such as colistin. However, there is concern regarding the emergence of isolates resistant to this agent. The report describes two patients with urinary tract infection (UTI) and ventilator-associated pneumonia (VAP) infection caused by CRKP strains. The first case was a 23-year-old male with UTI caused by a strain of ST16 co-harboring bla CTX-M, bla TEM, bla SHV, bla NDM, bla OXA-48-like genes. The second case was a 39-year-old woman with VAP due to hypervirulent ST337-K2 co-harboring bla SHV, bla NDM, bla OXA-48-like, iucA, rmpA2 and rmpA . The patients’ general condition improved after combination therapy with colistin (plus meropenem and rifampin, respectively) and both of them recovered and were discharged from the hospital. This study highlights the necessary prevention and control steps to prevent the further spread of CRKP strains should be a priority in our hospital.

Brucellosis is an infectious disease of animals that can infect humans. The disease causes significant economic losses and threatens human health. A timely and accurate disease diagnosis plays a vital role in the identification of brucellosis. In addition to traditional diagnostic methods, molecular methods allow diagnosis and typing of the causative agent of brucellosis. This review will discuss various methods, such as Bruce-lаdder, Suiladder, high-resolution melt analysis, restriction fragment length polymorphism, multilocus sequence typing, multilocus variable-number tandem repeat analysis, and whole-genome sequencing single-nucleotide polymorphism, for the molecular typing of Brucella and discuss their advantages and disadvantages.

Abstract Background Group B Streptococcus (GBS) causes invasive infections in newborns and elderly individuals, but is a noninvasive commensal bacterium in most immunocompetent people. Recently, the incidence of invasive GBS infections has increased worldwide, and there is growing interest in the molecular genetic characteristics of invasive GBS strains. Vaccines against GBS are expected in the near future. Here, we aimed to analyze the molecular epidemiology of GBS according to the invasiveness in South Korea. Methods We analyzed GBS isolates collected and stored in two hospitals in South Korea between January 2015 and December 2020. The invasiveness of these isolates was determined via a retrospective review of clinical episodes. Totally, 120 GBS isolates from 55 children and 65 adults were analyzed. Serotype and sequence type (ST) were determined using multiplex polymerase chain reaction (PCR) and multilocus sequence typing, respectively. Fourteen virulence factor-encoding genes of GBS were analyzed using multiplex PCR. Results Forty one (34.2%) were invasive infection-related GBS isolates (iGBS). The most frequently detected serotype was III (39/120, 32.5%), and it accounted for a high proportion of iGBS (21/41, 51.2%). The most frequent ST was ST19 (18/120, 15.0%), followed by ST2 (17/120, 14.2%). Serotype III/ST17 was predominant in iGBS (12/41, 29.3%), and all 17 ST2 strains were noninvasive. The distribution of most of the investigated virulence factors was not significantly related to invasiveness; noteworthily, most of the serotype III/ST17 iGBS carried pilus island (PI) 2b (10/12, 83.3%), and the prevalence of fbsB was significantly low compared with noninvasive GBS isolates (P = 0.004). Characteristically, the combination of bca(+)-cspA(+)-pavA(+)-fbsB(-)-rib(+)-bac(-) was predominant in iGBS (24.4%, 10/41). Conclusions Serotype III/ST17 GBS carrying PI-2b was frequently detected in iGBS. There was no significant association between invasiveness and the pattern of virulence factors; however, a specific combination of virulence factors was predominant in iGBS.

ObjectiveTo understand the diarrhea-type virulence genes， the antimicrobial resistance and the molecular typing of emetic Bacillus cereus from the pre-packaged pastries in Taizhou City.MethodsThe Polymerase Chain Reaction （PCR） method was used to identify the vomiting strains of 97 Bacillus cereus strains which were isolated from pre-packaged pastries in Taizhou and detecte whether the vomiting strains carry diarrhea virulence genes. The sensitivity of the vomiting strains to 19 antibiotics was tested by disk diffusion method， and the molecular typing was carried out by multilocus sequence typing technique.ResultsThe results showed that five emetic Bacillus cereus stains were detected of 97 Bacillus cereus strains and divided into ST 26 and ST 1186 two types. Non-hemolytic enterotoxin gene nhe， enterotoxin genes entFM and bceT were the main diarrhea-type virulence genes of the five strains. The five strains were completely resistant to penicillin， ampicillin and cefotaxime. The resistance rates to cotrimoxazole， rifampicin and cefazolin were 80%（4/5）， 40%（2/5） and 20%（1/5）， respectively. The sensitivity rates of amoxicillin， clindamycin and cefoxitin were 40%（2/5）， 80%（4/5） and 80%（4/5）， respectively， and they were completely sensitive to other antibiotics.ConclusionThis study showed that the detection rate of vomiting Bacillus cereus in pre-packaged pastries in Taizhou was 5.2%（5/97）， ST 26 was the main type， there were a variety of diarrhea-type virulence genes in the five vomiting Bacillus cereus strains.

Xiaojuan Ji,1,* Wenjie Zhu,1,* Hongfei Lu,2 Zhiwei Wu,1 Huaqing Chen,1 Chunli Lin,1 Zhaolin Zeng,1 Cong You,1 Longnian Li1 1Department of Dermatology, First Affiliated Hospital of Gannan Medical University, Joint Organization of Jiangxi Clinical Medicine Research Center for Dermatology, Ganzhou, People’s Republic of China; 2Clinical Laboratory, First Affiliated Hospital of Gannan Medical University, Ganzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Cong You; Longnian Li, Department of Dermatology, First Affiliated Hospital of Gannan Medical University, Ganzhou, People’s Republic of China, Email youcong1987@tmu.edu.cn; li_longnian@foxmail.comObjective: To analyze the antibiotic resistance profile, virulence genes, and molecular typing of Staphylococcus aureus (S. aureus) strains isolated in skin and soft tissue infections at the First Affiliated Hospital, Gannan Medical University, to better understand the molecular epidemiological characteristics of S. aureus.Methods: In 2023, 65 S. aureus strains were isolated from patients with skin and soft tissue infections. Strain identification and susceptibility tests were performed using VITEK 2 and gram-positive bacteria identification cards. DNA was extracted using a DNA extraction kit, and all genes were amplified using polymerase chain reaction. Multilocus sequence typing (MLST) was used for molecular typing.Results: In this study, of the 65 S. aureus strains were tested for their susceptibility to 16 antibiotics, the highest resistance rate to penicillin G was 95.4%. None of the staphylococcal isolates showed resistance to ceftaroline, daptomycin, linezolid, tigecycline, teicoplanin, or vancomycin. fnbA was the most prevalent virulence gene (100%) in S. aureus strains isolated in skin and soft tissue infections, followed by arcA (98.5%). Statistical analyses showed that the resistance rates of methicillin-resistant S. aureus isolates to various antibiotics were significantly higher than those of methicillin-susceptible S. aureus isolates. Fifty sequence types (STs), including 44 new ones, were identified by MLST.Conclusion: In this study, the high resistance rate to penicillin G and the high carrying rate of virulence gene fnbA and arcA of S.aureus were determine, and 44 new STs were identified, which may be associated with the geographical location of southern Jiangxi and local trends in antibiotic use. The study of the clonal lineage and evolutionary relationships of S. aureus in these regions may help in understanding the molecular epidemiology and provide the experimental basis for pathogenic bacteria prevention and treatment.Keywords: Staphylococcus aureus, skin and soft tissue infections, antibiotic resistance, multilocus sequence typing, virulence gene

Background: Commonly used approaches for genomic investigation of bacterial outbreaks, including SNP and gene-by-gene approaches, are limited by the requirement for background genomes and curated allele schemes, respectively. As a result, they only work on a select subset of known organisms, and fail on novel or less studied pathogens. We introduce refMLST, a gene-by-gene approach using the reference genome of a bacterium to form a scalable, reproducible and robust method to perform outbreak investigation. Results: When applied to multiple outbreak causing bacteria including 1263 Salmonella enterica, 331 Yersinia enterocolitica and 6526 Campylobacter jejuni genomes, refMLST enabled consistent clustering, improved resolution, and faster processing in comparison to commonly used tools like chewieSnake. Conclusions: refMLST is a novel multilocus sequence typing approach that is applicable to any bacterial species with a public reference genome, does not require a curated scheme, and automatically accounts for genetic recombination. Availability and implementation: refMLST is freely available for academic use at https://bugseq.com/academic. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a major public health problem, requiring the use of last-resort antibiotics such as colistin. However, there is concern regarding the emergence of isolates resistant to this agent. The report describes two patients with urinary tract infection (UTI) and ventilator-associated pneumonia (VAP) infection caused by CRKP strains. The first case was a 23-year-old male with UTI caused by a strain of ST16 co-harboring blaCTX-M, blaTEM, blaSHV, blaNDM, blaOXA-48-like genes. The second case was a 39-year-old woman with VAP due to hypervirulent ST337-K2 co-harboring blaSHV, blaNDM, blaOXA-48-like,iucA, rmpA2 and rmpA. The patients' general condition improved after combination therapy with colistin (plus meropenem and rifampin, respectively) and both of them recovered and were discharged from the hospital. This study highlights the necessary prevention and control steps to prevent the further spread of CRKP strains should be a priority in our hospital. [ABSTRACT FROM AUTHOR]

Brucellosis is an infectious disease of animals that can infect humans. The disease causes significant economic losses and threatens human health. A timely and accurate disease diagnosis plays a vital role in the identification of brucellosis. In addition to traditional diagnostic methods, molecular methods allow diagnosis and typing of the causative agent of brucellosis. This review will discuss various methods, such as Bruce-ladder, Suiladder, high-resolution melt analysis, restriction fragment length polymorphism, multilocus sequence typing, multilocus variable-number tandem repeat analysis, and whole-genome sequencing single-nucleotide polymorphism, for the molecular typing of Brucella and discuss their advantages and disadvantages. [ABSTRACT FROM AUTHOR]

Background: Group B Streptococcus (GBS) causes invasive infections in newborns and elderly individuals, but is a noninvasive commensal bacterium in most immunocompetent people. Recently, the incidence of invasive GBS infections has increased worldwide, and there is growing interest in the molecular genetic characteristics of invasive GBS strains. Vaccines against GBS are expected in the near future. Here, we aimed to analyze the molecular epidemiology of GBS according to the invasiveness in South Korea. Methods: We analyzed GBS isolates collected and stored in two hospitals in South Korea between January 2015 and December 2020. The invasiveness of these isolates was determined via a retrospective review of clinical episodes. Totally, 120 GBS isolates from 55 children and 65 adults were analyzed. Serotype and sequence type (ST) were determined using multiplex polymerase chain reaction (PCR) and multilocus sequence typing, respectively. Fourteen virulence factor-encoding genes of GBS were analyzed using multiplex PCR. Results: Forty one (34.2%) were invasive infection-related GBS isolates (iGBS). The most frequently detected serotype was III (39/120, 32.5%), and it accounted for a high proportion of iGBS (21/41, 51.2%). The most frequent ST was ST19 (18/120, 15.0%), followed by ST2 (17/120, 14.2%). Serotype III/ST17 was predominant in iGBS (12/41, 29.3%), and all 17 ST2 strains were noninvasive. The distribution of most of the investigated virulence factors was not significantly related to invasiveness; noteworthily, most of the serotype III/ST17 iGBS carried pilus island (PI) 2b (10/12, 83.3%), and the prevalence of fbsB was significantly low compared with noninvasive GBS isolates (P = 0.004). Characteristically, the combination of bca(+)-cspA(+)-pavA(+)-fbsB(-)-rib(+)-bac(-) was predominant in iGBS (24.4%, 10/41). Conclusions: Serotype III/ST17 GBS carrying PI-2b was frequently detected in iGBS. There was no significant association between invasiveness and the pattern of virulence factors; however, a specific combination of virulence factors was predominant in iGBS. [ABSTRACT FROM AUTHOR]

Little has been known about symbiotic relationships and host specificity for symbionts in the human gut microbiome so far. Bifidobacteria are a paragon of the symbiotic bacteria biota in the human gut. In this study, we characterized the population genetic structure of three bifidobacteria species from 58 healthy mother-infant pairs of three ethnic groups in China, geographically isolated, by Rep-PCR, multi-locus sequence analysis (MLSA), and in vitro carbohydrate utilization. One hundred strains tested were incorporated into 50 sequence types (STs), of which 29 STs, 17 STs, and 4 STs belong to B. longum subsp. longum, B. breve, and B. animalis subsp. lactis, respectively. The conspecific strains from the same mother-child pair were genetically very similar, supporting the vertical transmission of Bifidobacterium phylotypes from mother to offspring. In particular, results based on allele profiles and phylogeny showed that B. longum subsp. longum and B. breve exhibited considerable intraspecies genetic heterogeneity across three ethnic groups, and strains were clustered into ethnicity-specific lineages. Yet almost all strains of B. animalis subsp. lactis were incorporated into the same phylogenetic clade, regardless of ethnic origin. Our findings support the hypothesis of co-evolution between human gut symbionts and their respective populations, which is closely linked to the lifestyle of specific bacterial lineages. Hence, the natural and evolutionary history of Bifidobacterium species would be an additional consideration when selecting bifidobacterial strains for industrial and therapeutic applications. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae infection is a major public health concern with high morbidity and mortality rates. This study aimed to evaluate the serotype distribution, antimicrobial resistance changes, clonal composition, and virulence factors of S. pneumoniae isolates causing pneumococcal disease in northeast China from 2000 to 2021. A total of 1,454 S. pneumoniae isolates were included, with 568 invasive strains and 886 non-invasive strains. The patients from whom the S. pneumoniae were isolated ranged in age from 26 days to 95 years, with those ≤ 5 years old comprising the largest group (67.19%). 19 F, 19 A, 23 F, 14, and 6B were the most common serotypes, of which 19 A and 19 F were the main serotypes of invasive and non-invasive S. pneumoniae, respectively. CC271 was the most common multilocus sequence type. Serotype 14 had the lowest expression of cbpA, rrgA, and psrP genes, but expression levels of 19 A and 19 F genes were similar. All isolates were sensitive to ertapenem, moxifloxacin, linezolid, and vancomycin but highly resistant to macrolides, tetracyclines, and cotrimoxazole. Simultaneous resistance to erythromycin, clindamycin, tetracyclines, and trimethoprim/sulfamethoxazole was common pattern among multidrug-resistant isolates. Non-invasive S. pneumoniae had higher resistance to β-lactam antibiotics than invasive strains. 19 A and 19 F were the main strains of penicillin-resistant S. pneumoniae. The resistance rate of β-lactam antibiotics decreased from 2017 to 2021 compared to previous periods. Including PCV13 in the national immunization program can reduce the morbidity and mortality rates of pneumococcal disease effectively. [ABSTRACT FROM AUTHOR]

Simple Summary: Blastocystis spp. and Giardia duodenalis are two prevalent intestinal parasites with a worldwide distribution that can infect humans and animals, resulting in significant public health concerns and economic losses. For goats, infection with the two parasites can cause symptoms such as diarrhea, which is not conducive to the development of stockbreeding. Shanxi Province is one of the largest goats breeding provinces in China. However, the prevalence of Blastocystis spp. and G. duodenalis in black goats in Shanxi Province remains unknown. Thus, 1200 fecal samples of black goats were collected in five representative geographical locations in Shanxi Province to examine the presence and genotypes of G. duodenalis and Blastocystis spp. by using a molecular approach. The results showed that the total infection rates of G. duodenalis and Blastocystis spp. were 7.5% and 3.5%, respectively. The presence and genotypes of G. duodenalis were determined based on three established loci (tpi, bg, and gdh). Among the detected assemblages B and E of G. duodenalis, the most prevalent assemblage was E in black goats in the five study areas. One novel MLG (MLG-E12) was identified by multilocus genotypes (MLGs) analysis. Through DNA sequence analysis, four subtypes of Blastocystis spp. were found in black goats, namely ST5, ST10, ST14, and ST30, among which ST10 was the dominant subtype in this study. This is the first report of Blastocystis spp. and G. duodenalis infection in black goats in Shanxi Province, which not only enhances our understanding of the genetic diversity of Blastocystis spp. and G. duodenalis in black goats in China but also provides essential baseline data for the prevention and control of Blastocystis spp. and G. duodenalis infection in black goats in the study areas. Blastocystis spp. and Giardia duodenalis are two prevalent zoonotic intestinal parasites that can cause severe diarrhea and intestinal diseases in humans and many animals. Black goat (Capra hircus) farming is increasingly important in China due to the remarkable adaptability, high reproductive performance, rapid growth rate, and significant economic value of black goats. A number of studies have indicated that black goats are the potential reservoir of multiple zoonotic protozoans in China; however, the prevalence and zoonotic status of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province is still unknown. Thus, a total of 1200 fecal samples of black goats were collected from several representative regions at different altitudes in Shanxi Province and were examined for the presence and genotypes of G. duodenallis and Blastocystis spp. by amplifying the beta-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) loci of G. duodenalis and SSU rRNA of Blastocystis spp. using PCR and sequence analysis methods, respectively. The overall prevalence of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province were 7.5% and 3.5%, respectively. Two assemblages (B and E) of G. duodenalis and four subtypes (ST5, ST10, ST14, and ST30) of Blastocystis spp. were identified, with assemblage E and ST10 as the prevalent genotype and subtype in black goats, respectively. One novel multilocus genotype (MLG) was identified in MLG-E and was designated as MLG-E12. For both G. duodenalis and Blastocystis spp., the prevalence was significantly related to the region and age groups (p < 0.05). This is the first report on the prevalence of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province. These results not only provide baseline data for the prevention and control of both parasites in black goats in Shanxi Province, but also enhance our understanding of the genetic composition and zoonotic potential of these two parasites. [ABSTRACT FROM AUTHOR]

The increasing problem of antimicrobial resistance in N. gonorrhoeae necessitates the development of molecular typing schemes that are suitable for rapid and mass screening. The objective of this study was to design and validate a mini-MLST scheme for N. gonorrhoeae based on global pathogen population data. Using sequences of seven housekeeping genes of 21,402 isolates with known MLSTs from the PubMLST database, we identified eighteen informative polymorphisms and obtained mini-MLST nucleotide profiles to predict MLSTs of isolates. We proposed a new MLST grouping system for N. gonorrhoeae based on mini-MLST profiles. Phylogenetic analysis revealed that MLST genogroups are a stable characteristic of the N. gonorrhoeae global population. The proposed grouping system has been shown to bring together isolates with similar antimicrobial susceptibility, as demonstrated by the characteristics of major genogroups. Established MLST prediction algorithms based on nucleotide profiles are now publicly available. The mini-MLST scheme was evaluated using a MLST detection/prediction method based on the original hydrogel DNA microarray. The results confirmed a high predictive ability up to the MLST genogroup. The proposed holistic approach to gonococcal population analysis can be used for the continuous surveillance of known and emerging resistant N. gonorrhoeae isolates. [ABSTRACT FROM AUTHOR]

ABSTRACT Ocular syphilis is a serious complication of Treponema pallidum infection that can occur at any stage of syphilis and affect any eye structure. It remains unknown if certain T. pallidum strains are associated with ocular infections; therefore, we performed genotyping and whole genome sequencing (WGS) to characterize strains from patients with ocular syphilis. Seventy-five ocular or non-ocular specimens from 55 ocular syphilis patients in 14 states within the United States were collected between February 2016 and November 2020. Sufficient T. pallidum DNA was available from nine patients for genotyping and three for WGS. Genotyping was done using the augmented Centers for Disease Control and Prevention typing scheme, and WGS was performed on Illumina platforms. Multilocus sequence typing allelic profiles were predicted from whole genome sequence data. T. pallidum DNA was detected in various specimens from 17 (30.9%) of the 55 patients, and typing was done on samples from 9 patients. Four complete strain types (14d10/g, 14b9/g, 14d9/g, and 14e9/f) and five partial types were identified. WGS was successful on samples from three patients and all three strains belonged to the SS14 clade of T. pallidum. Our data reveal that multiple strain types are associated with ocular manifestations of syphilis. While genotyping and WGS were challenging due to low amounts of T. pallidum DNA in specimens, we successfully performed WGS on cerebrospinal fluid, vitreous fluid, and whole blood.IMPORTANCESyphilis is caused by the spirochete Treponema pallidum. Total syphilis rates have increased significantly over the past two decades in the United States, and the disease remains a public health concern. In addition, ocular syphilis cases has also been on the rise, coinciding with the overall increase in syphilis rates. We conducted a molecular investigation utilizing traditional genotyping and whole genome sequencing over a 5-year period to ascertain if specific T. pallidum strains are associated with ocular syphilis. Genotyping and phylogenetic analysis show that multiple T. pallidum strain types are associated with ocular syphilis in the United States.

ABSTRACT The clonal transmission of fluconazole-resistant Candida glabrata isolates within hospitals has seldom been analyzed by whole-genome sequencing (WGS). We performed WGS on 79 C. glabrata isolates, comprising 31 isolates from three premature infants with persistent C. glabrata bloodstream infection despite antifungal treatment in the same neonatal intensive care unit (NICU) in 2022 and 48 (27 fluconazole-resistant and 21 fluconazole-susceptible dose-dependent) bloodstream isolates from 48 patients in 15 South Korean hospitals from 2010 to 2022. Phylogenetic analysis based on WGS single-nucleotide polymorphisms (SNPs) distinguished the 79 isolates according to multilocus sequence typing (MLST) (17 sequence type [ST]3, 13 ST7, two ST22, 41 ST26, four ST55, and two ST59 isolates) and unveiled two possible clusters of nosocomial transmission among ST26 isolates. One cluster from two premature infants with overlapping NICU hospitalizations in 2022 encompassed 15 fluconazole-resistant isolates harboring pleiotropic drug-resistance transcription factor (Pdr1p) P258L (13 isolates) or N1086I (two isolates), together with 10 fluconazole-susceptible dose-dependent isolates lacking Pdr1p SNPs. The other cluster indicated unforeseen clonal transmission of fluconazole-resistant bloodstream isolates among five patients (four post-lung transplantation and one with diffuse interstitial lung disease) in the same hospital over 8 months. Among these five isolates, four obtained after exposure to azole antifungals harbored distinct Pdr1p SNPs (N1091D, E388Q, K365E, and R376Q). The findings reveal the transmission patterns of clonal bloodstream isolates of C. glabrata among patients undergoing antifungal treatment, exhibiting different levels of fluconazole susceptibility or distinct Pdr1p SNP profiles.IMPORTANCEThe prevalence of fluconazole-resistant bloodstream infections caused by Candida glabrata is increasing globally, but the transmission of these resistant strains within hospitals has rarely been documented. Through whole-genome sequencing and epidemiological analyses, this study identified two potential clusters of C. glabrata bloodstream infections within the same hospital, revealing the transmission of clonal C. glabrata strains with different levels of fluconazole susceptibility or distinct transcription factor pleiotropic drug resistance protein 1 (Pdr1p) single-nucleotide polymorphism profiles among patients receiving antifungal therapy.

Background: Clostridioides difficile infection (CDI) is an increasingly common disease in healthcare facilities and community settings. However, there are limited reports of community-onset CDI (CO-CDI) in China. Methods: We collected diarrheal stool samples from 3885 patients who went to outpatient department or emergency department in a tertiary hospital in China during 2010–2023, analyzed the correlation between patients’ basic information and the detection rate of CDI. Besides, all stool samples from 3885 outpatients included were tested by culturing. Moreover, we randomly selected 89 patients’ stools during the 14 years and isolated 126 C. difficile strains from them. The presence of toxin genes (tcdA, tcdB, cdtA, and cdtB) were confirmed by PCR. Toxigenic strains were typed using multilocus sequence typing (MLST). Susceptibility to 9 antimicrobials was evaluated using the E-test. Results: 528 of 3885 patients (13.6 %) with diarrhea were finally diagnosed as CDI. The median age of patients included was 51 years (6 months-95 years), while the median of patients with CDI was older than patients with negative results [55.5 years (6 months-93 years) vs. 50 years (9 months −95 years), p < 0.001]. In winter, patients with diarrhea might be more likely to have CDI. The detection rate of CDI of patients in emergency department was much higher than those in other outpatients (20.7 % vs. 12.4 %, p < 0.001), and did differ from each outpatient departments (p < 0.05). There were 95 isolated strains detected as toxigenic C. difficile. Among these strains, 82 (86.3 %) had the tcdA and tcdB genes (A+B+) and 5 of these 82 strains were positive for the binary toxin genes (cdtA and cdtB) (A+B+CDT+). There were 15 different sequence types (STs) by multilocus sequence typing (MLST), while the most ST was ST-54 (23.2 %). ST types composition was relatively stable over the time span of this study. Some strains had high resistance to ciprofloxacin, clindamycin, and erythromycin. Twenty-three isolates (24.2 %) were multidrug-resistant. Conclusions: Outpatients with CDI were common among patients having diarrhea during this period in our hospital. Elderly patients and patients went to emergency department may be susceptible to CDI. Based on MLST, the result revealed that the C. difficile isolates had high genetic diversity and maintained stability in this period. All isolates were susceptible to metronidazole and vancomycin, and nearly one quarter of all isolates had multidrug resistance.

ObjectiveTo understand the current drug resistance status and bacterial multilocus sequence typing （MLST） of human and avian Campylobacter jejuni in Jinshan District， Shanghai.MethodsFecal samples were collected from diarrhea patients in the annuity mountainous area from 2021 to 2022， and poultry and related samples were collected from 2 poultry farms in the Jinshan area for detection of C. jejuni. Minimal inhibitory concentration （MIC） drug sensitivity test was performed on the detected C. jejuni， and some strains were selected for whole genome sequencing and MLST analysis.ResultsA total of 823 samples of diarrhea disease were collected， and 32 strains of C. jejuni were detected， with a detection rate of 3.89%. Out of 600 poultry related samples， 62 strains of C. jejuni were detected， with a detection rate of 10.33%. Human multidrug resistance reached 93.75% （30/32）， while avian multidrug resistance reached 100.00%（62/62）. The top four drug resistance rates of human and avian C. jejuni were azithromycin （100.00% from humans and 100.00% from birds）， naphthoic acid （93.75% from humans and 87.10% from birds）， ciprofloxacin （90.63% from humans and 98.39% from birds）， and tetracycline （84.38% from humans and 98.39% from birds）. The relatively low resistance strains of human derived C. were erythromycin， chloramphenicol， and thalithromycin. The relatively low resistance strains of avian C. jejuni were erythromycin， clindamycin， and flufenicol. MLST analysis showed that the selected 16 strains of bacteria were divided into 9 ST types， among which the evolutionary relationship of avian C. jejuni was relatively concentrated， while human C. jejuni was relatively dispersed. It was found that one strain of avian C. jejuni was closely related to two strains of human C. jejuni.ConclusionsC. jejuni infection is severe in patients with diarrhea in this region， with a detection rate second only to salmonella and Vibrio parahaemolyticus. C. jejuni infection in poultry is relatively common， and both are highly resistant. Therefore， monitoring and control should be strengthened. MLST analysis shows new ST types in both avian and human sources of C. jejuni， indicating the emergence of new mutations that require continuous monitoring to avoid the epidemics caused by new strains. The isolated strains with close genetic relationships between avian and human sources reveal the evidence of the spread of C. jejuni from poultry to humans. Therefore it is necessary to strengthen the monitoring of C. jejuni in relevant samples from breeding farms.

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 M1–M17, 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.

Introduction. Borrelia miyamotoi is a pathogen of erythema-free ixodid tick-borne borreliosis (ITBB), a disease widespread in Russia. To date, there are no generally accepted methods for B. miyamotoi genotyping. The multilocus sequencing typing (MLST) scheme of Borrelia was originally developed for B. burgdorferi, and does not have the required discrimination power for monitoring the ITBB pathogens. The objective of this study is to develop the MLST scheme for B. miyamotoi. Materials and Methods. The whole genome sequences of 10 reference strains (GenBank) were analyzed for the selection of the house-keeping loci. The MLST scheme development was based on principles published by the authors of the method. For this experiment, 81 B. miyamotoi strains and positive clinical samples were used to test the MLST scheme. Results. After analyzing the genomic data, 8 house-keeping loci were chosen for MLST, for which the PCR and sequencing primers were designed. Each MLST loci was represented by several alleles (from 4 to 7) which form 15 sequence types. The genetic diversity of pathogens isolated from ITBB patients and ticks were characterized. Discussion. Based on pairwise distances between allelic profiles, the sequence types can be classified into four groups. The first two groups are clonal complexes; the other two groups are formed by once identified sequence types. The first clonal complex unites 11 sequence types (80 or 88% of the characterized B. miyamotoi), the second consists of 2 sequence types (9 or 9.8%). The genetic differences between B. miyamotoi are associated with the sources of strains and biological isolates. The MLST based classification confirms the previously described genetic heterogeneity of B. miyamotoi populations associated with ecologically unrelated vectors of ITBB pathogens. Conclusion. The proposed MLST scheme is an appropriate tool for ITBB pathogen classification and evolutionary change characterization within clonal complexes.

Objectives: Infection by carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a serious clinical problem worldwide. However, the molecular epidemiology of the clinical isolates varies depending on the region. This study was conducted to analyse the resistance phenotype and clarify the genetic and epidemiological properties of CRPA clinical isolates from southeast Shanxi, China. Methods: Fifty-seven isolates of CRPA were collected from a hospital in this region. These isolates were reidentified by MALDI-TOF and subjected to whole-genome sequencing by next-generation sequencing. Phylogenetic trees were constructed based on single nucleotide polymorphisms (SNPs), after which multilocus sequence typing (MLST) was performed and antimicrobial resistance genes were identified. Results: All the 57 CRPA isolates carried at least one kind of gene encoding carbapenemase, such as blaIMP-1, blaIMP-10, blaOXA-10, blaOXA-395, blaOXA-396, blaOXA-485, blaOXA-486, blaOXA-488, blaOXA-494, and blaOXA-50. The isolates harboured AIM-1, CMY-51, mecD, and NmcR genes and carried one kind of Pseudomonas-derived cephalosporinase (PDC) β-lactamase-encoding gene, such as blaPCD-1 to blaPCD-3, blaPCD-5, or blaPCD-7 to blaPCD-10. Two isolates were found to harbour the aminoglycoside-modifying enzyme genes aadA1 and aadA7; however, no isolates were found to harbour genes encoding 16S rRNA methylase or quinolone resistance-related genes. These CRPA isolates belonged to various sequence types (STs), two of which, namely, ST235 and ST277, were high-risk types. Conclusions: Our findings indicate that CRPA isolates carrying resistance genes with unique regional characteristics are spreading in this region, with a high diversity of STs, especially in high-risk clones. These findings highlight the necessity for further measures to prevent CRPA spread in Shanxi.

BackgroundAntimicrobial resistant infections continue to be a leading global public health crisis. Mobile genetic elements, such as plasmids, have been shown to play a major role in the dissemination of antimicrobial resistance (AMR) genes. Despite its ongoing threat to human health, surveillance of AMR in the United States is often limited to phenotypic resistance. Genomic analyses are important to better understand the underlying resistance mechanisms, assess risk, and implement appropriate prevention strategies. This study aimed to investigate the extent of plasmid mediated antimicrobial resistance that can be inferred from short read sequences of carbapenem resistant E. coli (CR-Ec) in Alameda County, California. E. coli isolates from healthcare locations in Alameda County were sequenced using an Illumina MiSeq and assembled with Unicycler. Genomes were categorized according to predefined multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) schemes. Resistance genes were identified and corresponding contigs were predicted to be plasmid-borne or chromosome-borne using two bioinformatic tools (MOB-suite and mlplasmids).ResultsAmong 82 of CR-Ec identified between 2017 and 2019, twenty-five sequence types (STs) were detected. ST131 was the most prominent (n = 17) followed closely by ST405 (n = 12). blaCTX-M were the most common ESBL genes and just over half (18/30) of these genes were predicted to be plasmid-borne by both MOB-suite and mlplasmids. Three genetically related groups of E. coli isolates were identified with cgMLST. One of the groups contained an isolate with a chromosome-borne blaCTX-M-15 gene and an isolate with a plasmid-borne blaCTX-M-15 gene.ConclusionsThis study provides insights into the dominant clonal groups driving carbapenem resistant E. coli infections in Alameda County, CA, USA clinical sites and highlights the relevance of whole-genome sequencing in routine local genomic surveillance. The finding of multi-drug resistant plasmids harboring high-risk resistance genes is of concern as it indicates a risk of dissemination to previously susceptible clonal groups, potentially complicating clinical and public health intervention.

Simple Summary: The current study aimed to investigate the virulence and resistance genes associated with Klebsiella pneumoniae isolated from the farms of four animal species in selected areas of Xinjiang, China, encompassing cows, sheep, chickens, and pigs. The sensitivity to antibiotics was evaluated. Genetic diversity was assessed using wzi allele typing and multilocus sequence typing. The findings shed light on the antibiotic resistance patterns and genetic diversity among K. pneumoniae isolates originating from different animal sources, highlighting the potential for inter-species transmission of this bacterium. Klebsiella pneumoniae (K. pneumoniae) is recognized as a zoonotic pathogen with an increasing threat to livestock and poultry. However, research on K. pneumoniae of animal origin remains limited. To address the gap, a comprehensive investigation was carried out by collecting a total of 311 samples from the farms of four animal species (dairy cow, chicken, sheep, and pig) in selected areas of Xinjiang, China. Isolates were identified by khe gene amplification and 16S rRNA gene sequencing. Genotyping of K. pneumonia isolates was performed using wzi typing and multilocus sequence typing (MLST). PCR was employed to identify virulence and resistance genes. An antibiotic susceptibility test was conducted using the Kirby–Bauer method. The findings revealed an isolation of 62 K. pneumoniae strains, with an average isolation rate of 19.94%, with the highest proportion originating from cattle sources (33.33%). Over 85.00% of these isolates harbored six virulence genes (wabG, uge, fimH, markD, entB, and ureA); while more than 75.00% of isolates possessed four resistance genes (blaTEM, blaSHV, oqxA, and gyrA). All isolates exhibited complete resistance to ampicillin and demonstrated substantial resistance to sulfisoxazole, amoxicillin/clavulanic acid, and enrofloxacin, with an antibiotic resistance rate of more than 50%. Furthermore, 48.39% (30/62) of isolates were classified as multidrug-resistant (MDR) strains, with a significantly higher isolation rate observed in the swine farms (66.67%) compared to other farms. Genetic characterization revealed the classification of the 62 isolates into 30 distinct wzi allele types or 35 different sequence types (STs). Notably, we identified K. pneumoniae strains of dairy and swine origin belonging to the same ST42 and wzi33-KL64 types, as well as strains of dairy and chicken origin belonging to the same wzi31-KL31-K31 type. These findings emphasize the widespread occurrence of drug-resistant K. pneumoniae across diverse animal sources in Xinjiang, underscoring the high prevalence of multidrug resistance. Additionally, our results suggest the potential for animal-to-animal transmission of K. pneumoniae and there was a correlation between virulence genes and antibiotic resistance genes. Moreover, the current study provides valuable data on the prevalence, antibiotic resistance, and genetic diversity of K. pneumoniae originating from diverse animal sources in Xinjiang, China. [ABSTRACT FROM AUTHOR]

Simple Summary: Infectious coryza is an acute respiratory infectious disease of chickens caused by Av. paragallinarum. Av. paragallinarum can be serotyped into A (A1–A4), B (B-1), and C (C1–C4) serovars. However, there is no effective molecular typing scheme to gain basic knowledge about the Av. paragallinarum population structure. MLST has played a major role in investigating the genetic structure of bacterial populations and has rapidly become the cornerstone technique for the molecular typing of pathogenic microorganisms. In this study, we are the first to establish this method and applied to PubMLST for a public MLST site for Av. paragallinarum. More samples and the ST sequence data can help to clearly observe the genetic evolution and epidemic patterns of Av. paragallinarum. Infectious coryza is an acute respiratory infection caused by Avibacterium paragallinarum, which is widely distributed throughout the world. However, there is no effective molecular typing scheme to obtain basic knowledge about the Av. paragallinarum population structure. This study aimed to develop a multilocus sequence typing (MLST) scheme for Av. paragallinarum that allows for the worldwide comparison of sequence data. For this purpose, the genetic variability of 59 Av. paragallinarum strains from different geographical origins and serovars was analyzed to identify correlations. The MLST scheme was developed using seven conserved housekeeping genes, which identified eight STs that clustered all of the strains into three evolutionary branches. The analytical evaluation of the clone group relationship between the STs revealed two clone complexes (CC1 and CC2) and three singletons (ST2, ST5, and ST6). Most of the isolates from China belonged to ST1 and ST3 in CC1. ST8 from Peru and ST7 from North America together formed CC2. Our results showed that the Av. paragallinarum strains isolated from China had a distant genetic relationship with CC2, indicating strong regional specificity. The MLST scheme established in this study can monitor the dynamics and genetic differences of Av. paragallinarum transmission. [ABSTRACT FROM AUTHOR]

Introduction: This study aimed to investigate the prevalence and molecular characterization of Leptospira species in Belgrade, Serbia, an area where this disease is underexplored. Specifically, the study sought to employ molecular and multilocus sequence typing analyses to fill the gap in understanding the diversity and distribution of Leptospira species within the region. Methods: A comprehensive molecular analysis was conducted on kidney samples obtained from Norway rats (Rattus norvegicus) in the urban environment. The study utilized molecular diagnostic techniques including real-time PCR targeting the lipL32 gene and performing sequence-based typing schemes utilizing adk, icdA, lipL32, lipL41, rrs2, and secY genes. These methodologies were applied to ascertain the presence and characterize different Leptospira species and serovars, respectively. Results: The findings revealed the presence of two Leptospira species and three separate serovars in the Belgrade area. This study identified the presence of L. kirschneri serovar Mozdok in Serbia for the first time, a significant discovery previously undocumented in the region. This pioneering investigation sheds light on the molecular diversity and prevalence of Leptospira species in Serbia. Discussion: The study underscores the importance of employing molecular typing methods to gain insights into the epidemiology and characterization of Leptospira species. These findings significantly contribute to both local and global perspectives on leptospirosis epidemiology, providing vital insights for the development of effective control strategies and interventions. Summary: In our recent study, we explored the presence and performed molecular typing of the Leptospira species, the bacteria responsible for leptospirosis, in wild rats in Serbia. This was the first time such a study was conducted in the region. Leptospirosis is a serious disease that affects both animals and humans, often transmitted through contact with water contaminated by infected animals. Our focus was on understanding which types of Leptospira were present in these animals. Excitingly, we discovered a particular strain of Leptospira, known as L. kirshneri serovar Mozdok, for the first time in Serbia. This finding is significant because it sheds light on the presence and spread of different Leptospira serovars in Serbia. It also raises awareness about the potential health risks associated with this serovar, which was previously unknown in the area. Our work fits into a broader context of disease surveillance and public health. By identifying the types of Leptospira present in a specific region, we can better understand the risks to public health and take steps to prevent and control the spread of leptospirosis. This discovery is not just important for scientists studying infectious diseases; it has real implications for public health officials, veterinarians, and anyone concerned with preventing and treating leptospirosis. Our findings highlight the need for ongoing monitoring of Leptospira in wildlife and synanthropic fauna, to protect both animal and human health. [ABSTRACT FROM AUTHOR]

Background and Objectives: Multi-drug-resistant pathogens pose a significant threat as they can rapidly spread, leading to severe healthcare-associated invasive infections. In developing countries, diarrheagenic Escherichia coli (DEC) is a major bacterial pathogen responsible for causing diarrhea. However, the outbreak of resistant strains has made the treatment of DEC infections much more challenging. This study aimed to investigate the relationship between antibiotic resistance genes and other virulence categories in E. coli strains that cause diarrhea, particularly DEC. Materials and Methods: The phylogenetic grouping was defined using PCR and multi-locus sequence type (MLST) methods. Results: Among the isolates analyzed, 14 were identified as resistant and were classified into eight distinct sequence types: ST3, ST53, ST77, ST483, ST512, ST636, ST833, and ST774, indicating genetic diversity among the resistant strains. Certain sequence types, notably ST512 and ST636, were found to be associated with multiple antibiotic resistance in DEC. Regarding antibiotic susceptibility, strains showed the highest resistance to amoxicillin, suggesting that this antibiotic may not be effective in treating DEC infections. On the other hand, the isolates demonstrated susceptibility to amikacin and chloramphenicol, implying that these antibiotics could be more suitable treatment options for DEC infections. Conclusion: The findings underscore the importance of promptly identifying antibiotic resistance patterns and their correlation with specific pathogenic virulence categories, as this knowledge can aid in selecting the most appropriate antibiotics for treating DEC infections. Considering the antibiotic resistance profiles and associated resistance genes is crucial in managing and containing diarrheal outbreaks and in selecting effective antibiotic therapies for DEC infections. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is an important public health parasitic infection, which is endemic in many parts of the world, including Iran. We aimed to investigate genetic diversity and phylogenetic relationship among different Leishmania isolates using multilocus sequence typing (MLST). Methods: Totally, 41 isolates collected either from patients referred to Leishmaniasis Diagnostics and Treatment Center at Tehran University of Medical Sciences, Tehran, Iran or from animals during 2019-2021, were subjected to the study. They included L. major and L. tropica from human, L. infantum from canine, and L. turanica from rodents from different endemic foci of Iran analyzed using MLST including gp63, g6pdh, lack, nagt, and hsp70 genes. Results: A total of 5010 bps was analyzed from each isolate. The three targets, nagt, lack, and g6pdh, generated better topology comparing to the other genes. In the 44 isolates, 22 haplotypes (STs) were identified. Leishmania tropica contained the highest number of haplotypes (n=12) comparing to L. major (n=8), L. infantum (n=1) and L. turanica (n=1). All five genomic loci caused separation of Iranian Leishmania species at the species level, indicating conservation of these genes in the Leishmania parasite. Conclusion: The highest number of haplotypes belonged to L. tropica, indicating that the genetic diversity of this species is higher than that of L. major. It was further confirmed that the MLST is a suitable method to examine genetic variation of Leishmania parasites with respect to evolutionary and epidemiological studies. [ABSTRACT FROM AUTHOR]

Objective To understand the distribution, serotype, and drug resistance of Salmonella in domestic sewage in Jining city, and to analyze the whole genome sequencing so as to provide scientific basis for the prevention and control of Salmonella. Methods From February to March in 2023, two samples of sewage from the intake of 3 urban sewage plants with 500 ml each were collected continuously for 7 weeks. A total of 42 sewage samples were collected for culture, isolation, and identification of Salmonella. The isolated Salmonella was tested for serum agglutination test and fluorescent quantitative PCR serotyping. In total 41 strains of Salmonella were tested for minimum inhibitory concentration (MIC) of 17 drugs in 9 classes by microbroth dilution method, followed by genome sequencing and phylogenetic analysis. Results The isolation rate of Salmonella from domestic sewage in Jining city was high. Among 24 positive samples, 41 strains of Salmonella were isolated, belonging to 9 serotypes, mainly S.Agona and S.Kentucky. Liying and Jidong sewage treatment plants had their own unique serotypes. Only one strain of S.Give was not resistant, and the other 40 strains of Salmonella were multi-drug resistant. The resistance rates to sulfamethoxazole, tetracycline, ampicillin, and streptomycin were more than 90%, and the resistance rate to ampicillin/sulbactam was more than 80%. There were 19 kinds of drug resistance spectra, of which SXT-TET-STR-AMP-AMS ranked the first place. The resistance spectra of different serotypes of Salmonella were different. S.Kentucky, S.Gold Coast, and S.Infant were multi-drug resistant seriously. The resistance spectrum of the same serotype was also inconsistent in different sewage plants, and the resistance was the most serious in Liying sewage plant. The CARD database was used to prediet drug resistance genes, mainly predicting 18 drug resistance genes in 8 types of drug resistance families, and the drug resistance phenotypes and drug resistance genes were almost all consistent. The ST types of different serotypes of Salmonella were different, and ST13 S.Agona was dominant. The phylogenetic tree was divided into 6 clusters, and strains in different sewage plants were in different branches, indicating that their genomes were different and had different sources. Conclusion There are various serotypes of salmonella in domestic sewage in Jining city, suggesting that they might have exist and spread in the population. Almost all strains are multi-drug resistant bacteria, carrying resistance genes extensively. Some types of strains live persistently and distribute across regions, leading to the potential risk to population. It is necessary to carry out continuous monitor, and scientific prevention and control measures need to be formulated to control the epidemic of salmonella and the aggravation of drug resistance. [ABSTRACT FROM AUTHOR]

Isolation on culture media followed with macroscopic, microscopic, molecular and phylogenetic analyses and pathogenicity tests allowed us to identify Pythium capillosum to be capable of destroying dagger nematode through endozoic parasitism from ingested oomycete zoospores. Although endoparasitism of free-living nematodes is found to be common among oomycetes in nature, this is the first finding of such occurrence in Bosnia and Herzegovina soils. The obtained results could open the door to future studies for controlling nematodes in an environmentally safe and sustainable way. [ABSTRACT FROM AUTHOR]

Little has been known about symbiotic relationships and host specificity for symbionts in the human gut microbiome so far. Bifidobacteria are a paragon of the symbiotic bacteria biota in the human gut. In this study, we characterized the population genetic structure of three bifidobacteria species from 58 healthy mother–infant pairs of three ethnic groups in China, geographically isolated, by Rep-PCR, multi-locus sequence analysis (MLSA), and in vitro carbohydrate utilization. One hundred strains tested were incorporated into 50 sequence types (STs), of which 29 STs, 17 STs, and 4 STs belong to B. longum subsp. longum, B. breve, and B. animalis subsp. lactis, respectively. The conspecific strains from the same mother–child pair were genetically very similar, supporting the vertical transmission of Bifidobacterium phylotypes from mother to offspring. In particular, results based on allele profiles and phylogeny showed that B. longum subsp. longum and B. breve exhibited considerable intraspecies genetic heterogeneity across three ethnic groups, and strains were clustered into ethnicity-specific lineages. Yet almost all strains of B. animalis subsp. lactis were incorporated into the same phylogenetic clade, regardless of ethnic origin. Our findings support the hypothesis of co-evolution between human gut symbionts and their respective populations, which is closely linked to the lifestyle of specific bacterial lineages. Hence, the natural and evolutionary history of Bifidobacterium species would be an additional consideration when selecting bifidobacterial strains for industrial and therapeutic applications.

Background: Leishmaniasis is an important public health parasitic infection, which is endemic in many parts of the world, including Iran. We aimed to investigate genetic diversity and phylogenetic relationship among different Leishmania isolates using multilocus sequence typing (MLST). Methods: Totally, 41 isolates collected either from patients referred to Leishmaniasis Diagnostics and Treatment Center at Tehran University of Medical Sciences, Tehran, Iran or from animals during 2019-2021, were subjected to the study. They included L. major and L. tropica from human, L. infantum from canine, and L. turanica from rodents from different endemic foci of Iran analyzed using MLST including gp63, g6pdh, lack, nagt, and hsp70 genes. Results: A total of 5010 bps was analyzed from each isolate. The three targets, nagt, lack, and g6pdh, generated better topology comparing to the other genes. In the 44 isolates, 22 haplotypes (STs) were identified. Leishmania tropica contained the highest number of haplotypes (n=12) comparing to L. major (n=8), L. infantum (n=1) and L. turanica (n=1). All five genomic loci caused separation of Iranian Leishmania species at the species level, indicating conservation of these genes in the Leishmania parasite. Conclusion: The highest number of haplotypes belonged to L. tropica, indicating that the genetic diversity of this species is higher than that of L. major. It was further confirmed that the MLST is a suitable method to examine genetic variation of Leishmania parasites with respect to evolutionary and epidemiological studies.

Abstract Background Cats are the primary reservoirs of the bacterium Bartonella henselae, the main cause of cat-scratch disease in humans. The main vector of the bacterium is the cat flea, Ctenocephalides felis. In southeastern Europe, data are lacking on the prevalence of B. henselae infection in cats, the strains of B. henselae involved and the risk factors associated with the infection. Methods Blood samples collected in ethylenediaminetetraacetic acid-containing tubes from 189 domestic cats (156 pet cats and 33 stray cats) from Zagreb, the capital city of Croatia, and 10 counties throughout Croatia were cultured for Bartonella spp. Following culture, bacterial isolates were genotyped at eight loci after using PCR to amplify 16S ribosomal RNA (rRNA) and the internal transcribed spacer region between the 16S and 23S rRNA sequences. Univariate and multivariate logistic regression were used to identify risk factors for B. henselae infection in cats. Results Bartonella spp. was detected in 31 cats (16.4%), and subsequent genotyping at the eight loci revealed B. henselae in all cases. Thirty complete multilocus sequence typing profiles were obtained, and the strains were identified as four sequence types that had been previously reported, namely ST5 (56.7%), ST6 (23.3%), ST1 (13.3%) and ST24 (3.3%), as well as a novel sequence type, ST33 (3.3%). The univariate analysis revealed a significantly higher risk of B. henselae infection in cats residing in coastal areas of Croatia (odds ratio [OR] 2.592, 95% confidence interval [CI] 1.150–5.838; P = 0.0191) and in cats with intestinal parasites (OR 3.207, 95% CI 1.088–9.457; P = 0.0279); a significantly lower risk was identified in cats aged > 1 year (OR 0.356, 95% CI 0.161–0.787; P = 0.0247) and in cats sampled between April and September (OR 0.325, 95% CI 0.147–0.715; P = 0.005). The multivariate analysis that controlled for age showed a positive association with the presence of intestinal parasites (OR 4.241, 95% CI 1.243–14.470; P = 0.0119) and coastal residence (OR 2.567, 95% CI 1.114–5.915; P = 0.0216) implying increased risk of infection, and a negative association with sampling between April and September (OR 0.379, 95% CI 0.169–0.848; P = 0.018) implying a decreased risk of infection. After controlling for the season, an increased risk of infection remained for the coastal region (OR 2.725, 95% CI 1.200–6.186; P = 0.012). Conclusions Bartonella henselae is prevalent throughout Croatia and is a public health threat. Environmental and host factors can significantly affect the risk of infection, and these should be explored in more detail. The presence of intestinal parasites highlights the need to eliminate the flea vector, Ctenocephalides felis, as the most effective approach to control infections in cats and humans. Graphical Abstract

Objective(s): Difficult-to-treat Streptococcus agalactiae infections are increasingly described in patients with urinary tract infections (UTIs). This occurrence could be due to the production of virulence determinants. This study aimed to characterize the molecular features of S. agalactiae responsible for UTIs.Materials and Methods: In this cross-sectional study, 70 S. agalactiae isolated from UTIs were examined. Antibiotic susceptibility testing was performed using the disk diffusion method. All S. agalactiae isolates were confirmed by atr and dltS PCR assays. Virulence, alpha protein-like, and pilus island genes were detected by PCR. Isolates were characterized using the multilocus sequence typing method. Results: Multidrug resistance was observed in 80% of isolates. Five virulence profiles were detected, wherein cylE, lmb, bca, rib (35.7%), cylE, lmb, alp3 (27.1%), and cylE, lmb, bac, rib, alp2 (21.4%) were the most frequent detected profiles. S. agalactiae was isolated and categorized within three clonal complexes (CCs) including CC22 (40%), CC17 (25.7%), and CC23 (20%). The main sequence types (STs) found were ST22 (27.1%), ST23 (17.1%), ST17 (12.9%), ST31 (8.7%), ST40 (8.7%), ST74 (7.1%), ST48 (4.3%), ST890 (4.3%), ST189 (2.8%), ST38 (2.8%), ST52 (2.8%), and ST155 (1.4%). ST74 and ST38 were reported for the first time in Tehran-Iran. Conclusion: This study highlights the predominance of the CC22 lineage among S. agalactiae strains isolated from UTIs in Tehran, Iran, and highlights the significant penetration of this lineage into hospitals. MDR patterns among these strains appear to be becoming a major concern in the management of infections.