Mapping Heterogeneous Population Structure of Mannheimia haemolytica Associated with Pneumonic Infection of Sheep in Southern State Karnataka, India.

Mannheimia haemolytica is recognized as principal pathogen associated with pneumonic pasteurellosis leading to huge economic losses to small ruminant farmers. Even though the disease causes huge economic losses, epidemiology of M. haemolytica is less studied, hindering the formulation of effective control strategies. Current study aimed to highlight molecular characterisation of M. haemolytica strains isolated from ovine pneumonic infection. M. haemolytica 27 isolates with two reference strains were characterised using capsular and virulence gene typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) methods. M. haemolytica serotype A2 recognized as predominant serotype (74%) followed by A6 (11%) and A1 (5%) serotypes. Virulence gene profiling by PCRs showed dominance of all five virulent genes [such as adh and gcp (100% each)] followed by gs60 (88.8%), lktC (85.2%), tbpB (51.9%) and least nmaA gene (14.8%). MLST profiling delineated M. haemolytic isolates into 11 sequence types (STs) with most prevalent being ST37 (27.9%) and ST16 (23%) and nine new STs (ST37, 38, 39, 40, 41, 42, 47, 48, and 49). These new STs did not belong to any of the three clonal complexes (CC4, CC8 and CC28). ST16 was exclusively noted in A1 and A6 serotypes. Amongst 25 isolates, 22 pulsotypes (GD 0.88) recorded indicated variability of the M. haemolytica isolates in PFGE analysis. In conclusion, the study suggested dominance of M. haemolytica serotype A2 harbouring different virulent genes, diverse STs and pulsotypes responsible for pneumonic pasteurellosis frequently encountered in sheep.

An evaluation of a Stenotrophomonas maltophilia outbreak due to commercial arterial blood gas collection kit.

BACKGROUND: Stenotrophomonas maltophilia is a gram-negative bacterium that can cause hospital infections and outbreaks within hospitals. This study aimed to evaluate an outbreak of Stenotrophomonas maltophilia, caused by ready-to-use commercial syringes containing liquid lithium and heparin for arterial blood gas collection in a university hospital. METHODS: Upon detecting an increase in Stenotrophomonas maltophilia growth in blood cultures between 15.09.2021 and 19.11.2021, an outbreak analysis and a case-control study (52 patients for the case group, 56 patients for the control group) were performed considering risk factors for bacteremia. Samples from possible foci for bacteremia were also cultured. Growing bacteria were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The genetic linkage and clonal relationship isolates were investigated with pulsed-field gel electrophoresis (PFGE) in the reference laboratory. RESULTS: In the case-control study, the odds ratio for the central venous catheter [3.38 (95% confidence interval [CI]: 1.444, 8.705 ; p = 0.006)], for surgery [3.387 (95% confidence interval [CI]: 1.370, 8.373 ; p = 0.008)] and for arterial blood gas collection history [18.584 (95% confidence interval [CI]:4.086, 84.197; p < 0.001)] were identified as significant risk factors. Stenotrophomonas maltophilia growth was found in ready-to-use commercial syringes used for arterial blood gas collection. Molecular analysis showed that the growths in the samples taken from commercial syringes and the growths from blood cultures were the same. It was decided that the epidemic occurred because the method for sterilization of heparinized liquid preparations were not suitable. After discontinuing the use of the kits with this lot number, the outbreak was brought under control. CONCLUSIONS: According to our results, disposable or sterile medical equipment should be included as a risk factor in outbreak analyses. The method by which injectors containing liquids, such as heparin, are sterilized should be reviewed. Our study also revealed the importance of the cooperation of the infection control team with the microbiology laboratory.

Molecular characterization of Escherichia coli isolates recovered from broilers with cellulitis.

Avian cellulitis in broilers, caused by avian pathogenic Escherichia coli, is a major cause for carcass rejections during meat inspection, resulting in significant economic losses. In this study, we analysed E. coli isolates obtained from broiler chickens affected by cellulitis for their genetic relatedness and antimicrobial resistance phenotype and genotype. The objective was to determine whether there is a clonal spread or whether these clinical isolates differ. For this purpose, E. coli was isolated from swab samples collected from diseased broilers across 77 poultry farms in Germany, resulting in 107 isolates. These isolates were subjected to serotyping, PCR-based phylotyping and macrorestriction analysis with subsequent pulsed-field gel-electrophoresis for typing purposes. In addition, the presence of virulence genes associated with avian pathogenic E. coli (APEC) was investigated by PCR. Antimicrobial susceptibility of the isolates was examined by the disk diffusion method according to CLSI guidelines and subsequently, the presence of corresponding resistance genes was investigated by PCR. Typing results revealed that a significant proportion of the isolates belonged to serotype O78:K80, which is one of the major APEC serotypes. Phylogenetic grouping showed that phylogenetic group D was most commonly represented (n = 49). Macrorestriction analysis showed overall heterogenous results, however, some clustering of closely related isolates was observed. The level of antimicrobial resistance was high, with 83.8% of isolates non-susceptible to at least one class of antimicrobial agents and 40% of isolates showing resistance to at least three classes. The most frequently observed resistance was to ampicillin, mediated by blaTEM (n = 56). However, few isolates were non-susceptible to ciprofloxacin (n = 8) and none of the isolates was resistant to 3rd generation cephalosporins or carbapenems. Overall, the results show that genetically diverse APEC associated with avian cellulitis can be found among and within German poultry farms. While most isolates were antimicrobial resistant, resistance levels to high(est) priority critically important antimicrobials were low.

Usefulness and Limitations of PFGE Diagnosis and Nucleotide Sequencing Method in the Analysis of Food Poisoning Pathogens Found in Cooking Employees.

In the case of a food poisoning outbreak, it is essential to understand the relationship between cooking workers and food poisoning. Many biological diagnostic methods have recently been developed to detect food poisoning pathogens. Among these diagnostic tools, this study presents PCR-based pulsed-field gel electrophoresis and nucleotide sequencing diagnostic analysis results for diagnosing food poisoning outbreaks associated with cooking employees in Chungcheongnam-do, Republic of Korea. Pulsed-field gel electrophoresis was useful in identifying the food poisoning outbreaks caused by Staphylococcus aureus and Enteropathogenic Escherichia coli. In the case of Norovirus, nucleotide sequencing was used to identify the relationship between cooking workers and the food poisoning outbreak. However, it is difficult to determine whether cooking employees directly caused the food poisoning outbreaks based on these molecular biological diagnostic results alone. A system is needed to integrate epidemiological and diagnostic information to identify a direct correlation between the food poisoning outbreak and cooking employees.

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

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

Epidemiological and Whole Genome Sequencing Analysis of Restaurant Salmonella Enteritidis Outbreak Associated with an Infected Food Handler in Jiangxi Province, China, 2023.

In China, Salmonella is one of the most frequent causes of bacterial gastroenteritis, and food handlers in restaurants as an important contaminated source were rarely reported. In May 2023, an outbreak of Salmonella enterica serovar Enteritidis infection in a restaurant in Jiangxi Province, China, was investigated. Cases were interviewed. Stool samples from cases, anal swabs from restaurant employees, suspicious raw food materials, and semifinished food were collected and examined. Pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) were performed to determine the relatedness of the pathogen isolates. Antimicrobial resistance genes and virulence genes of isolates were analyzed by WGS. The antimicrobial profile of the isolates was detected by broth microdilution, which involved 20 different antibiotics. Among the 31 patrons, 26 showed gastrointestinal symptoms. Five Salmonella Enteritidis strains were isolated from patients (2), semifinished food (2), and food handler (1). The results of PFGE and single-nucleotide polymorphism showed that these five isolates were identical clones. These findings demonstrated that this outbreak was a restaurant Salmonella Enteritidis outbreak associated with an infected food handler. The rates of resistance to nalidixic acid and colistin and intermediate resistance to ciprofloxacin were 100%, 80%, and 100%, respectively. These outbreak isolates harbored point mutation gyrA p.D87G. The cause of inconsistency between the genotype and phenotype of resistance was deeply discussed. A total of 107 virulence genes were found in each isolate, with many being associated with Salmonella pathogenicity island (SPI)-1 and SPI-2. As an overlooked contamination source, infected food handlers can easily cause large-scale outbreaks. This outbreak highlighted that the government should enhance the training and supervision of food hygiene and safety for food handlers to prevent foodborne outbreaks.

Foodborne Outbreak by Salmonella enterica Serovar Weltevreden in West Bengal, India.

Foodborne gastroenteritis outbreaks owing to Salmonella enterica serovar Weltevreden (Salmonella Weltevreden) represent a significant global public health problem. In the past two decades, Salmonella Weltevreden has emerged as a dominant foodborne pathogen, especially in South-East Asian countries. This report describes a community foodborne outbreak of gastroenteritis caused by Salmonella Weltevreden in August 2022 following consumption of panipuri from a street vendor in the Polba block in Hooghly district, West Bengal, India. This food item was consumed by 185 people, of whom 129 had acute watery diarrhea with other clinical symptoms and 65 of them were admitted to different District hospitals for treatment. Stool specimens collected from hospitalized cases were positive for S. enterica, and further serotyped as Salmonella Weltevreden. All the Salmonella Weltevreden strains possessed the Salmonella pathogenicity islands associated genes (invA/E, orgA, ttrc, ssaQ, mgtC, misL, spi4D), the enterotoxin (stn), and hyperinvasive locus gene (hilA). Except erythromycin, all the strains were susceptible for commonly used antimicrobials in the treatment of diarrhea. The XbaI-based pulsed-field gel electrophoresis analysis indicated that all the isolates responsible for the recent outbreak were similar, but diverged from other Salmonella Weltevreden that were previously reported in West Bengal. This report indicates that foodborne infection is a major public health concern in India and demands to strengthen capacity-building measures at the local health care levels for linking causative agents of outbreaks.

Angle modulated two-dimensional single cell pulsed-field gel electrophoresis for detecting early symptoms of DNA fragmentation in human sperm nuclei.

We here developed a novel angle-modulated two-dimensional single cell pulsed-field gel electrophoresis (2D-SCPFGE). Variations in current-application-time and rotation angle generated different alignments of DNA fibers and segments. After the first run, the specimen was turned by 150° (2D-SCPFGE-0-150) to detect naturally occurring the earliest stage of DNA fragmentation or 75° (2D-SCPFGE-0-75) to analyze artificially induced cleavage. The former revealed that a part of long chain fibers remained at the origin and long segments were still tangled in the bundle of elongated fibers after the first run. The latter visualized the dose-dependent cleavage of DNA by EcoR1. Multicycle 2D-SCPFGE was useful for generating 2D-alignments of single nuclear DNA fibers, which is the first step for visualization of single-strand breaks on stretched fibers. To date, many articles have accepted the pathogenetic significances of DNA fragmentation in human sperm for male infertility and congenital anomaly. It is necessary to perform multivariate analyses of not only earliest-stage DNA fragmentation but also other types of damage, including single-strand breaks, in sequential DNA fibers. 2D-SCPFGE is the fundamental tool for understanding single nuclear DNA damages.

Antimicrobial resistance in E. Coli of animal origin and discovery of a novel ICE mobile element in Northeast China.

BACKGROUND: Multidrug resistance in Enterobacteriaceae including resistance to quinolones is rising worldwide. The development of resistance may lead to the emergence of new transmission mechanisms. In this study, the collection of different E. coli was performed from animals and subjected to subsequent procedures including pulsed-field gel electrophoresis, micro-broth dilution method, polymerase chain reaction. Whole genome sequencing of E. coli C3 was performed to detect the affinity, antimicrobial resistance and major carriers of the isolates. RESULTS: A total of 66 E. coli were isolated and their antibiotic resistance genes, frequency of horizontal transfer and genetic environment of E. coli C3 were determined. The results showed there were both different and same types in PFGE typing, indicating clonal transmission of E. coli among different animals. The detection of antimicrobial resistance and major antibiotic resistance genes and the plasmid transfer results showed that strains from different sources had high levels of resistance to commonly used clinical antibiotics and could be spread horizontally. Whole-genome sequencing discovered a novel ICE mobile element. CONCLUSION: In summary, the antimicrobial resistance of E. coli in northeast China is a serious issue and there is a risk of antimicrobial resistance transmission. Meanwhile, a novel ICE mobile element appeared in the process of antimicrobial resistance formation.

[Comparative study on three molecular typing methods of Vibrio parahaemolyticus].

OBJECTIVE: To evaluate the correlation among the three molecular typing method of pulsed field gel electrophoresis(PFGE), repetitive extragenic palindromic(REP)-PCR and en-terobacterial repetitive intergenic consensus(ERIC)-PCR, and to explore the genetic relationship among strains, and to further understand the distribution and epidemic trend of Vibrio parahaemolyticus in Liaoning Province by combining Serotype analysis. METHODS: Serum typing, PFGE, REP-PCR, and ERIC-PCR molecular typing and cluster analysis were performed on 150 VP isolates from Liaoning Province in 2018. RESULTS: 118 isolates could be divided into 14 Serotype, and 32 isolates could not be classified. The main serotypes were O3, O1 and O2. The resolution(DI) of PFGE is 0.969, the resolution(DI) of REP-PCR is 0.948, and the resolution(DI) of ERIC-PCR is 0.927. The Serotype O3 group strains are highly similar to the molecular types of O1 group strains. CONCLUSION: In 2018, the epidemic Serotype of clinical VP isolates in Liaoning Province is still O3: K6, and the epidemic serotype of food VP isolates is still O2. The result of PFGE, REP-PCR, and ERIC-PCR typing method are consistent, and the resolution and reproducibility of PFGE typing method are superior to the other two method. The Serotype O3 group is closely related to O1 group.

Optical Genome Mapping for the Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy: Advancement and Challenges.

Facioscapulohumeral muscular dystrophy (FSHD) is the second most common muscular dystrophy in adults, and it is associated with local D4Z4 chromatin relaxation, mostly via the contraction of the D4Z4 macrosatellite repeat array on chromosome 4q35. In this study, we aimed to investigate the use of Optical Genome Mapping (OGM) as a diagnostic tool for testing FSHD cases from the UK and India and to compare OGM performance with that of traditional techniques such as linear gel (LGE) and Pulsed-field gel electrophoresis (PFGE) Southern blotting (SB). A total of 6 confirmed and 19 suspected FSHD samples were processed with LGE and PFGE, respectively. The same samples were run using a Saphyr Genome-Imaging Instrument (1-color), and the data were analysed using custom EnFocus FSHD analysis. OGM was able to confirm the diagnosis of FSHD1 in all FSHD1 cases positive for SB (n = 17), and D4Z4 sizing highly correlated with PFGE-SB (p < 0.001). OGM correctly identified cases with mosaicism for the repeat array contraction (n = 2) and with a duplication of the D4Z4 repeat array. OGM is a promising new technology able to unravel structural variants in the genome and seems to be a valid tool for diagnosing FSHD1.

Rapid Genotyping of Campylobacter coli Strains from Poultry Meat by PFGE, Sau-PCR, and fla-DGGE.

The genotyping of Campylobacter coli was done using three methods, pulsed-field gel electrophoresis (PFGE), Sau-polymerase chain reaction (Sau-PCR), and denaturing gradient gel electrophoresis assay of flagellin gene (fla-DGGE) and the characteristics of these assays were compared. The results showed that a total of 53 strains of C. coli were isolated from chicken and duck samples in three markets. All isolates were clustered into 31, 33, and 15 different patterns with Simpson's index of diversity (SID) values of 0.972, 0.974, and 0.919, respectively. Sau-PCR assay was simpler, more rapid, and had higher discriminatory power than PFGE assay. Fla-DGGE assay could detect and illustrate the number of contamination types of C. jejuni and C. coli without cultivation, which saved more time and cost than Sau-PCR and PFGE assays. Therefore, Sau-PCR and fla-DGGE assays are both rapid, economical, and easy to perform, which have the potential to be promising and accessible for primary laboratories in genotyping C. coli strains.

Virulence potential of Salmonella 1,4, [5],12:i:- strains isolated during decades from different sources in the Southeast region of Brazil.

Salmonella 1,4, [5],12:i:- is one of the most prevalent serovars associated with gastroenteritis in several countries, including Brazil. However, few studies have analyzed the virulence potential of this variant in this country. Therefore, this study aimed to characterize S. 1,4, [5],12:i:- strains isolated in Southeast Brazil. To this end, 113 S. 1,4, [5],12:i:- strains isolated from different sources between 1983 and 2020 were analyzed. For all strains, the frequencies of 11 virulence genes were investigated using PCR and the molecular typing was performed using pulsed-field gel electrophoresis (PFGE). Furthermore, 40 strains isolated from human and non-human sources were characterized by survival under acid and oxidative stress, and virulence analysis in Galleria mellonella was performed for 20 selected strains. All virulence genes were detected in more than 91% of the strains. The studied strains were grouped into four clusters using PFGE. Most strains were present in one cluster, named PFGE-A, with a genetic similarity of ≥ 79.5%. All 40 strains survived acid stress after 10 min and 1 h of exposure. Under oxidative stress, all 40 strains survived after 10 min, and 36 survived after 1 h of exposure. In the G. mellonella assay, nine isolates from non-human sources and six isolates from human showed high-to-intermediate virulence profiles. In conclusion, the pathogenic potential of the strains studied was corroborated by the high frequency of all the virulence genes identified. The PFGE results suggested that most strains belonged to one main cluster that has been prevailing in the São Paulo State, Brazil. The S. 1,4, [5],12:i:- strains isolated from human and non-human sources successfully survived the unfavorable conditions in the human gastrointestinal tract. Finally, strains isolated from non-human sources showed a higher proportion of isolates with high to intermediate virulence profiles in G. mellonella than in human isolates, suggesting a possible difference between isolates from different origins.

Impact of genetic diversity and antibiotic-resistance of Salmonella isolated from feral cats: One Health approach.

Free-living cats usually live in colonies in urban areas, especially close to parks and neighbourhoods where people feed them without any sanitary control. This can pose a human, animal and environmental health concern due to the close contact between uncontrolled colonies, the population and other domestic and/or wild animals. Thus, this study aimed to assess the genetic diversity and antimicrobial resistance (AMR) among Salmonella enterica subsp. enterica strains isolated from feral cats in a previous epidemiological study in the Gran Canaria island (Spain). A total of nineteen Salmonella isolates were obtained from November 2018 to January 2019 in a Salmonella epidemiological study in feral cats. All isolates obtained were genotyped by pulsed-field gel electrophoresis (PGFE) and were tested for antimicrobial susceptibility, in accordance with Decision 2013/652/EU. PFGE analysis revealed isolates clustering by serovar, with identical clones for serovars Bredeney and Grancanaria, while differing pulsotypes were observed for serovars Florida (88.89 % similarity) and Nima (83.23 % similarity). All but two isolates were resistant to at least one antimicrobial. The results obtained demonstrate that feral cats in the region investigated are a reservoir of Salmonella strains resistant to gentamicin (94.1 %) and of the critically important antimicrobial tigecycline (23.5 %). Hence, they could excrete AMR strains through their faeces and contaminate the environment, favoring the spread of such bacteria to cohabiting pets. Moreover, this widespread presence of AMR Salmonella clones across various serovars highlights the urgent need to implement efficient antimicrobial stewardship and control programs by the local governments due to the ongoing need to protect human and animal health under a One Health concept.

Validating the Utility of Multilocus Variable Number Tandem-repeat Analysis (MLVA) as a Subtyping Strategy to Monitor Listeria monocytogenes In-built Food Processing Environments.

Listeria monocytogenes is a serious human pathogen and an enduring challenge to control for the ready-to-eat food processing industry. Cost-effective tools that can be deployed by commercial or in-house laboratories to rapidly investigate and resolve contamination events in the built food processing environment are of value to the food industry. Multilocus variable number tandem-repeat analysis (MLVA) is a molecular subtyping method, which along with other same-generation methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) is being superseded in disease tracking and outbreak investigations by whole-genome sequencing (WGS). In this paper, it is demonstrated that MLVA can continue to play a valuable role as a valid, fast, simple, and cost-effective method to identify and track Listeria monocytogenes subtypes in factory environments, with the method being highly congruent with MLST. Although MLVA does not have the discriminatory power of WGS to identify truly persistent clones, with careful interpretation of results alongside isolate metadata, it remains a powerful tool in situations and locations where WGS may not be readily available to food business operators.

Etiological Survey and Traceability Analysis of a Foodborne Disease Outbreak of Salmonella Senftenberg in Guizhou Province.

To conduct a study that examined the molecular epidemiology and pathogenesis of Salmonella Senftenberg isolates associated with an outbreak of foodborne disease in Guizhou Province and to provide a reference basis for the traceability of foodborne salmonellosis outbreaks and clinical diagnosis and treatment in the province. Fourteen strains of suspected Salmonella isolated from patient stool and food samples were used for pathogenic identification and serotyping by biochemical and mass spectrometry methods. Fourteen types of antibiotics were tested for drug sensitivity by the microbroth dilution method, and molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS). After the sequencing data were spliced by SPAdes, the gene protein sequences were compared with the Comprehensive Antibiotic Research Database and Virulence Factor Database, drug resistance and virulence genes were predicted, and whole genome multilocus sequence typing (wgMLST) was performed. The results were compared with those for Salmonella strains of the same serotype from the past 5 years in China detailed on the TraNet website. All 14 strains were identified as Salmonella Senftenberg (with the antigenic formula 1,3,19:g,s,t:-), and in the PFGE cluster tree, the strains were divided into two band types, with a similarity of 88.9%. The 14 strains were sensitive to the 14 antibiotics. WGS analysis showed that the 14 strains carried the same drug resistance and virulence genes and that all strains carried 3 aminoglycoside and lipopeptide drug resistance genes, including 114 virulence genes. The wgMLST results showed that the strains were distributed on the same small branch as those obtained from previous outbreaks of infection in Tianjin and Jilin. Salmonella Senftenberg, which caused the outbreak, carries a variety of virulence genes, which suggests that the strain is highly pathogenic. These pathogenic bacteria may be associated with the Salmonella strain in Tianjin, Jilin, and other places and have caused foodborne disease outbreaks as a result of imported contamination.

Genotypic and phenotypic analysis of Elizabethkingia meningoseptica in bullfrog Rana catesbeiana isolated in Taiwan.

Elizabethkingia meningoseptica is a hazardous bacterium for agriculture production and human health. The present study identified E. meningoseptica from the bullfrog, human and reference strain BCRC 10677 by API 20NE, 50S ribosome protein L27 sequencing and pulse field gel electrophoresis to differentiate isolates of E. meningoseptica from aquatic animals and humans. All isolates from bullfrogs and humans were identified as E. meningoseptica by DNA sequencing with 98.8%-100% sequence identity. E. meningoseptica displayed significant genetic diversity when analysed using pulsed-field gel electrophoresis (PFGE). There were six distinct pulsotypes, including one pulsotype found in bullfrog isolates and five pulsotypes found in human isolates. However, E. meningoseptica from bullfrog exhibited one genotype only by PFGE. Overall, molecular epidemiological analysis of PFGE results indicated that the frog E. meningoseptica outbreaks in Taiwan were produced by genetically identical clones. The bullfrog isolates were not genetically related to other E. meningoseptica from human and reference isolates. This research provided the first comparisons of biochemical characteristics and genetic differences of E. meningoseptica from human and bullfrog isolates.

[Drug resistance and genomic characteristics of Salmonella enterica serovar London from clinical and food sources in Hangzhou City from 2017 to 2021].

Objective: To analyze the drug resistance and genomic characteristics of Salmonella enterica serovar London isolated from clinical and food sources in Hangzhou City from 2017 to 2021. Methods: A total of 91 Salmonella enterica serovar London strains isolated from Hangzhou City from 2017 to 2021 were analyzed for drug susceptibility, pulsed field gel electrophoresis (PFGE) typing and whole genome sequencing. Multilocus sequence typing (MLST), core genome multilocus sequence typing (cgMLST) and detection of drug resistance genes were performed by using the sequencing data. Phylogenetic analysis was conducted to compare the 91 genomes from Hangzhou City with 347 genomes from public databases. Results: No significant difference in the drug resistance rate was observed between clinical strains and food strains to 18 drugs in Hangzhou City(all P>0.05), and the multidrug resistance (MDR) rate was 75.8% (69/91). Most strains were resistant to 7 drug classes simultaneously. One strain was resistant to Polymyxin E as well as positive for mcr-1.1, and 50.5% (46/91) of the strains were resistant to Azithromycin and were positive for mph(A). All 91 Salmonella enterica serovar London strains were ST155, which were subdivided into 44 molecular types by PFGE and 82 types by cgMLST. Phylogenetic analysis showed that most strains from Hangzhou City (83/91) were clustered together, and a small number of human isolates from Europe, North America and pork isolates from Hubei and Shenzhen were mixed in the cluster. Other strains from Hangzhou City (8/91) were closely related to strains from Europe, America and Southeast Asia. Strains isolated from pork were the most closely related to clinical strains. Conclusion: The epidemic of Salmonella enterica serovar London in Hangzhou City is mainly caused by the spread of ST155 strains, which is mainly transmitted locally. At the same time, cross-region transmission to Europe, North America, Southeast Asia, and other provinces and cities in China may also occur. There is no significant difference in the drug resistance rate between clinical strains and food strains, and a high level of MDR is found in the strains. Clinical infection of Salmonella enterica serovar London may be closely related to pork consumption in Hangzhou City.

Geographic distribution of the major clone of extended-spectrum beta-lactamase-producing Escherichia coli infection in a pediatric community in southern Taiwan.

BACKGROUND: The geographic distribution of the major clone of sequence type 131 (ST131) in extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) infections is not known. We analyzed the clinical features, resistance mechanisms, and geographic distribution of ESBL-producing E. coli clones in 120 children. METHODS: We studied the 120 ESBL-producing E. coli strains from children younger than 18 years. A VITEK 2 automated system was used to determine bacterial identification and ESBL production. Sequence type was determined by multi-locus sequence typing (MLST). The genetic relationship of the ESBL-producing strains was studied using pulsed-field gel electrophoresis (PFGE). Phylogenetic group and blaCTX-M group was performed using polymerase chain reaction (PCR). Multiplex PCR for detecting the common group 9 variant, CTX-M-14, and group 1 variant, CTX-M-15, was also performed. The addresses of the 120 children were collected, and plotted on the Taiwan map. RESULTS: The groups in the center of Kaohsiung City lived mainly in urban areas with a population density of over 10,000 people per square kilometer, and the majority of the Kaohsiung groups on the outskirts of the city center lived in suburban areas with a population density of under 6000 people per square kilometer. There was no statistically significant difference between the city center and outskirt groups in terms of clinical presentation, laboratory, and imaging data. However, more ST131 clones, major pulsotype groups, and phylogenetic group B2 strains were found in the center of Kaohsiung than on the outskirts. CONCLUSION: ESBL-producing E. coli clones may be more challenging to treat clinically. Most infections were community-acquired, and there appeared to be major pulsotype clones, mainly in urban areas. This reinforces the necessity of environmental surveillance and sanitary procedures for ESBL-producing E. coli.

Phenotypic and genotypic characterization of Listeria monocytogenes in clinical ruminant cases in Korea.

Listeria monocytogenes, a foodborne human and veterinary pathogen, is associated with high mortality rates in ruminants. However, no studies have investigated the antimicrobial resistance of L. monocytogenes isolates from clinical ruminant cases. This study aimed to determine the phenotypic and genotypic characteristics of L. monocytogenes isolates from clinical cases of Korean ruminants. We collected 24 L. monocytogenes isolates from aborted bovine fetuses and goats presenting with listeriosis-related symptoms. The isolates were subjected to PCR serogrouping, conventional serotyping, virulence gene detection, and antimicrobial susceptibility testing. Furthermore, pulsed-field gel electrophoresis and multilocus sequence typing were used to classify and compare genetic diversity among the isolates, including human L. monocytogenes isolates. The most prevalent L. monocytogenes serotypes were 4b (Ⅳb), 1/2a (Ⅱa; Ⅱc), and 1/2b (Ⅱb). All isolates harbored the virulence genes; however, llsX-encoding listeriolysin were identified only in serotypes 4b and 1/2b. All isolates, including two found in humans, formed three genetically diverse pulsed-field gel electrophoresis clusters according to serotype, lineage, and sequence type. The most prevalent sequence type was ST1, followed by ST365 and ST91. The isolates from ruminants with listeriosis were resistant to oxacillin and ceftriaxone and showed diverse lineage, serotype (serogroup), and sequence type characteristics. Considering that the atypical sequence types exhibited clinical manifestations and histopathological lesions, further study is needed to elucidate the pathogenicity of genetically diverse ruminant L. monocytogenes isolates. Furthermore, continuous monitoring of antimicrobial resistance is required to prevent the emergence of L. monocytogenes strains resistant to common antimicrobials.