Multilocus Sequence Typing Results Research Articles

Relevance of genetic causes and environmental adaptation of Cronobacter spp. isolated from infant and follow-up formula production factories and retailed products in China: A 7-year period of continuous surveillance based on genome-wide analysis

Relevance of genetic causes and environmental adaptation of Cronobacter spp. isolated from infant and follow-up formula production factories and retailed products in China: A 7-year period of continuous surveillance based on genome-wide analysis

Multi-locus sequence typing, antimicrobials resistance and virulence profiles of Salmonella enterica isolated from bovine carcasses in Minas Gerais state, Brazil.

The aim of this study was to identify virulence and antimicrobial resistance profiles and determine the sequence type (ST) by multilocus sequence typing (MLST) of Salmonella enterica isolates from bovine carcasses from slaughterhouse located in Minas Gerais state, Brazil, and its relationship with bovine isolates obtained on the American continent based on sequence type profile. The MLST results were compared with all Salmonella STs associated with cattle on American continent, and a multi-locus sequence tree (MS tree) was built. Among the 17 S. enterica isolates, five ST profiles identified, and ST10 were the most frequent, grouping seven (41.2%) isolates. The isolates presented 11 different profiles of virulence genes, and six different antibiotics resistance profiles. The survey on Enterobase platform showed 333 Salmonella STs from American continent, grouped into four different clusters. Most of the isolates in the present study (13/17), were concentrated in a single cluster (L4) composed by 74 STs. As a conclusion, five different STs were identified, with ST10 being the most common. The isolates showed great diversity of virulence genes and antibiotics resistance profiles. Most of the isolates of this study were grouped into a single cluster composed by 74 STs formed by bovine isolates obtained on the American continent.

Automating the Illumina DNA library preparation kit for whole genome sequencing applications on the flowbot ONE liquid handler robot.

Whole-genome sequencing (WGS) is currently making its transition from research tool into routine (clinical) diagnostic practice. The workflow for WGS includes the highly labor-intensive library preparations (LP), one of the most critical steps in the WGS procedure. Here, we describe the automation of the LP on the flowbot ONE robot to minimize the risk of human error and reduce hands-on time (HOT). For this, the robot was equipped, programmed, and optimized to perform the Illumina DNA Prep automatically. Results obtained from 16 LP that were performed both manually and automatically showed comparable library DNA yields (median of 1.5-fold difference), similar assembly quality values, and 100% concordance on the final core genome multilocus sequence typing results. In addition, reproducibility of results was confirmed by re-processing eight of the 16 LPs using the automated workflow. With the automated workflow, the HOT was reduced to 25min compared to the 125min needed when performing eight LPs using the manual workflow. The turn-around time was 170 and 200min for the automated and manual workflow, respectively. In summary, the automated workflow on the flowbot ONE generates consistent results in terms of reliability and reproducibility, while significantly reducing HOT as compared to manual LP.

High levels of multidrug-resistant isolates of genetically similar Salmonella 1,4, [5],12:I:- from Brazil between 1983 and 2020.

Introduction. Salmonella 1,4, [5],12:i:- strains with different antimicrobial resistance profiles have been associated with foodborne disease outbreaks in several countries. In Brazil, S. 1,4, [5],12:i:- was identified as one of the most prevalent serovars in São Paulo State during 2004-2020.Gap Statement. However, few studies have characterized this serovar in Brazil.Aim. This study aimed to determine the antimicrobial resistance profiles of S. 1,4, [5],12:i:- strains isolated from different sources in Southeast Brazil and compare their genetic diversity.Methodology. We analysed 113 S. 1,4, [5],12:i:- strains isolated from humans (n=99), animals (n=7), food (n=5) and the environment (n=2) between 1983 and 2020. Susceptibility testing against 13 antimicrobials was performed using the disc diffusion method for all the strains. Plasmid resistance genes and mutations in the quinolone resistance-determining regions were identified in phenotypically fluoroquinolone-resistant strains. Molecular typing was performed using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) for all strains and multilocus sequence typing (MLST) for 40 selected strains.Results. Of the 113 strains, 54.87 % were resistant to at least one antimicrobial. The highest resistance rates were observed against ampicillin (51.33 %), nalidixic acid (39.82 %) and tetracycline (38.05 %). Additionally, 39 (34.51 %) strains were classified as multidrug-resistant (MDR). Nine fluoroquinolone-resistant strains exhibited the gyrA mutation (Ser96→Tyr96) and contained the qnrB gene. The 113 strains were grouped into two clusters using ERIC-PCR, and most of strains were present in one cluster, with a genetic similarity of ≥80 %. Finally, 40 strains were typed as ST19 using MLST.Conclusion. The prevalence of MDR strains is alarming because antimicrobial treatment against these strains may lead to therapeutic failure. Furthermore, the ERIC-PCR and MLST results suggested that most strains belonged to one main cluster. Thus, a prevalent subtype of Salmonella 1,4, [5],12:i:- strains has probably been circulating among different sources in São Paulo, Brazil, over decades.

Molecular confirmation of the systemic toxoplasmosis in cat.

Systemic toxoplasmosis with tissue-spread parasites occurring in intermediate hosts may also occur in immunocompromised cats (e.g., infected with FLV or FIV). To the best of our knowledge, no reports have been published on the detection and genotyping of T. gondii DNA in cats with extraintestinal toxoplasmosis in Poland. The article describes the case of the sudden death of 3 out of 4 cats in a cattery, and the detection and molecular characterization of T. gondii DNA detected in the tissues of one of the dead cats. Samples of brain, lungs, heart, and liver of the cat that died suddenly were examined for the presence of T. gondii DNA (B1 gene) by nested PCR and real-time PCR. DNA positive samples were also genotyped at 12 genetic markers using multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP) and multilocus sequence typing (MLST). A total of 9 out of the 20 DNA samples were successfully amplified with nested and/or Real-time PCR. DNA from 3 out of 5 types of tested samples were genotyped (brain, heart and muscle). Mn-PCR-RFLP and MLST results revealed type II (and II/III at SAG1) alleles at almost all loci, except a clonal type I allele at the APICO locus. This profile corresponds to the ToxoDB#3 genotype, commonly identified amongst cats in Central Europe. To the best of our knowledge, this is the first study describing the genetic characteristics of T. gondii population determined in a cat in Poland. These data confirm the importance of this host as a reservoir for this pathogen, and demonstrate the genotypic variation of this parasite. Veterinarians should take into account that cats may develop disseminated toxoplasmosis, and that it is a systemic disease which may lead to the death of the cat, and to transmission of the pathogen to other domestic animals and to humans.

Study on the features of Clostridioides difficile infection among diarrhea patients in Kunming from 2018 to 2020

Objective: We analyze the characteristics of Clostridioides difficile (C. difficile) infection among diarrhea patients in Kunming from 2018 to 2020 and provide evidence for follow-up surveillance and prevention. Methods: A total of 388 fecal samples of diarrhea patients from four sentinel hospitals in Yunnan Province from 2018 to 2020 were collected. Real-time quantitative PCR was used to detect the fecal toxin genes of C. difficile. The positive fecal samples isolated the bacteria, and isolates were identified by mass spectrometry. The genomic DNA of the strains was extracted for multi-locus sequence typing (MLST). The fecal toxin, strain isolation, and clinical patient characteristics, including co-infection with other pathogens, were analyzed. Results: Among the 388 fecal samples, 47 samples with positive reference genes of C. difficile were positive, with a total positive rate of 12.11%. There were 4 (8.51%) non-toxigenic and 43 (91.49%) toxigenic ones. A total of 18 strains C. difficile were isolated from 47 positive specimens, and the isolation rate of positive specimens was 38.30%. Among them, 14 strains were positive for tcdA, tcdB, tcdC, tcdR, and tcdE. All 18 strains of C. difficile were negative for binary toxins. The MLST results showed 10 sequence types (ST), including 5 strains of ST37, accounting for 27.78%; 2 strains of ST129, ST3, ST54, and ST2, respectively; and 1 strain of ST35, ST532, ST48, ST27, and ST39, respectively. Fecal toxin gene positive (tcdB+) results were statistically associated with the patient's age group and with or without fever before the visit; positive isolates were only statistically associated with the patient's age group. In addition, some C. difficile patients have co-infection with other diarrhea-related viruses. Conclusions: The infection of C. difficile in diarrhea patients in Kunming is mostly toxigenic strains, and the high diversity of strains was identified using the MLST method. Therefore, the surveillance and prevention of C. difficile should be strengthened.

First report of Staphylococcus pseudintermedius ST71-SCCmec III and ST45-ΨSCCmec57395 from canine pyoderma in Argentina

Staphylococcus pseudintermedius is an opportunistic pathogen commonly associated with skin infections in dogs. Twenty-three methicillin-resistant S. pseudintermedius (MRSP) isolated in Argentina from dogs with pyoderma were analyzed using whole genome sequencing (WGS) and classified into sequence types (ST) by multilocus sequence typing (MLST) and staphylococcal chromosome cassette mec (SCCmec) types.Based on the WGS analysis, MLST, and SCCmec type results, we report for the first time in Argentina two MRSP strains, one each, belonging to ST71-SCCmec III and ST45-ΨSCCmec57395 from dogs with pyoderma. We also identified seven isolates with ST339, which had been previously reported in only two isolates in Argentina. Additionally, we identified ten MRSP isolates harboring variants of the SCCmec V found in S. aureus, seven SCCmec V (5C2&5) with two ccrC1 recombinases, and three SCCmec V (5C2) with one ccrC1 recombinase.Our findings provide important insights into the evolution and geographic spread of these hypervirulent dominant clones that threaten the health of our companion animals and represent a significant risk for zoonotic infections.

Antimicrobial resistance and genomic characterization of Salmonella enterica serovar Senftenberg isolates in production animals from the United States.

In the USA, Salmonella enterica subspecies enterica serovar Senftenberg is among the top five serovars isolated from food and the top 11 serovars isolated from clinically ill animals. Human infections are associated with exposure to farm environments or contaminated food. The objective of this study was to characterize S. Senftenberg isolates from production animals by analyzing phenotypic antimicrobial resistance profiles, genomic features and phylogeny. Salmonella Senftenberg isolates (n = 94) from 20 US states were selected from NVSL submissions (2014-2017), tested against 14 antimicrobial drugs, and resistance phenotypes determined. Resistance genotypes were determined using whole genome sequencing analysis with AMRFinder and the NCBI and ResFinder databases with ABRicate. Plasmids were detected using PlasmidFinder. Integrons were detected using IntFinder and manual alignment with reference genes. Multilocus-sequence-typing (MLST) was determined using ABRicate with PubMLST database, and phylogeny was determined using vSNP. Among 94 isolates, 60.6% were resistant to at least one antimicrobial and 39.4% showed multidrug resistance. The most prevalent resistance findings were for streptomycin (44.7%), tetracycline (42.6%), ampicillin (36.2%) and sulfisoxazole (32.9%). The most commonly found antimicrobial resistance genes were aac(6')-Iaa (100%), aph(3″)-Ib and aph(6)-Id (29.8%) for aminoglycosides, followed by bla TEM-1 (26.6%) for penicillins, sul1 (25.5%) and sul2 (23.4%) for sulfonamides and tetA (23.4%) for tetracyclines. Quinolone-resistant isolates presented mutations in gyrA and/or parC genes. Class 1 integrons were found in 37 isolates. Thirty-six plasmid types were identified among 77.7% of the isolates. Phylogenetic analysis identified two distinct lineages of S. Senftenberg that correlated with the MLST results. Isolates were classified into two distinct sequence types (ST): ST14 (97.9%) and ST 185 (2.1%). The diversity of this serotype suggests multiple introductions into animal populations from outside sources. This study provided antimicrobial susceptibility and genomic characteristics of S. Senftenberg clinical isolates from production animals in the USA during 2014 to 2017. This study will serve as a base for future studies focused on the phenotypic and molecular antimicrobial characterization of S. Senftenberg isolates in animals. Monitoring of antimicrobial resistance to detect emergence of multidrug-resistant strains is critical.

Discovery of Rickettsia spp. in mosquitoes collected in Georgia by metagenomics analysis and molecular characterization.

Arthropods have a broad and expanding worldwide presence and can transmit a variety of viral, bacterial, and parasite pathogens. A number of Rickettsia and Orientia species associated with ticks, fleas, lice, and mites have been detected in, or isolated from, patients with febrile illness and/or animal reservoirs throughout the world. Mosquitoes are not currently considered vectors for Rickettsia spp. pathogens to humans or to animals. In this study, we conducted a random metagenome next-generation sequencing (NGS) of 475 pools of Aedes, Culex, and Culiseta species of mosquitoes collected in Georgia from 2018 to 2019, identifying rickettsial gene sequences in 33 pools of mosquitoes. We further confirmed the findings of the Rickettsia by genus-specific quantitative PCR (qPCR) and multi-locus sequence typing (MLST). The NGS and MLST results indicate that Rickettsia spp. are closely related to Rickettsia bellii, which is not known to be pathogenic in humans. The results, together with other reports of Rickettsia spp. in mosquitoes and the susceptibility and transmissibility experiments, suggest that mosquitoes may play a role in the transmission cycle of Rickettsia spp.

Penicillium vietnamense sp. nov., the First Novel Marine Fungi Species Described from Vietnam with a Unique Conidiophore Structure and Molecular Phylogeny of Penicillium Section Charlesia

Penicillium vietnamense sp. nov. was isolated from Nha Trang Bay, Vietnam in June 2017. It is phylogenetically distinct from the sister species of Penicillium section Charlesia series Indica based on multi-locus sequence typing results using internal transcribed spacer, large subunit ribosomal RNA, β-tubulin, calmodulin, and RNA polymerase II second largest subunit regions. It showed strong growth on Czapek yeast autolysate agar at 37 °C, a strong acid production on Creatine sucrose agar, and produced short stipes, small vesicles, and subglobose to globose conidia delicately roughened with very short ridges. As the first novel marine fungi species described from Vietnam and discovered in a unique environment, the data could be significant for understanding the taxonomy and geographical distribution of marine fungi in tropical coastal systems such as Vietnam.

Handling practice as a critical point influencing the transmission route of campylobacter throughout a commercial restaurant kitchen in China

Foodborne illness constitutes a substantial health and economic burden on society, and most cases are believed to be due to mishandling of food practices. This study aimed to establish the relationship between food handling practices and Campylobacter contamination in commercial restaurant kitchens. The handling practices of chefs during chicken preparation were observed and recorded. Thirty-three chicken samples and 39 environmental samples were collected to detect the presence of Campylobacter. In total, 75 Campylobacter jejuni isolates and 14 Campylobacter coli isolates were identified. All C. jejuni isolates were characterized by whole genome sequencing (WGS) and multilocus sequence typing (MLST). Campylobacter was positive on chickens with a ratio from 50% to 66.67%. In washing and cutting areas, the Campylobacter positive ratio varied from 33.33% to 100%. Hands, boards, and countertop showed the highest loads of bacterial counts. WGS and MLST results indicated that all isolates were grouped into 17 genetic clusters. Isolates belonging to the same sequence type were highly homologous and our findings indicated that C. jejuni was likely transmitted from chicken to other areas within the kitchen. Improper handling practices (i.e., using cloths, washing hands without soap, cleaning boards with knives) caused the surfaces contamination of Campylobacter. Our results indicated that handling practices were a critical point of Campylobacter cross contamination in commercial kitchen premises. Therefore, more attention should be focused on proper handling practices in commercial kitchens to reduce the risk of foodborne illness.

High Prevalence of Antimicrobial Resistance and Integron Gene Cassettes in Multi-Drug-Resistant Klebsiella pneumoniae Isolates From Captive Giant Pandas (Ailuropoda melanoleuca).

Multi-drug-resistant Klebsiella pneumoniae (MDR K. pneumonia) is increasingly being reported with corresponding increase in morbidity and mortality all over the world. However, limited information is available concerning MDR K. pneumonia in giant pandas. The objective of this study was to grasp the drug resistance profile of MDR K. pneumonia isolated from giant pandas. A total of 182 K. pneumoniae isolates were collected from fresh feces of 94 captive giant pandas of different ages and sex and separated by season. We performed a standard disk diffusion antimicrobial susceptibility test with the isolates and further evaluated the antibiotic resistance genes (ARGs) of multi-drug-resistant strains by high-throughput quantitative PCR. In addition, we then analyzed mobile genetic elements (MGEs), integron gene cassettes, and the multi-locus sequence typing of multi-drug-resistant strains by PCR. Antimicrobial susceptibility testing results demonstrated that a total of 30 (16.5%) K. pneumoniae isolates showed multiple drug resistances. The thirty MDR K. pneumonia isolates were mainly resistant to amoxicillin (100.0%), doxycycline (86.7%), chloramphenicol (60.0%), compound trimethoprim (60.0%) and trimethoprim (56.7%). Fifty different types of antibiotic resistance genes were found, which included a total of 671 antibiotic resistance genes, in the 30 multi-drug-resistant isolates. The top ten resistance genes were: vanTC-02, aacC, blaCTX-M-04, blaSHV-01, blaSHV-02, ampC-04, blaOXY, tetD, blaTEM and tetA-02. Thirteen mobile genetic elements were detected, of which IS26 (96.67%) and intI1 (96.67%) had the highest frequency. The thirty MDR K. pneumonia isolates were negative for the traA, traF, tnsA, IS1133, ISpa7, ISkpn6, intI2 and intI3 genes. Moreover, a further investigation of integrons revealed that two types of specific gene cassettes (dfrA12 + orfF + aadA2 and dfrA12 + orfF) were identified in class 1 integrons. Multi-locus sequence typing results showed that 22 STs in the thirty MDR K. pneumonia isolates were identified, the main type was ST37 (5/30). Our results illustrate that effective surveillance and strict biosecurity strategies should be taken to prevent the spread of multi-drug-resistant bacteria, and monitor the emergence of mobile genetic elements and integrons.

Genetic diversity of Listeria monocytogenes isolates from farm products in Shanghai, China

Abstract Farm products are considered important vehicles for the transmission of Listeria monocytogenes (L. monocytogenes). The typing of L. monocytogenes from farm products contributes to the surveillance and source tracing of the pathogen. In this study, 77 L. monocytogenes strains from seven farm product categories in Shanghai were analyzed by serological typing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and whole-genome nucleotide polymorphism (wgSNP). The results showed that the 77 isolates were classified into four serovars (1/2a, 1/2c, 1/2b, and 4b), and of these, 1/2a (n=47) was the most prevalent. Seventeen sequence types (STs) were generated by MLST with two novel STs (ST1402 and ST1403), and 20 of 77 L. monocytogenes isolates had high genetic identity with previously documented outbreak isolates according to the minimum spanning tree from the MLST results. Moreover, 34 PFGE patterns (PF1–PF34) were differentiated, and based on a similarity value higher than 80% by the unweighted pair group method dendrogram, the discriminatory index was relatively low (equal to 0.775). Furthermore, 14 isolates were chosen and further analyzed by wgSNP based on the previous typing results, which demonstrated that wgSNP and MLST yielded mostly consistent typing results but higher resolution than PFGE. In conclusion, 77 L. monocytogenes isolates from farm products collected in nine districts in Shanghai were highly genetically diverse, and 20 of these isolates had high relatedness with previously documented outbreak strains worldwide. The results indicate a possible cross-contamination risk of L. monocytogenes and a potential public health concern resulting from farm products during the supply chain in Shanghai, China.

Investigation of possible transmission of a susceptible microorganism through a contaminated duodenoscope; a case report

BackgroundDespite compliance to extensive reprocessing protocols, duodenoscopes have been linked to outbreaks of susceptible and multi-drug resistant organisms (MDRO) due to persistent duodenoscope contamination. Duodenoscope-associated infections (DAIs) based on transmission of susceptible microorganisms are likely to be underreported due to detection bias.Case presentationWe describe the retrospective detection of a DAI case caused by a susceptible microorganism which at the time of clinical infection was not recognized as such. During 2017 and 2018, duodenoscopes were cultured on a daily basis due to research activities. While analyzing this data, it was found that a duodenoscope had been contaminated with Enterobacter cloacae complex over a period of 3 months. We checked whether patients treated with this duodenoscope had developed infections and found one patient with an E. cloacae cholangitis 3 months after the ERCP (Endoscopic retrograde cholangiopancreaticography) procedure. The isolates on the duodenoscope and in the patients’ blood culture were indistinguishable by amplified fragment length polymorphism (AFLP). By classical multi-locus sequence typing (MLST), both strains were of the same (but novel) sequence type. Application of whole genome MLST showed 93 (out of 3757) allelic differences.ConclusionThis case report describes a plausible link between a contaminated duodenoscope and a patient infection with E. cloacae. Transmission of susceptible E. cloacae was highly suspected from AFLP and MLST results; by WGS, 93 allelic differences were found which proves closely related strains. This report shows that DAIs by susceptible microorganisms can be easily missed and therefore its true prevalence remains underscored.

Co-occurrence of Carbapenemase-encoding Genes Among Klebsiella pneumoniae Clinical Isolates: Positive Relationship of bla NDM and bla SIM with Imipenem Resistance

Background: Carbapenem-resistant Klebsiella pneumoniae (CR-KP), known as a significant public health threat, is the most common causative agent of nosocomial and community-acquired infections. Objectives: This study aimed to evaluate resistance to carbapenems and determine the prevalence of carbapenemase genes and multilocus sequence typing (MLST) of K. pneumoniae clinical isolates. Methods: One-hundred K. pneumoniae isolates were evaluated. The minimum inhibitory concentrations (MIC) of imipenem and meropenem were assessed by the broth microdilution method. Multiplex-polymerase chain reaction (PCR) was applied to detect 11 carbapenemase-encoding genes belonging to different classes. The alleles and sequence types (ST) of three isolates were identified by MLST. Results: The MIC of carbapenems for the isolates ranged from 0.062 to 32 µg/mL. Overall, resistance rates to imipenem and meropenem were reported 11% and 34%, respectively. The bla IMP gene was the most abundant (78.4%), followed by bla OXA-48 (48.6%), bla GIM (27%), bla KPC (27%), bla SIM (21.6%), bla BIC (21.6%), bla NDM (16.2%), bla AIM (16.2%), bla VIM (16.2%), bla DIM (8.1%), and bla SPM (8.1%). The co-existence of carbapenemase genes was observed in 81.8% of the isolates. A positive relationship was found between the presence of bla NDM and bla SIM and resistance to imipenem. Multilocus sequence typing results showed three different sequence types, including ST14, ST5188, and ST1861. Conclusions: This study revealed a high prevalence of CR-KP isolates that suggests a high risk of horizontal gene transfer and potential to spread resistance among other strains. Since STs are reported for the first time in Iran, they can be considered as emerging strains.

Phylogenomic analysis for Campylobacter fetus ocurring in Argentina.

Background and Aim:Campylobacter fetus is one of the most important pathogens that severely affects livestock industry worldwide. C. fetus mediated bovine genital campylobacteriosis infection in cattle has been associated with significant economic losses in livestock production in the Pampas region, the most productive area of Argentina. The present study aimed to establish the genomic relationships between C. fetus strains, isolated from the Pampas region, at local and global levels. The study also explored the utility of multi-locus sequence typing (MLST) as a typing technique for C. fetus.Materials and Methods:For pangenome and phylogenetic analysis, whole genome sequences for 34 C. fetus strains, isolated from cattle in Argentina were downloaded from GenBank. A local maximum likelihood (ML) tree was constructed and linked to a Microreact project. In silico analysis based on MLST was used to obtain information regarding sequence type (ST) for each strain. For global phylogenetic analysis, a core genome ML-tree was constructed using genomic dataset for 265 C. fetus strains, isolated from various sources obtained from 20 countries.Results:The local core genome phylogenetic tree analysis described the presence of two major clusters (A and B) and one minor cluster (C). The occurrence of 82% of the strains in these three clusters suggested a clonal population structure for C. fetus. The MLST analysis for the local strains revealed that 31 strains were ST4 type and one strain was ST5 type. In addition, a new variant was identified that was assigned a novel ST, ST70. In the present case, ST4 was homogenously distributed across all the regions and clusters. The global analysis showed that most of the local strains clustered in the phylogenetic groups that comprised exclusively of the strains isolated from Argentina. Interestingly, three strains showed a close genetic relationship with bovine strains obtained from Uruguay and Brazil. The ST5 strain grouped in a distant cluster, with strains obtained from different sources from various geographic locations worldwide. Two local strains clustered in a phylogenetic group comprising intercontinental Campylobacter fetus venerealis strains.Conclusion:The results of the study suggested active movement of animals, probably due to economic trade between different regions of the country as well as with neighboring countries. MLST results were partially concordant with phylogenetic analysis. Thus, this method did not qualify as a reliable subtyping method to assess C. fetus diversity in Argentina. The present study provided a basic platform to conduct future research on C. fetus, both at local and international levels.

Tracing Clostridium perfringens strains along the chicken production chain from farm to slaughter by multilocus sequence typing.

The current study is undertaken to characterize the prevalence, genotypes distribution, antibiotic resistance and genetic diversity of Clostridium perfringens (C.perfringens) collected from different stages of a chicken production chain. In total, 579 samples from a broiler farm and 378 samples from the slaughterhouse were collected from a large-scale rearing and slaughter one-stop enterprise in Weifang, China, between June and July 2019, of which 30.40% of the samples from farm and 54.50% of samples from slaughterhouse were determined to be positive for C.perfringens, respectively. The contamination of chicken products was relatively serious, with the total positive rate of carcasses at 59.73%; the positive rate of carcass samples was the highest in the evisceration process, which might be the critical point of C.perfringens contamination. A total of 476 isolates of C.perfringens were recovered; and 99.58% of recovered isolates were identified as type A, with the remaining isolates being type G. Antimicrobial susceptibility testing revealed that 97.15% of the isolates showed multiple antibiotic resistance and 67.89% of them were resistant to at least five classes of commonly used antibiotics. Multilocus sequence typing results of 91 representative isolates showed that the isolates can be divided into 74 sequences types (STs); 40.66% of the isolates can be included into seven clonal complexes (CCs). Although most of the isolates were classified as type A, considerable genetic diversity was observed, with the Simpson's diversity index of ST up to 0.9902. Some isolates from farm stage and slaughter stage were distributed in the same ST or CC, indicating that chicken products may be contaminated by the same ST or CC of C.perfringens originated from the farm stage. The high contamination rates of chicken products and the widespread multiple antibiotic resistance of isolates indicated potential public health risks, control measures at rearing and slaughtering stage should be considered to reduce this risk.

Characterization of Extended-Spectrum β-Lactamase-Producing and AmpC β-Lactamase-Producing Enterobacterales Isolated from Companion Animals in Korea.

The emergence of extended-spectrum cephalosporin (ESC)-resistant Gram-negative bacteria is of great concern in both human and veterinary medicine. The aim of this study was to investigate ESC-resistant bacterial isolates from companion animals in South Korea between 2017 and 2019. Isolates with ESC resistance genes, which were identified by PCR, were assessed for genetic relatedness by multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). In total, 91 ESC-resistant Escherichia coli, Klebsiella spp., Serratia spp., and Enterobacter cloacae isolates harbored the blaTEM gene. Among other ESC resistance genes, blaCTX-M-15, blaCIT, and blaCTX-M-55 were predominantly detected in E. coli isolates, whereas blaSHV and blaDHA were more frequently detected in Klebsiella pneumoniae isolates. In addition, all blaEBC-positive isolates were classified as E. cloacae. From the MLST results, blaCTX-M-9-carrying ST131, blaCIT-carrying ST405, and blaCTX-M-1-carrying ST3285 strains were dominant among E. coli isolates. ST273 and ST275 strains harboring blaSHV were frequently detected in K. pneumoniae isolates. Various sequence types were obtained in E. cloacae and Klebsiella oxytoca isolates. All isolates demonstrated unique PFGE profiles (<57–98% similarity) and were unlikely to be derived from a single clone. The present study reveals the presence and wide genetic distribution of ESC-resistant bacterial species in South Korean companion animals.

Carbapenem and Colistin Resistant Klebsiella Pneumoniae ST14 and ST2096 Dominated in Two Hospitals in Turkey

The determination of clonal interactions between microorganisms is very important in epidemiological studies. In the present study, we aimed to evaluate the resistance mechanisms and genetic relationships of carbapenem and colistin resistant Klebsiella pneumoniae (K. pneumoniae) strains isolated from inpatients at two university hospitals in Turkey. A total of 38 K. pneumoniae clinical isolates were included in the study. Identification of isolates was confirmed by 16S rRNA sequencing. Antimicrobial susceptibility testing was performed with VITEK-2 system (bio-Mérieux, France). Modified Hodge test was used for the detection of carbapenemase activity in isolates. Carbapenem resistance genes (blaOXA-48, blaNDM, blaKPC, blaIMP, blaVIM) and colistin resistance genes (mcr-1, mcr-2 and mcr-3) were investigated by PCR. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and multilocus sequence typing (MLST) analysis were used to determine the genetic relatedness among the isolates. We detected that 58% of isolates were positive for only blaOXA-48, 5% were only positive for blaNDM, and 34% were positive for both blaOXA-48 and blaNDM. blaKPC, blaIMP, blaVIM, mcr-1, mcr-2, and mcr-3 were not detected among the isolates. Only one carbapenem resistant isolate was negative for the carbapenemase genes tested. A total of nine profiles were found by ERIC-PCR, and MLST results showed seven different sequence types-ST14, ST16, ST79, ST101, ST1543, ST2096, and ST2832. The seven STs were grouped by PHYLOVIZ Online into four clonal complexes. The most common ST was ST14 (81%) in Center 1 and ST2096 (94%) in Center 2. We determined MLST types of carbapenem and colistin resistant K. pneumoniae isolates from two different centers. Although the most common ST types were different in these centers, both ST types were clustered in CC14. To the best of our knowledge this is the first report of ST14 and ST2096 outbreaks in Turkey.

Benchmarking Long-Read Assemblers for Genomic Analyses of Bacterial Pathogens Using Oxford Nanopore Sequencing.

Oxford Nanopore sequencing can be used to achieve complete bacterial genomes. However, the error rates of Oxford Nanopore long reads are greater compared to Illumina short reads. Long-read assemblers using a variety of assembly algorithms have been developed to overcome this deficiency, which have not been benchmarked for genomic analyses of bacterial pathogens using Oxford Nanopore long reads. In this study, long-read assemblers, namely Canu, Flye, Miniasm/Racon, Raven, Redbean, and Shasta, were thus benchmarked using Oxford Nanopore long reads of bacterial pathogens. Ten species were tested for mediocre- and low-quality simulated reads, and 10 species were tested for real reads. Raven was the most robust assembler, obtaining complete and accurate genomes. All Miniasm/Racon and Raven assemblies of mediocre-quality reads provided accurate antimicrobial resistance (AMR) profiles, while the Raven assembly of Klebsiella variicola with low-quality reads was the only assembly with an accurate AMR profile among all assemblers and species. All assemblers functioned well for predicting virulence genes using mediocre-quality and real reads, whereas only the Raven assemblies of low-quality reads had accurate numbers of virulence genes. Regarding multilocus sequence typing (MLST), Miniasm/Racon was the most effective assembler for mediocre-quality reads, while only the Raven assemblies of Escherichia coli O157:H7 and K. variicola with low-quality reads showed positive MLST results. Miniasm/Racon and Raven were the best performers for MLST using real reads. The Miniasm/Racon and Raven assemblies showed accurate phylogenetic inference. For the pan-genome analyses, Raven was the strongest assembler for simulated reads, whereas Miniasm/Racon and Raven performed the best for real reads. Overall, the most robust and accurate assembler was Raven, closely followed by Miniasm/Racon.