Multilocus Sequence Typing of Non-JP2 Serotype b Aggregatibacter actinomycetemcomitans Strains of Ghanaian and Swedish Origin

Objective and MethodsThe Gram-negative bacterium, Aggregatibacter actinomycetemcomitans is associated with periodontitis affecting young individuals. The geographic dissemination of the highly leukotoxic JP2 genotype of serotype b of this species was previously studied by multilocus sequence typing (MLST). Here, we have used MLST to genetically characterize non-JP2 genotype strains of serotype b, isolated from individuals living in Ghana (n=41), and in Sweden (n=13), respectively.ResultsThe MLST analysis revealed a total of nine sequence types (ST). Both Ghanaian and Swedish isolates were distributed in ST 1-3. ST 5 and 6 were only identified among the Ghanaian strains, whereas ST 4, 7, 8 and 9 were uniquely represented among the Swedish strains. Previously, we characterized these non-JP2 genotype strains of A. actinomycetemcomitans serotype b by arbitrarily-primed (AP)-PCR, which distributed them into three groups, AP-PCR type 1, 2, and 3, respectively. AP-PCR type 1 strains are generally highly leukotoxic, and are associated with progression of periodontal attachment loss. As AP-PCR type 1 includes both JP2 genotype strains and a proportion of non-JP2 genotype strains of serotype b, a straightforward diagnostic procedure has been sought. This has revealed a gene, cagE, which appears to be conserved only in this AP-PCR type. According to our results, MLST was not a highly discriminatory method to identify AP-PCR type 1, as strains of this AP-PCR type could be found within three different ST: ST 2, ST 3 and ST 8.ConclusionAccording to MLST, a geographic dissemination of non-JP2 genotype A. actinomycetemcomitans serotype b appears to exist. However, aiming to identify carriers of AP-PCR type 1, non-JP2 genotype serotype b, PCR with cagE-specific primers is likely the most efficient diagnostic procedure known today.

Identification and genetic characterization of Pseudomonas syringae pv. syringae from sweet cherry in Turkey

Pseudomonas syringae pv. syringae (Pss), which causes bacterial canker, is the most polyphagous bacterium in the P. syringae complex due to its broad host range. This pathogen is considered the major bacterial disease in cherry orchards. In this study, several samples were collected from infected sweet cherry trees in different locations of the Marmara region in Turkey between 2016-2018. Sixty-three isolates were identified as Pss by pathogenicity, LOPAT, GATTa, and MALDI-TOF MS tests. Total genomic DNA was extracted to confirm identity, followed by PCR amplification of syrB and cfl genes. Out of 63 isolates, 12 were randomly selected for Repetitive Element Sequence-based PCR (rep-PCR) and Multilocus Sequence Typing (MLST) analysis to gain insight into the relationships of those isolates. The cluster analysis of rep-PCR (ERIC-, REP- and BOX-PCR) could classify the isolates into two distinct clusters. Phylogenetic analysis was carried out to obtain the relation between isolates and the location.The MLST analysis of gyrB, rpoDp, rpoDs, and gltA genes allowed a clear allocation of the isolates into two separate main clusters. The relationship among the isolates were also evaluated by constructing a genealogical median-joining network (MJN). The isolates from six locations produced 11 haplotypes that were illustrated in the MJN. The results of this study proved that location could not be an indicator for showing the genetic diversity of Pss from cherry orchards. As the genetic variability of Pseudomonads has been demonstrated, the current study also showed high diversity among different isolates even within the populations. While more research is recommended, the results of this study contributed to a better understanding of the Pss evolutionary progress and genetic diversity of sweet cherry isolates.

740. Elucidation of the Mode of Clostridioides difficile Transmission Based on One Health Approach

Background Community-onset Clostridioides difficile (C. difficile) infection (CACDI) has been increasing in recent years. To explore the transmission route of CACDI, we performed the whole-genome sequencing of C. difficile isolated from CACDI patients and compared it to the isolates from livestock, companion animals, and soil. Methods From October 2020 until April 2021, fecal specimens of cattle, poultry, swine, felines, canines, CACDI patients, their families, and soil from the CACDI patients' living environment were applied for isolation of C.difficile. Whole-genome sequencing of C. difficile was performed on the MiSeq system (Illumina). Using the draft genome obtained from these analyses, the house-keeping gene (tpi), MLST, toxin genes (tcdA, tcdB, cdtA, cdtB), and resistance genes (gyrA, gyrB, rpoA, rpoB, rpoC) were comprehensively analyzed. Results As of March 31, 2021, 275 specimens were collected. Forty-five fecal specimens of companion animal origin (23 feline and 22 canines) were collected and the positive rate of C.difficile was 28.9% (2 felines, 11 canines). In MLST analysis, ST 15 (4 strains), ST 26 (2 strains), ST 42, ST 3, ST 28, ST 100, and ST 185 were detected in canines, and ST 203 and ST 297 strains were detected in felines. Samples of livestock origin were collected from 135 cattle, 41 poultries, and 20 swine. The detection rate in cattle was 11%, toxin-gene positivity was 60%. MLST analysis of 9 strains revealed ST 11 (5 strains), ST 2, ST 15, ST 58, and ST 101. No isolates were found from poultry or swine. Patient-derived strains of CACDI were collected from 14 patients at 2 sites. MLST analysis revealed ST42, ST37, ST100, and ST203(two isolates, respectively), ST 224, ST 81, ST 28, and ST 47. 2 isolates were unclassifiable. One case was a healthy 1-year-old girl, whose family revealed no isolation of C.difficile. Impressively, the soil in the parks (A and B) related to the child detected C.difficile from 4/4 samples (toxin-gene positivity; 75%) in Park A and 1/4 samples (toxin-gene positive) in Park B. MLST analysis demonstrated ST 42, the same as that in the affected child and core-genome single-nucleotide polymorphisms(SNPs) analysis suggested closely related strain. Conclusion Our results suggest one health approach is fundamental to prevent the transmission of C.difficile. Disclosures All Authors: No reported disclosures

Genetic Diversity and Population Structure of the Xanthomonas campestris pv. campestris Strains Affecting Cabbages in China Revealed by MLST and Rep-PCR Based Genotyping

<i>Xanthomonas campestris</i> pv. <i>campestris</i> (<i>Xcc</i>) is the causal agent of black rot for cruciferous vegetables worldwide, especially for the cole crops such as cabbage and cauliflower. Due to the lack of resistant cabbage cultivars, black rot has brought about considerable yield losses in recent years in China. Understanding of the pathogen features is a key step for disease prevention, however, the pathogen diversity, population structure, and virulence are largely unknown. In this study, we studied 50 <i>Xcc</i> strains including 39 <i>Xcc</i> isolates collected from cabbage in 20 regions across China, using multilocus sequence genotyping (MLST), repetitive DNA sequence-based PCR (rep-PCR), and pathogenicity tests. For MLST analysis, a total of 12 allelic profiles (AP) were generated, among which the largest AP was AP1 containing 32 strains. Further cluster analysis of rep-PCR divided all strains into 14 DNA groups, with the largest group DNA I comprising of 34 strains, most of which also belonged to AP1. Inoculation tests showed that the representative <i>Xcc</i> strains collected from diverse regions performed differential virulence against three brassica hosts compared with races 1 and 4. Interestingly, these results indicated that AP1/DNA I was not only the main pathotype in China, but also a novel group that differed from the previously reported type races in both genotype and virulence. To our knowledge, this is the first extensive genetic diversity survey for <i>Xcc</i> strains in China, which provides evidence for cabbage resistance breeding and opens the gate for further cabbage-<i>Xcc</i> interaction studies.

Serotyping, MLST, and Core Genome MLST Analysis of Salmonella enterica From Different Sources in China During 2004–2019

Salmonella enterica (S. enterica) is an important foodborne pathogen, causing food poisoning and human infection, and critically threatening food safety and public health. Salmonella typing is essential for bacterial identification, tracing, epidemiological investigation, and monitoring. Serotyping and multilocus sequence typing (MLST) analysis are standard bacterial typing methods despite the low resolution. Core genome MLST (cgMLST) is a high-resolution molecular typing method based on whole genomic sequencing for accurate bacterial tracing. We investigated 250 S. enterica isolates from poultry, livestock, food, and human sources in nine provinces of China from 2004 to 2019 using serotyping, MLST, and cgMLST analysis. All S. enterica isolates were divided into 36 serovars using slide agglutination. The major serovars in order were Enteritidis (31 isolates), Typhimurium (29 isolates), Mbandaka (23 isolates), and Indiana (22 isolates). All strains were assigned into 43 sequence types (STs) by MLST. Among them, ST11 (31 isolates) was the primary ST. Besides this, a novel ST, ST8016, was identified, and it was different from ST40 by position 317 C → T in dnaN. Furthermore, these 250 isolates were grouped into 185 cgMLST sequence types (cgSTs) by cgMLST. The major cgST was cgST235530 (11 isolates), and only three cgSTs contained isolates from human and other sources, indicating a possibility of cross-species infection. Phylogenetic analysis indicated that most of the same serovar strains were putatively homologous except Saintpaul and Derby due to their multilineage characteristics. In addition, serovar I 4,[5],12:i:- and Typhimurium isolates have similar genomic relatedness on the phylogenetic tree. In conclusion, we sorted out the phenotyping and genotyping diversity of S. enterica isolates in China during 2004–2019 and clarified the temporal and spatial distribution characteristics of Salmonella from different hosts in China in the recent 16 years. These results greatly supplement Salmonella strain resources, genetic information, and traceability typing data; facilitate the typing, traceability, identification, and genetic evolution analysis of Salmonella; and therefore, improve the level of analysis, monitoring, and controlling of foodborne microorganisms in China.

Molecular epidemiology of blaCTX-M gene-producing uropathogenic Escherichia coli among Iranian kidney transplant patients: clonal dissemination of CC131 and CC10

Background This study aimed to investigate the phylogenetic characterization and virulence traits of uropathogenic Escherichia coli (UPEC) isolated from kidney transplant patients (KTPs) as well as non-KTPs and analyze the clonal distribution of Extended spectrum β-lactamases (ESBLs)-producing UPEC containing blaCTX-M gene. Methods To this end, we determined virulence marker and the phylogenetic characterization of UPEC in non-KTPs (n = 65) and KTPs (n = 46). The non-KTPs were considered the control group of the study. Also, according to the Achtman scheme, we performed multilocus sequence typing to assess the relationship between twenty-nine of ESBL-producing isolates containing blaCTX-M gene. Results According to the results of PCR assay, the prevalence of virulence factor genes ranged from 0% (cnf and papG III) to 93.7% (fimH). Also, KTP isolates significantly differed from non-KTP isolates only in terms of the prevalence of pap GI elements. Moreover, the most frequent UPEC isolates were in phylogenetic group B2, followed by group D (18.9%), and group A (13.5%). Furthermore, except for phylogenetic group C, there was no significant correlation between phylogenetic distribution in KTPs and non-KTPs. Additionally, MLST analysis of blaCTX-M carrying isolates identified 18 unique sequence types (ST) the most common of which was ST131 (24.1%), followed by ST1193 (10.3%), while fourteen STs were detected only once. Conclusions The results further revealed significant differences between the UPEC isolates from KTPs and non-KTPs regarding the phylogroups C and PAI gene. Based on MLST analysis, we also observed a relatively high diversity in UPEC isolates obtained from KTPs and non-KTPs. Moreover, clonal complex (CC) 131 and ST131 were found to be the most prevalent clones and ST types, respectively. Besides, for the first time, ST8503 were reported in KTPs. These results suggested regular studies on characterization of UPEC isolates among KTPs.

Increasing frequency of OXA-48-producing Enterobacterales worldwide and activity of ceftazidime/avibactam, meropenem/vaborbactam and comparators against these isolates

Objectives To investigate the increase in the rates of OXA-48-like-producing isolates during 3 years of global surveillance. Methods Among 55?&gt;162 Enterobacterales isolates, 354 carbapenem-resistant isolates carried genes encoding OXA-48-like enzymes. Isolates were susceptibility tested for ceftazidime/avibactam and comparators by broth microdilution methods. Analysis of β-lactam resistance mechanisms and MLST was performed in silico using WGS data. Results OXA-48-like-producing isolates increased from 0.5% (94/18 656) in 2016 to 0.9% (169/18?&gt;808) in 2018. OXA-48 was the most common variant; isolates primarily were Klebsiella pneumoniae (318/354 isolates) from Europe and adjacent countries. MLST analysis revealed a diversity of STs, but K. pneumoniae belonging to ST395, ST23 and ST11 were observed most frequently. Thirty-nine isolates harboured MBLs and were resistant to most agents tested. The presence of blaCTX-M-15 (258 isolates), OmpK35 nonsense mutations (232) and OmpK36 alterations (316) was common among OXA-48 producers. Ceftazidime, cefepime and aztreonam susceptibility rates, when applying CLSI breakpoints, were 12%–15% lower for isolates carrying ESBLs alone and with either or both OmpK35 stop codons and OmpK36 alterations. Meropenem and, remarkably, meropenem/vaborbactam were affected by specific OmpK36 alterations when a deleterious mutation also was observed in OmpK35. These mechanisms caused a decrease of 12%–42% in the susceptibility rates for meropenem and meropenem/vaborbactam. Ceftazidime/avibactam susceptibility rates were &gt;98.9%, regardless of the presence of additional β-lactam resistance mechanisms. Conclusions Guidelines for the treatment of infections caused by OXA-48-producing isolates are scarce and, as the dissemination of these isolates continues, studies are needed to help physicians understand treatment options for these infections.

The Emergence and Molecular Characteristics of New Delhi Metallo β-Lactamase-Producing Escherichia coli From Ducks in Guangdong, China

This study aimed to determine the prevalence and transmission characteristics of New Delhi metallo β-lactamase (NDM)-producing Escherichia coli from ducks in Guangdong, China. In this study, a total of 28 NDM-producing E. coli isolates were recovered from 88 unduplicated diseased duck samples (31.8%) from veterinary clinics in Guangzhou, Foshan, Qingyuan, and Huizhou. Two variants, blaNDM−1 and blaNDM−5, were detected and the latter was present in 89.6% of the isolates (25/28). Multilocus sequence typing (MLST) analysis indicated that these E. coli isolates possessed six distinct STs, and ST156 was the most prevalent followed by ST648, ST746, ST354, ST10, and ST162. In addition, phylogenomic analysis found that two of the isolates that were recovered from a single sample possessed different genomes, and the blaNDM-carrying IncX3 plasmids may be horizontal transfer between E. coli isolates in the intestinal tracts of ducks. Whole-genome sequencing (WGS) analysis further revealed that blaNDM co-existed with other 25 types of antimicrobial resistance genes (ARGs), of which 16 ARGs were highly prevalent with detection rates &gt;50%, and a high incidence of coproducing blaNDM and mcr-1 E. coli isolates (22/88, 25.0%) was detected in ducks. This study underscores the importance of surveillance for blaNDM-harboring microbes in ducks.

Molecular Characterization of German Acinetobacter baumannii Isolates and Multilocus Sequence Typing (MLST) Analysis Based on WGS Reveals Novel STs

Acinetobacter baumannii (A. baumannii) is a major cause of severe nosocomial infections worldwide. The emergence of infections associated with A. baumannii poses a significant health risk in Germany. A. baumannii is part of the ACB complex and is difficult to distinguish from other species phenotypically, necessitating its reliable identification. The current study analyzed 89 A. baumannii strains from human and non-human origins by matrix-assisted laser desorption/ionization (MALDI–TOF) and PCR detection of intrinsic blaOXA-51-like carbapenemase, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and ISAba 1 genes. Whole-genome sequencing (WGS) was applied for species confirmation and strain type determination. Combining the molecular detection of the intrinsic blaOXA-51-like carbapenemase gene together with MALDI–TOF with a score value of >2.300 proved to be a suitable tool for A. baumannii identification. WGS data for all of the sequenced strains confirmed the identity of all A. baumannii strains. The Pasteur scheme successfully assigned 79.7% of the strains into distinct STs, while the Oxford scheme succeeded in allocating only 42.7% of isolates. Multilocus sequence typing (MLST) analysis based on the Pasteur scheme identified 16 STs. ST/241 was the most prevalent in samples from non-human origin, whereas ST/2 was predominant in human samples. Furthermore, eight isolates of non-human origin were allocated to seven new STs (ST/1410, ST/1414, ST/1416, ST/1417, ST/1418, ST/1419, and ST/1421). Ten isolates from non-human origin could not be typed since new alleles were observed in the loci Pas_cpn60, Pas_rpoB, and Pas_gltA. MLST analysis based on the Pasteur scheme was more appropriate than the Oxford scheme for the current group of A. baumannii.