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Yu e Xue,1,* Dongmei Zhang,1,* Shuaixian Du,2,* Du Chen,3,* Shihan Liu,2 Tianfeng Peng,4 Chong Li,5 Jianchu Zhang,1 Xiaorong Wang1 1Department of Respiratory and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China; 2Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China; 3Department of Neurology, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, People’s Republic of China; 4Emergency Department, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China; 5Gastrointestinal Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiaorong Wang; Jianchu Zhang, Department of Respiratory and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Number 1277, Jie Fang Rode, Wuhan, Hubei, 430022, People’s Republic of China, Tel + 86-27-85726707, Email rong-100@163.com; zsn0928@163.comPurpose: To investigate the molecular epidemiology and risk factors of carbapenem-resistant Klebsiella pneumoniae (CRKP) infection.Patients and Methods: Patient’s clinical data and CRKP strains were collected from November 2017 to December 2018 at a tertiary hospital in Wuhan, China. The antimicrobial susceptibilities, carbapenem-resistant genes, multi-locus sequence typing (MLST), homologous analysis, and risk factors for CRKP were determined.Results: A total of 203 CRKP strains were isolated, and 98.5% (200/203) of patients were nosocomially infected. The mortality rate was 17.7% (36/203). All 203 strains were confirmed as carbapenemases -producing strains. The most predominant carbapenemase gene was blaIMP-4 (81.3%, 165/203), followed by blaKPC-2 (25.1%, 51/203) and blaNDM-1 (23.2%, 47/205). Of the 203 strains, 28 (13.8%) had both blaKPC-2 and blaIMP-4 genes, 23 (11.3%) had both blaIMP-4 and blaNDM-1 genes, 20 (9.9%) had blaKPC-2, blaIMP-4 and blaNDM-1 three genes. MLST analysis showed that there were 48 ST typologies (including 7 new STs), of which ST-11 was the most prevalent (59.6%, 121/203). Phylogenetic analysis showed that 203 CRKP isolates came from 7 clusters and exhibited a strong correlation with the isolation source. eBURST analyses indicated that CRKP isolates have undergone different evolutionary processes. Patients with ST-11 CRKP underwent more mechanical ventilation (50% vs 32.9%, P=0.020) and gastric catheterization (15.7% vs 6.1%, P=0.042) within 3 months before sample collection, and also had higher drug-resistance rate than non-ST-11 CRKP. Comparing with CSKP (carbapenem-sensitive Klebsiella pneumoniae), gastrointestinal disease (odds ratio [OR]=6.168, P=0.003), nosocomial infection (OR=5.573, P=0.012), antibiotic exposure (OR=4.131, P=0.004), urinary catheterization (OR=3.960, P=0.031) and venous/arterial catheterization (OR=2.738, P=0.026) within the preceding 3 months were independent risk factors for CRKP infection.Conclusion: The IMP-4 was the most predominant carbapenemase and blaIMP-4 bearing Klebsiella pneumoniae ST-11 was spreading in the hospital. Nosocomial infections, antibiotic exposure, and urinary and venous/arterial catheterization within 3 months were the risk factors for developing CRKP infection.Keywords: Klebsiella pneumoniae, carbapenemase, multi-locus sequence typing, MLST, ST-11, IMP-4

Transmission of pathogens commonly carried by domestic sheep and goats poses a serious threat to bighorn sheep (Ovis canadensis) populations. All-age pneumonia die-offs usually ensue, followed by asymptomatic carriage of Mycoplasma ovipneumoniae by some of the survivors. Lambs born into these chronically infected populations often succumb to pneumonia, but adults are usually healthy. Surprisingly, we found that introduction of a new genotype (strain) of M. ovipneumoniae into a chronically infected bighorn sheep population in the Hells Canyon region of Washington and Oregon was accompanied by adult morbidity (100%) and pneumonia-induced mortality (33%) similar to that reported in epizootics following exposure of naïve bighorn sheep. This suggests an immune mismatch occurred that led to ineffective cross-strain protection. To understand the broader context surrounding this event, we conducted a retrospective analysis of M. ovipneumoniae strains detected in 14 interconnected populations in Hells Canyon over nearly 3 decades. We used multi-locus sequence typing of DNA extracts from 123 upper respiratory tract and fresh, frozen, and formalin-fixed lung samples to identify 5 distinct strains of M. ovipneumoniae associated with all-age disease outbreaks between 1986 and 2014, a pattern consistent with repeated transmission events (spillover) from reservoir hosts. Phylogenetic analysis showed that the strain associated with the outbreak observed in this study was likely of domestic goat origin, whereas strains from other recent disease outbreaks probably originated in domestic sheep. Some strains persisted and spread across populations, whereas others faded out or were replaced. Lack of cross-strain immunity in the face of recurrent spillovers from reservoir hosts may account for a significant proportion of the disease outbreaks in bighorn sheep that continue to happen regularly despite a century of exposure to domestic sheep and goats. Strain-specific immunity could also complicate efforts to develop vaccines. The results of our study support existing management direction to prevent contacts that could lead to pathogen transmission from domestic small ruminants to wild sheep, even if the wild sheep have previously been exposed. Our data also show that under current management, spillover is continuing to occur, suggesting that enhanced efforts are indicated to avoid introducing new strains of M. ovipneumoniae into wild sheep populations. We recommend looking for new management approaches, such as clearing M. ovipneumoniae infection from domestic animal reservoirs in bighorn sheep range, and placing greater emphasis on existing strategies to elicit more active cooperation by the public and to increase vigilance on the part of resource managers.

Multi‐Locus Sequence Typing (MLST) is a key method for allocation of Sequence Types (STs) for bacterial isolates. Traditionally, this is performed by the Sanger sequencing method, which can be highly time‐consuming and laborious. In this study, we present NanoMLST, a high‐throughput MLST workflow using multiplex PCR, Oxford Nanopore Technologies Next‐Generation Sequencing, and the Krocus program for typing ESKAPE + E pathogens (Enterococcus faecium [E. faecium], Staphylococcus aureus, Klebsiella pneumoniae [K. pneumoniae], Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli). Bacterial isolates were obtained from the Hospital Universitario La Paz's Microbiology Department and the Centro Nacional de Microbiología. Primers that can be multiplexed in a single PCR reaction were designed for the seven housekeeping genes for each species. DNA was extracted from single colonies by heating at 95°C for 10 min, mechanical lysis at 4.20 m/s for 2 min, and then by the MagCore extraction system. Multiplex PCRs were then performed with the respective primer mixes for each species, and libraries were prepared for sequencing by ONT Flongle cells. The Krocus program was then used to determine the STs from the raw FastQ reads. STs for 221 isolates were obtained through this workflow with an average time of 12 h per 24 isolates. In line with local data, the K. pneumoniae and E. faecium isolates were relatively oligoclonal, while the rest were polyclonal. STs from representative isolates showed 100% concordance between Sanger sequencing and the proposed workflow. NanoMLST offers a fast, cheaper, and less labor‐intensive alternative for large‐scale MLST applications targeting clinically important pathogens. NanoMLST is a workflow suggested as a viable alternative to high‐throughput ST determination for ESKAPE + E pathogens using ONT next‐generation sequencing.

Background Clostridioides difficile infection (CDI) in patients with inflammatory bowel disease (IBD) is of increasing concern. This study aimed to investigate the molecular epidemiology and antimicrobial susceptibilities of toxigenic C. difficile isolated from IBD patients and to evaluate the risk factors for CDI in IBD population. Methods Loose or watery stools from IBD patients were tested for glutamate dehydrogenase, C. difficile toxins A&B and anaerobic culture. Toxigenic C. difficile isolates were characterized by multi‐locus sequence typing, ribotyping and antimicrobial susceptibility testing. Results The prevalence of CDI in IBD patients was 13.6% (43/317). The dominant sequence types (STs) were ST35 (20.9%), ST2 (18.6%) and ST37 (16.3%). The most common ribotypes (RTs) were RT 017 (18.6%), RT 012 (14.0%), and RT 220 (14.0%), whereas RT 027 and RT 078 were not detected in this study. All the isolates were susceptible to vancomycin and metronidazole. The multidrug resistance rate of C. difficile RT 017 was higher (p < 0.01) than that of other RT strains. Recent hospitalization, use of corticosteroids and proton pump inhibitors were related to increased risk of CDI in IBD patients; of these, recent hospitalization and proton pump inhibitors use were independent risk factors. Conclusion Patients with IBD have a relatively high incidence rate of CDI. C. difficile RT 017 is most frequently isolated from IBD patients in this region and warrants more attention to its high resistance rate. Clinicians should pay greater attention to CDI testing in IBD patients with diarrhea to ensure early diagnosis and initiation of effective treatment. Toxigenic Clostridioides difficile isolated from patients with IBD was mostly attributed to clade1, containing 7 STs and 33 strains. Clade 4 was split into two branches, including ST37 and ST375. Clade 3 consists of 2 STs and 2 strains. There is a correspondence between ST and RT, while one ST may contain several RTs. ST types of C. difficile isolated from patients with UC and CD did not show significant aggregation or predisposition.

This study was to identify and analyze the pathogen responsible for food poisoning in a tourist group traveling from Macao to Zhuhai. Samples were obtained from 27 patients of 96 cases, as well as samples of contaminated food in Macau. The collected samples were subjected to serological identification, drug sensitivity analysis, drug resistance gene identification, virulence factor analysis, and tracing. Twenty-six isolates and the salad isolate were ST11. Isolates from patients were exhibited significant resistance to Penicillin AMP (Ampicillin) and quinolones NAL (Nalidixic acid). Among these isolates, 21 strains were resistant to two or more antibiotics, indicating the multi-drug resistance (MDR). Genomic characteristics and phylogenetic analysis were performed on 9 of the isolates using whole genome sequencing (WGS). The analysis revealed that the resistance to AMP and NAL was primarily caused by a gryA mutation D87Y (9/9, 100%), and the presence of beta-lactam resistance genes blaOXA-1 (1/9, 11.11%), blaTEM-141 (1/9, 11.11%), and blaTEM-1B (8/9, 88.89%). It was also found a strains isolated from patients had two resistance genes to quinolones or beta-lactam drugs (1/8, 12.5%), respectively. The strains were found to possess 165 virulence genes, one adherence class virulence factor, one invasion class virulence factor and various pathogenicity islands, including SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-9, SPI-10, SPI-13, SPI-14, SPI-15, SGI 1, CS54_island, and C63PI-1. Additionally, the virulence plasmids were detected, including IncFIB(s)-IncFII(s)-IncX1 (55.56%), IncFIB(s)-IncFII(s) (33.33%), and IncFIB(s)-IncFII(s)-IncHI2-IncHI2A (11.11%). PFGE (Pulsed Field Gel Electrophoresis) and phylogenetic tree analysis revealed a high degree of similarity between Salmonella isolates from patients and food samples from Macao. This study identified Salmonella ST11 as the cause of the food poisoning outbreak. The findings highlight the importance of phenotypic characterization and next-generation sequencing (NGS) tools in epidemiological studies and emphasize the potential risk of a new emerging multi-antibiotic ST11 clone for .

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.

Antimicrobial resistance associated with the spread of plasmid-encoded extended-spectrum β-lactamase (ESBL) genes conferring resistance to third generation cephalosporins is increasing worldwide. However, data on the population of ESBL producing E. coli in different animal sources and their antimicrobial characteristics are limited. The purpose of this study was to investigate potential reservoirs of ESBL-encoded genes in E. coli isolated from swine, beef, dairy, and poultry collected from different regions of the United States using whole-genome sequencing (WGS). Three hundred isolates were typed into different phylogroups, characterized by BOX AIR-1 PCR and tested for resistance to antimicrobials. Of the 300 isolates, 59.7% were resistant to sulfisoxazole, 49.3% to tetracycline, 32.3% to cephalothin, 22.3% to ampicillin, 20% to streptomycin, 16% to ticarcillin; resistance to the remaining 12 antimicrobials was less than 10%. Phylogroups A and B1 were most prevalent with A (n = 92, 30%) and B1 (87 = 29%). A total of nine E. coli isolates were confirmed as ESBL producers by double-disk synergy testing and multidrug resistant (MDR) to at least three antimicrobial drug classes. Using WGS, significantly higher numbers of ESBL-E. coli were detected in swine and dairy manure than from any other animal sources, suggesting that these may be the primary animal sources for ESBL producing E. coli. These isolates carry plasmids, such as IncFIA(B), IncFII, IncX1, IncX4, IncQ1, CollRNAI, Col440I, and acquired ARGs aph(6)-Id, aph(3″)-Ib, aadA5, aph(3′)-Ia, blaCTX-M-15, blaTEM-1B, mphA, ermB, catA1, sul1, sul2, tetB, dfrA17. One of the E. coli isolates from swine with ST 410 was resistant to nine antibiotics and carried more than 28 virulence factors, and this ST has been shown to belong to an international high-risk clone. Our data suggests that ESBL producing E. coli are widely distributed in different animal sources, but swine and dairy cattle may be their main reservoir.

This study aims to assess contamination with Legionella spp. in water from dental chair units (DCUs) of a hospital dental ward and to perform its molecular characterization by whole-genome sequencing (WGS). We collect eight water samples (250 mL) from four DCUs (sink and water-syringe). Samples are tested for the presence of Legionella spp. (CFUs/mL) by culturing according to the Nederland Norm (NEN) 6265. Three DCUs are found positive for Legionella anisa, and four isolates are cultured (sink n = 2, water-syringe n = 1; two isolates from the same chair) with 1 × 102 CFU/mL. Whole-genome multi-locus sequence typing (wgMLST) results indicate that all strains belong to the same cluster with two to four allele differences. Classical culture combined with WGS allows the identification of a unique clone of L. anisa in several DCUs in the same hospital dental ward. This may indicate a common contamination source in the dental unit waterlines, which was fixed by replacing the chairs and main pipeline of the unit. Our results reveal tap water contamination in direct contact with patients and the usefulness of WGS to investigate bacterial molecular epidemiology.

Evidence supports the role of adherent invasive Escherichia coli (AIEC) in the pathogenesis of inflammatory bowel disease (IBD). However, little is known about the phylogenetic structure and origin of this group of bacteria. Multi-locus sequence typing (MLST), and fimH sequence analysis were performed to elucidate the phylogenetic relationships between E. coli strains isolated from IBD tissue.MethodsThirty-six E. coli isolated from IBD patients and healthy individuals were used. MLST analysis of the adk, fumC, gyrB, icd, mdh, purA, and recA housekeeping genes was performed. The fimH gene was also sequenced and phylogenetically analyzed. Biochemical profiling of strains were performed using the API 20 E system.ResultsMLST analysis distinguished 9 new alleles and 11 new sequence types, nearly all of which belonged to IBD isolates. E. coli isolated from IBD patients were more likely to be grouped into separate clonal clusters by eBURST analysis of allelic profiles (P = 0.02). Sequencing of fimH placed putative AIEC strains into the same cluster with the uro-pathogenic E. coli CFT073 and the avian-pathogenic E. coli O1:K1:H7.ConclusionsMLST analysis suggested that E. coli isolated from IBD patients did not evolve from a unique ancestral background. Together with the fimH sequence we conclude that AIEC represent a group of bacteria that have been able to take advantage of an “IBD microenvironment” and likely shares common genes with extraintestinal pathogens like uro-pathogenic CFT073 and avian-pathogenic O1:K1:H7 E. coli. Future research should focus on genes that are unique to AIEC.

•Most comprehensive review of Group B Streptococcal serotypes through 2018.•First systematic review of Group B Streptococcal strain type and protein data.•Theoretically candidate vaccines may protect against 93-99% disease-causing strains.•More studies on GBS strains in low- and middle-income countries are needed. 21 million pregnant women worldwide (18%) are estimated to carry Group B Streptococcus (GBS), which is a risk for invasive disease in newborns, pregnant women, and stillbirths. Adults ≥ 60 years or with underlying health conditions are also vulnerable to invasive GBS disease. We undertook systematic reviews on GBS organism characteristics including: capsular polysaccharide (serotype), sequence type (multi-locus sequence types (MLST)), and virulence proteins. We synthesised data by at-risk populations, to inform vaccine development. We conducted systematic reviews and meta-analyses to estimate proportions of GBS serotypes for at risk populations: maternal colonisation, invasive disease in pregnant women, stillbirths, infants 0–90 days age, and older adults (≥60 years). We considered regional variation and time trends (2001–2018). For these at-risk population groups, we summarised reported MLST and surface proteins. Based on 198 studies (29247isolates), 93–99% of GBS isolates were serotypes Ia, Ib, II, III, IV and V. Regional variation is likely, but data gaps are apparent, even for maternal colonisation which has most data. Serotype III dominates for infant invasive disease (60%) and GBS-associated stillbirths (41%). ST17 accounted for a high proportion of infant invasive disease (41%; 95%CI: 35–47) and was found almost exclusively in serotype III strains, less present in maternal colonisation (9%; 95%CI:6–13),(4%; 95%CI:0–11) infant colonisation, and adult invasive disease (4%, 95%CI:2–6). Percentages of strains with at least one of alp 1, alp2/3, alpha C or Rib surface protein targets were 87% of maternal colonisation, 97% infant colonisation, 93% infant disease and 99% adult invasive disease. At least one of three pilus islands proteins were reported in all strains. A hexavalent vaccine (serotypes Ia, Ib, II, III, IV and V) might provide comprehensive cover for all at-risk populations. Surveillance of circulating, disease-causing target proteins is useful to inform vaccines not targeting capsular polysaccharide. Addressing data gaps especially by world region and some at-risk populations (notably stillbirths) is fundamental to evidence-based decision-making during vaccine design.