Multiple Replicons Research Articles

Molecular Characteristics of Salmonella Spp. Responsible for Bloodstream Infections in a Tertiary Hospital in Nanjing, China, 2019-2021.

To investigate the clinical and molecular characteristics of Salmonella spp. causing bloodstream infections (BSIs) in our hospital. We studied 22 clinical Salmonella isolates from BSIs and 16 from non-BSIs, performing antimicrobial susceptibility testing (AST) and whole genome sequencing (WGS). The analysis included serovars, antibiotic resistance genes (ARGs), virulence factors (VFs), sequence types (STs), plasmid replicons, and genetic relationships. We also assessed pathogenicity of the isolates causing BSIs through growth, biofilm formation, and anti-serum killing assays. WGS analysis identified 13 Salmonella serovars, with four responsible for BSIs. S. Enteritidis was the most prevalent serovar, involved in 19 (50.0%) cases. BSIs were caused by 17S. Enteritidis, two S. Typhimurium, two S. Munster and one S. Diguel. Of the 38 isolates, 27 (71.1%) exhibited high resistance to ampicillin, and 24 (63.2%) to ampicillin/sulbactam. Thirty-six types of ARGs were identified, with blaTEM-1B (n = 25, 65.8%) being the most frequent. Ten plasmid replicons were found; the combination of IncFIB(S)-IncFII(S)-IncX1 was the most common in S. Enteritidis (94.7%). Fifteen STs were identified, among which ST11 was the most prevalent and clonally disseminated, primarily responsible for BSIs. A total of 333 different VFs were detected, 177 of which were common across all strains. No significant differences were observed between the BSI and non-BSI isolates in terms of resistance rates, ARGs, plasmid replicons, and VFs, except for seven VFs. No strong pathogenicity was observed in the BSI-causing isolates. BSIs were predominantly caused by clonally disseminated S. Enteritidis ST11, the majority of which carried multiple ARGs, VFs and plasmid replicons. This study provides the first data on clonally disseminated S. Enteritidis ST11 causing BSIs, highlighting the urgent need for enhanced infection control measures.

Curing of a field strain of Salmonella enterica serovar Infantis isolated from poultry from its highly stable pESI like plasmid

Salmonella enterica serovar Infantis (S. infantis) is an important emerging pathogen, associated with poultry and poultry products and related to an increasing number of human infections in many countries. A concerning trend among S. infantis isolates is the presence of plasmid-mediated multidrug resistance. In many instances, the genes responsible for this resistance are carried on a megaplasmid known as the plasmid of emerging S. infantis (pESI) or pESI like plasmids. Plasmids can be remarkably stable due to the presence of multiple replicons and post-segregational killing systems (PSKs), which contribute to their maintenance within bacterial populations. To enhance our understanding of S. infantis and its multidrug resistance determinants toward the development of new vaccination strategies, we have devised a new method for targeted plasmid curing. This approach effectively overcomes plasmid addiction by leveraging the temporal overproduction of specific antitoxins coupled with the deletion of the partition region. By employing this strategy, we successfully generated a plasmid-free strain from a field isolate derived from S. infantis 119,944.This method provides valuable tools for studying S. infantis and its plasmid-borne multidrug resistance mechanisms and can be easily adopted for plasmid curing from other related bacteria.

Extended-spectrum β-lactamase-producing bacteria and their resistance determinants in different wastewaters and rivers in Nepal

Wastewaters serve as significant reservoirs of antibiotic resistant bacteria. Despite the evidence of antimicrobial resistance in wastewaters and river water in Kathmandu, direct linkage between them is not discussed yet. This study investigated the prevalence of extended-spectrum β-lactamase (ESBL)-producing bacteria and associated resistance genes in wastewaters and river water. Out of 246 bacteria from wastewaters, 57.72% were ESBL producers and 77.64% of them were multidrug resistant (MDR). ESBL producing E. coli was dominant in municipal and hospital wastewaters (HWW) as well as in river water while K. pneumoniae was common in pharmaceutical wastewater. The blaSHV and blaTEM genes were prevalent and commonly co-occurred with aac(6')-Ib-cr in K. pneumoniae isolated pharmaceutical wastewater. blaCTX-M carrying E. coli from hospital co-harbored aac(6')-Ib-cr while that from municipal influent and river water co-harbored qnrS. Whole genome sequencing data revealed the presence of diverse ARGs in bacterial isolates against multiple antibiotics. In average, an E. coli and a K. pneumoniae isolate contained 55.75 ± 0.96 and 40.2 ± 5.36 ARGs, respectively. Multi-locus sequence typing showed the presence of globally high-risk clones with wider host range such as E. coli ST10, and K. pneumoniae ST15 and ST307 in HWW and river indicating frequent dissemination of antimicrobial resistance in wastewater of Kathmandu. Whole genome sequence data aligned with phenotypic antibiograms and resistance genes detected by PCR in selected isolates. The presence of significant plasmid replicons (IncF, IncY) and mobile genetic elements (IS903, IS26) indicate high frequency of spreading antibiotic resistance. These findings indicate burden and dissemination of antimicrobial resistance in the environment and highlight the need for effective strategies to mitigate the antibiotic resistance.

Dissemination of Ceftriaxone-Resistant Salmonella Enteritidis Harboring Plasmids Encoding blaCTX-M-55 or blaCTX-M-14 Gene in China.

Salmonella Enteritidis was the primary foodborne pathogen responsible for acute gastroenteritis. The growing ceftriaxone resistance poses a significant threat to public health. Infection with S. Enteritidis has emerged as a major public health concern, particularly in developing countries. However, research on ceftriaxone-resistant S. Enteritidis (CRO-RSE) remains limited, particularly concerning its resistance mechanism, plasmid structure, and transmission characteristics. This study aims to address these gaps comprehensively. We collected 235 S. Enteritidis isolates from Hangzhou First People's Hospital between 2010 and 2020. Among these, 8.51% (20/235) exhibited resistance to ceftriaxone. Whole-genome analysis revealed that 20 CRO-RSE isolates harbored blaCTX-M-55 or blaCTX-M-14 on the plasmid. Moreover, the dissemination of the blaCTX-M-type gene was associated with IS26 and ISEcp1. Plasmid fusion entailing the integration of the p1 plasmid with antibiotic resistance genes and the p2 (pSEV) virulence plasmid was observed in certain CRO-RSE. Additionally, the structural analysis of the plasmids unveiled two types carrying the blaCTX-M-type gene: type A with multiple replicons and type B with IncI1 (Alpha) replicon. Type B plasmids exhibited superior adaptability and stability compared to type A plasmids within Enterobacteriaceae. Interestingly, although the type B (S808-p1) plasmid displayed the potential to spread to Acinetobacter baumannii, it failed to maintain stability in this species.

Rapid Dissemination of blaNDM-5 Gene among Carbapenem-Resistant Escherichia coli Isolates in a Yellow-Feather Broiler Farm via Multiple Plasmid Replicon.

Although carbapenems have not been approved for animal use, carbapenem-resistant Escherichia coli (CREC) strains are increasingly being detected in food-producing animals, posing a significant public health risk. However, the epidemiological characteristics of CREC isolates in yellow-feather broiler farms remain unclear. We comprehensively investigated the genetic features of carbapenem-resistance genes among E. coli isolates recovered from a yellow-feather broiler farm in Guangdong province, China. Among the 172 isolates, 88 (51.2%) were recovered from chicken feces (88.5%, 54/61), the farm environment (51.1%, 24/47), and specimens of dead chickens (15.6%, 41/64). All CREC isolates were positive for the blaNDM-5 gene and negative for other carbapenem-resistance genes. Among 40 randomly selected isolates subjected to whole-genome sequencing, 10 belonged to distinct sequence types (STs), with ST167 (n = 12) being the most prevalent across different sources, suggesting that the dissemination of blaNDM-5 was mainly due to horizontal and clonal transmission. Plasmid analysis indicated that IncX3, IncHI2, and IncR-X1-X3 hybrid plasmids were responsible for the rapid transmission of the blaNDM-5 gene, and the genetic surrounding of blaNDM-5 contained a common mobile element of the genetic fragment designated "IS5-△ISAba125-blaNDM-5-bleMBL-trpF-dsbC". These findings demonstrate a critical role of multiple plasmid replicons in the dissemination of blaNDM-5 and carbapenem resistance.

Hybrid Sequencing-Based Genomic Analysis of Klebsiella pneumoniae from Urinary Tract Infections Among Inpatients at a Tertiary Hospital in Beijing.

Urinary tract infection (UTI) associated with Klebsiella pneumoniae poses a serious threat for inpatients. This study aimed to describe the genomic characteristics of K. pneumoniae causing UTI in a tertiary-care hospital in Beijing, China. A total of 20 K. pneumoniae strains collected from 2020 to 2021 were performed whole-genome sequencing. The Antibiotic susceptibility of 19 common antimicrobial agents was tested against all strains. The multi-locus sequence types (MLSTs) and serotypes were determined from the WGS data. De novo assemblies were used to identify resistance and virulence genes. The presence and characteristics of the plasmids were detected using hybrid assembly of long and short-read data. These K. pneumoniae strains were clustered into nine sequence types (STs) and twelve K-serotypes. All the carbapenem-resistant K. pneumoniae (CRKP) strains acquired carbapenemase blaKPC-2 (n=7). Two CRKP strains exhibited increased resistance to Polymyxin B with MIC ≥ 4 mg/L due to insertion of an IS5-like sequence in the mgrB gene, and they were also involved in a transmission event in Intensive Care Unit. Long-read assemblies identified many plasmids co-carrying multiple replicons. Acquisition of a new IncM2_1 type blaCTX-M-3 positive plasmid was observed after transfer from ICU to neurovascular surgery by comparing the two strains collected from the same patient. K. pneumoniae is a significant pathogen responsible for urinary tract infections. The ST11-KL47 strain, prevalent at our hospital, exhibits a combination of high drug resistance and hypervirulence. It is imperative to enhance ongoing genomic surveillance of urinary tract infection-causing pathogens.

Genomic and clinical characterization of Klebsiella pneumoniae carrying the pks island.

The pks island and its production of the bacterial secondary metabolite genotoxin, colibactin, have attracted increasing attention. However, genomic articles focusing on pks islands in Klebsiella pneumoniae, as well as comparative genomic studies of mobile genetic elements, such as prophages, plasmids, and insertion sequences, are lacking. In this study, a large-scale analysis was conducted to understand the prevalence and evolution of pks islands, differences in mobile genetic elements between pks-negative and pks-positive K. pneumoniae, and clinical characteristics of infection caused by pks-positive K. pneumoniae. The genomes of 2,709 K. pneumoniae were downloaded from public databases, among which, 1,422 were from NCBI and 1,287 were from the China National GeneBank DataBase (CNGBdb). Screening for virulence and resistance genes, phylogenetic tree construction, and pan-genome analysis were performed. Differences in mobile genetic elements between pks-positive and pks-negative strains were compared. The clinical characteristics of 157 pks-positive and 157 pks-negative K. pneumoniae infected patients were investigated. Of 2,709 K. pneumoniae genomes, 245 pks-positive genomes were screened. The four siderophores, type VI secretion system, and nutritional factor genes were present in at least 77.9% (191/245), 66.9% (164/245), and 63.3% (155/245) of pks-positive strains, respectively. The number and fragment length of prophage were lower in pks-positive strains than in pks-negative strains (p < 0.05). The prevalence of the IS6 family was higher in pks-negative strains than in pks-positive strains, and the prevalence of multiple plasmid replicon types differed between the pks-positive and pks-negative strains (p < 0.05). The detection rate of pks-positive K. pneumoniae in abscess samples was higher than that of pks-negative K. pneumoniae (p < 0.05). The pks-positive strains had abundant virulence genes. There were differences in the distribution of mobile genetic elements between pks-positive and pks-negative isolates. Further analysis of the evolutionary pattern of pks island and epidemiological surveillance in different populations are needed.

Genomic and functional portrait of multidrug-resistant, hydrogen sulfide (H2S)-producing variants of Escherichia coli.

Atypical Escherichia coli forms exhibit unusual characteristics compared to typical strains. The H2S-producing variants of some atypical E. coli strains cause a wide range of illnesses in humans and animals. However, there are sparse reports on such strains worldwide. We performed whole-genome sequencing (WGS) and detailed characterization of four H2S-producing E. coli variants from poultry and human clinical sources in Dhaka, Bangladesh. All four isolates were confirmed as E. coli using biochemical tests and genomic analysis, and were multidrug-resistant (MDR). WGS analysis including an additional Chinese strain, revealed diverse STs among the five H2S-producing E. coli genomes, with clonal complex ST10 being detected in 2 out of 5 genomes. The predominant phylogroup detected was group A (n = 4/5). The blaTEM1B (n = 5/5) was the most predominant extended-spectrum beta-lactamase (ESBL) gene, followed by different alleles of blaCTX-M (blaCTX-M -55,-65,-123; n = 3/5). Multiple plasmid replicons were detected, with IncX being the most common. One E. coli strain was classified as enteropathogenic E. coli. The genomes of all five isolates harbored five primary and four secondary function genes related to H2S production. These findings suggest the potential of these isolates to cause disease and spread antibiotic resistance. Therefore, such atypical E. coli forms should be included in differential diagnosis to understand the pathogenicity, antimicrobial resistance and evolution of H2S-producing E. coli.

Characterization of antibiotic resistance genes and mobile elements in extended-spectrum β-lactamase-producing Escherichia coli strains isolated from hospitalized patients in Guangdong, China.

This study aimed to investigate the high-resolution phenotypic and genotypic characterization of extended-spectrum β-lactamase (ESBL)-producing E. coli strains isolated from hospitalized patients to explore the resistance genes and mobile genetic elements (MGEs) involved in horizontal dissemination. Between May and September 2021, a total of 216 ESBL-producing E. coli isolates were recovered from multiple departments. The identification of strains was performed using MALDI-TOF mass spectrometry and PCR, while antibiotic susceptibility testing was carried out using the Vitek 2 COMPACT system to determine resistance patterns, while PCR was used to detect different resistance genes and MGEs. In addition, a conjugation assay was performed to investigate the horizontal gene transfer of resistance genes. Selected isolates underwent whole-genome sequencing using the Illumina MiSeq platform. A total of 216 out of 409 E. coli isolates recovered from a tertiary hospital were observed to be ESBL-producing, giving a carriage rate of 52.8%, as determined by phenotypic screening. The most frequent sources of ESBL-producing E. coli isolates were urine (129/216, 59.72%,) and blood (50/216, 23.14%). The most prevalent ESBL genes identified were blaCTX-M (60.18%), blaTEM (40.27%), and blaSHV (18.05%). Three E. coli isolates were found to carry the genes blaNDM, mcr-1, and fosA3 genes. The most prevalent MGEs were IS26 (95.37%), Int (87.03%), and IncFIB (76.85%). Whole-genome sequencing analysis of eight MDR E. coli strains revealed that these isolates belonged to eight different sequence types (STs) and serotypes and were found to harbor multiple plasmid replicons and virulence factors. This study highlights a high incidence of antibiotic resistance genes and MGEs associated with the dissemination of ESBLs and other resistance genes.

Long time persistence and evolution of carbapenemase-producing Enterobacterales in the wastewater of a tertiary care hospital in Germany

BackgroundWorldwide observations revealed increased frequencies of multi-resistant Enterobacterales and resistance genes in hospital wastewater compared to any other type of wastewater. Despite the description of clonal lineages possibly adapted to hospital wastewater, little is known about long term persistence as well as evolution of these lineages. MethodsIn this study, wastewater isolates of different Enterobacterales species from a tertiary care hospital were investigated with 2.5 years distance. Whole Genome Sequencing (WGS) and resistance gene identification were performed for E. coli, C. freundii, S. marcescens, K. pneumoniae, K. oxytoca, and E. cloacae isolates (n = 59), isolated in 2022 and compared with strains isolated from the same wastewater pipeline in 2019 (n = 240). ResultsIndividual clonal lineages with highly related isolates could be identified in all species identified more than once in 2022 that appear to persist in the wastewater drainage. A common motif of all persistent clonal lineages was the carriage of mobile genetic elements encoding carbapenemase genes with hints for horizontal gene transfer in persistent clones in this environment observed over the 2.5-year period. Multiple plasmid replicons could be detected in both years. In 2022 isolates blaVIM-1 replaced blaOXA-48 as the most common carbapenemase gene compared to 2019. Interestingly, despite a similar abundance of carbapenemase genes (>80% of all isolates) at both time points genes encoding extended spectrum β-lactamases decreased over time. ConclusionsThis data indicates that hospital wastewater continuously releases genes encoding carbapenemases to the urban wastewater system. The evolution of the resident clones as well as the reasons for the selection advantage in this specific ecological niche needs to be further investigated in the future.

PKpQIL-like plasmid contributes to the dissemination of blaNDM-1 and plasmid mediated quinolone resistance determinants among multi drug resistant Klebsiella pneumoniae in Assiut university hospital, Egypt.

Concomitant carriage of blaNDM-1 and plasmid mediated quinolone resistance determinants (PMQRs) by multi drug resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) has increased globally, often related to their presence on transmissible plasmids. In this study, we hypothesized the presence of blaNDM-1 and PMQRs on a single conjugative plasmid that circulates among K. pneumoniae strains isolated from Assiut University Hospital. Twenty-two clinical MDR K. pneumoniae strains harboring both blaNDM-1 and PMQRs were genotyped using pulsed field gel electrophoresis. Horizontal transfer of blaNDM-1 and PMQRs was evaluated by conjugation and trans-conjugants were screened for the presence of both genes and integron by PCR. Trans-conjugant's plasmid DNA bands were purified using agarose gel electrophoresis and different DNA bands were screened for blaNDM-1 and PMQRs. Plasmids carrying blaNDM-1 and PMQRs were typed by PCR based replicon typing. All MDR K. pneumoniae contained class 1 integron and belonged to 15 pulsotypes. BlaNDM-1 and PMQRs were co-transferred in each conjugation process. Multiple replicons (5-9 types) were detected in each trans-conjugant; with IncFIIK and IncFIB-KQ replicons being common among all trans-conjugants. Both blaNDM-1 and PMQRs were detected on a pKpQIL-like multi-replicon plasmid that was present in all K. pneumoniae strains. In view of these results, the presence of blaNDM-1 and PMQRs on pKpQIL-like plasmid that existed in multiple unrelated K. pneumoniae isolates is highly suggestive of the circulation of pKpQIL-like MDR plasmids in our hospitals. Moreover, carriage of integrons by the-circulating MDR plasmids increases the risk of dissemination of antimicrobial resistance among pathogens.

Diversity of Bacterial Clones and Plasmids of NDM-1 Producing Escherichia coli Clinical Isolates in Central Greece.

The objective of the present study was to genetically characterize ten NDM-1 producing Escherichia coli isolates, recovered from patients in a hospital in Central Greece during the period 2017 to 2021.The isolates were studied by whole genome sequencing to obtain multi-locus sequencing typing (MLST), identification of blaNDM1-environment, resistome and plasmid content. MLST analysis showed the presence of eight sequence types: ST46* (two isolates), ST46, ST744, ST998, ST410, ST224, ST4380, ST683 and ST12 (one isolate each). Apart of the presence of blaNDM-1, the isolates carried a combination of various to β-lactams encoding resistance genes: blaTEM-1B, blaCTX-15, blaOXA-1, blaVIM-1, blaSHV-5, blaOXA-16, blaOXA-10 and blaVEB-1. Additionally, plurality of resistance genes to aminoglycosides, macrolides, rifamycin, phenicols, sulfonamides and tetracycline was detected. The presence of multiple replicons was observed, with predominance of IncFII and IncFIB. Analysis of blaNDM-1 genetic environment of the isolates showed that seven had 100% identity with the pS-3002cz plasmid (Accession Number KJ 958927), two with the pB-3002cz plasmid (Accession Number KJ958926) and one with the pEc19397-131 plasmid (Accession Number MG878866). Τhis latter plasmid was derived by the fusion of two, previously identified, plasmids, pAMPD2 and pLK75 (Accession Numbers CP078058 and KJ440076, respectively). The diversity of clones and plasmids of NDM-1 producing E. coli isolated from patients in Greece indicates a continuous horizontal gene transfer.

In silico analyses of diversity and dissemination of antimicrobial resistance genes and mobile genetics elements, for plasmids of enteric pathogens.

The antimicrobial resistance (AMR) mobilome plays a key role in the dissemination of resistance genes encoded by mobile genetics elements (MGEs) including plasmids, transposons (Tns), and insertion sequences (ISs). These MGEs contribute to the dissemination of multidrug resistance (MDR) in enteric bacterial pathogens which have been considered as a global public health risk. To further understand the diversity and distribution of AMR genes and MGEs across different plasmid types, we utilized multiple sequence-based computational approaches to evaluate AMR-associated plasmid genetics. A collection of 1,309 complete plasmid sequences from Gammaproteobacterial species, including 100 plasmids from each of the following 14 incompatibility (Inc) types: A/C, BO, FIA, FIB, FIC, FIIA, HI1, HI2, I1, K, M, N, P except W, where only 9 sequences were available, was extracted from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST tools. The extracted FASTA files were analyzed using the AMRFinderPlus web-based tools to detect antimicrobial, disinfectant, biocide, and heavy metal resistance genes and ISFinder to identify IS/Tn MGEs within the plasmid sequences. In silico prediction based on plasmid replicon types showed that the resistance genes were diverse among plasmids, yet multiple genes were widely distributed across the plasmids from enteric bacterial species. These findings provide insights into the diversity of resistance genes and that MGEs mediate potential transmission of these genes across multiple plasmid replicon types. This notion was supported by the observation that many IS/Tn MGEs and resistance genes known to be associated with them were common across multiple different plasmid types. Our results provide critical insights about how the diverse population of resistance genes that are carried by the different plasmid types can allow for the dissemination of AMR across enteric bacteria. The results also highlight the value of computational-based approaches and in silico analyses for the assessment of AMR and MGEs, which are important elements of molecular epidemiology and public health outcomes.

A clinical KPC-producing Klebsiella michiganensis strain carrying IncFII/IncFIA (HI1)/IncFIB (K) multiple replicon plasmid.

Klebsiella michiganensis is an increasingly important bacterial pathogen causing nosocomial infections in clinical patients. In this study, we described the molecular and genomic characteristics of a carbapenem-resistant K. michiganensis strain KM166 cultured from a one-month premature baby's blood sample. KM166 showed lower biofilm forming ability in optical density (OD) than K. pneumoniae NTUH-K2044 (0.271 ± 0.027 vs. 0.595 ± 0.054, p = 0.001), and the median lethal dose (0.684 lg CFU/mL) was lower than K. pneumoniae strain NTUH-K2044 (6.679 lg CFU/mL). A IncFII/IncFIA(HI1)/IncFIB(K) multiple replicon plasmid in KM166 was identified carrying three replicon types. It has low homology to Escherichia coli pMRY09-581ECO_1 and the highest homology similarity to the INcFIA/INcFII(p14)-type plasmid in K. michiganensis strain fxq plasmid pB_KPC, suggesting that this multiple replicon plasmid was unlikely to have been transmitted from E. coli and probably a transfer of repFIB replicon genes from other K. michiganensis strains into the INcFIA/INcFII(p14)-type plasmid of KM166 had occurred. Mapping of the gene environment revealed that bla KPC-2 in KM166 plasmid 3 had high identity and same Tn3-tnpR-IS481-bla KPC-2-klcA_1 genomic context structure with K. pneumoniae strain JKP55, plasmid pKPC-J5501, and bla KPC-2-carrying plasmid proved to be autonomously transferred under the help of mobile genetic elements into Escherichia coli 600 by plasmid conjugation experiment. In conclusion, we have characterized a K. michiganensis strain carrying multi-replicon IncFII/IncFIA(HI1)/IncFIB(K) plasmid and bla KPC-2-carrying IncFII(p14)/IncFIA plasmid in this study, which provided insights about the evolutionary diversity of plasmids carried by K. michiganensis.

Coexistence of bla NDM-5 and tet(X4) in international high-risk Escherichia coli clone ST648 of human origin in China.

The emergence of pathogens is conferring resistance to last-resort therapies such as tigecycline, colistin, and carbapenems, limiting the therapeutic options, and raising concerns about the emergence of new "superbugs." This study reports the first incident of a bla NDM-5 and tet(X4) co-harboring Escherichia coli with resistance to carbapenem and tigecycline recovered as the causative agent of a urinary tract infection in a 94-year-old patient. The E. coli strain ECCL209 carries multiple resistance genes [i.e., bla TEM-1B , bla NDM-5, bla CMY-2, aadA22, florR, erm(B), mph(A), erm(42), lnuG, qnrS1, and sul2] and exhibits resistance to almost all clinically used antibiotics. MLST analysis found that the strain belongs to ST648, considered a worldwide high-risk pandemic clone. Moreover, multiple plasmid incompatibility types were detected, i.e., IncHI1A, IncHI1B, IncFII, IncFIA, IncFIB, IncQ1, Col, and IncX4. Genetic analysis revealed that bla NDM-5 and tet(X4) genes were localized on two hybrid plasmids with multiple replicons. Continuous monitoring studies are suggested to quantify the antimicrobial resistance and assess the dissemination of such superbugs into a human healthcare setting.

Discordant population structure among rhizobium divided genomes and their legume hosts.

Symbiosis often occurs between partners with distinct life history characteristics and dispersal mechanisms. Many bacterial symbionts have genomes comprising multiple replicons with distinct rates of evolution and horizontal transmission. Such differences might drive differences in population structure between hosts and symbionts and among the elements of the divided genomes of bacterial symbionts. These differences might, in turn, shape the evolution of symbiotic interactions and bacterial evolution. Here we use whole genome resequencing of a hierarchically structured sample of 191 strains of Sinorhizobium meliloti collected from 21 locations in southern Europe to characterize population structures of this bacterial symbiont, which forms a root nodule symbiosis with the host plant Medicago truncatula. S. meliloti genomes showed high local (within-site) variation and little isolation by distance. This was particularly true for the two symbiosis elements, pSymA and pSymB, which have population structures that are similar to each other, but distinct from both the bacterial chromosome and the host plant. Given limited recombination on the chromosome, compared to the symbiosis elements, distinct population structures may result from differences in effective gene flow. Alternatively, positive or purifying selection, with little recombination, may explain distinct geographical patterns at the chromosome. Discordant population structure between hosts and symbionts indicates that geographically and genetically distinct host populations in different parts of the range might interact with genetically similar symbionts, potentially minimizing local specialization.

Large-Scale Sequencing of Borreliaceae for the Construction of Pan-Genomic-Based Diagnostics.

The acceleration of climate change has been associated with an alarming increase in the prevalence and geographic range of tick-borne diseases (TBD), many of which have severe and long-lasting effects—particularly when treatment is delayed principally due to inadequate diagnostics and lack of physician suspicion. Moreover, there is a paucity of treatment options for many TBDs that are complicated by diagnostic limitations for correctly identifying the offending pathogens. This review will focus on the biology, disease pathology, and detection methodologies used for the Borreliaceae family which includes the Lyme disease agent Borreliella burgdorferi. Previous work revealed that Borreliaceae genomes differ from most bacteria in that they are composed of large numbers of replicons, both linear and circular, with the main chromosome being the linear with telomeric-like termini. While these findings are novel, additional gene-specific analyses of each class of these multiple replicons are needed to better understand their respective roles in metabolism and pathogenesis of these enigmatic spirochetes. Historically, such studies were challenging due to a dearth of both analytic tools and a sufficient number of high-fidelity genomes among the various taxa within this family as a whole to provide for discriminative and functional genomic studies. Recent advances in long-read whole-genome sequencing, comparative genomics, and machine-learning have provided the tools to better understand the fundamental biology and phylogeny of these genomically-complex pathogens while also providing the data for the development of improved diagnostics and therapeutics.

Centromere Interactions Promote the Maintenance of the Multipartite Genome in Agrobacterium tumefaciens.

ABSTRACTMany pathogens or symbionts of animals and plants contain multiple replicons, a configuration called a multipartite genome. Multipartite genomes enable those species to replicate their genomes faster and better adapt to new niches. Despite their prevalence, the mechanisms by which multipartite genomes are stably maintained are poorly understood. Agrobacterium tumefaciens is a plant pathogen that contains four replicons: a circular chromosome (Ch1), a linear chromosome (Ch2), and two large plasmids. Recent work indicates that their replication origins are clustered at the cell poles in a manner that depends on their ParB family centromeric proteins: ParB1 for Ch1 and individual RepB paralogs for Ch2 and the plasmids. However, understanding of these interactions and how they contribute to genome maintenance is limited. By combining genome-wide chromosome conformation capture (Hi-C) assays, chromatin-immunoprecipitation sequencing (ChIP-seq), and live cell fluorescence microscopy, we provide evidence here that centromeric clustering is mediated by interactions between these centromeric proteins. We further show that the disruption of centromere clustering results in the loss of replicons. Our data establish the role of centromeric clustering in multipartite genome stability.

Pan-genome and resistome analysis of extended-spectrum ß-lactamase-producing Escherichia coli: A multi-setting epidemiological surveillance study from Malaysia

This study profiled the prevalence of extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC) in the community and compared their resistome and genomic profiles with isolates from clinical patients through whole-genome sequencing. Fecal samples from 233 community dwellers from Segamat, a town in southern Malaysia, were obtained between May through August 2018. Putative ESBL strains were screened and tested using antibiotic susceptibility tests. Additionally, eight clinical ESBL-EC were obtained from a hospital in the same district between June through October 2020. Whole-genome sequencing was then conducted on selected ESBL-EC from both settings (n = 40) for pan-genome comparison, cluster analysis, and resistome profiling. A mean ESBL-EC carriage rate of 17.82% (95% CI: 10.48%- 24.11%) was observed in the community and was consistent across demographic factors. Whole-genome sequences of the ESBL-EC (n = 40) enabled the detection of multiple plasmid replicon groups (n = 28), resistance genes (n = 34) and virulence factors (n = 335), with no significant difference in the number of genes carried between the community and clinical isolates (plasmid replicon groups, p = 0.13; resistance genes, p = 0.47; virulence factors, p = 0.94). Virulence gene marker analysis detected the presence of extraintestinal pathogenic E. coli (ExPEC), uropathogenic E. coli (UPEC), and enteroaggregative E. coli (EAEC) in both the community and clinical isolates. Multiple blaCTX-M variants were observed, dominated by blaCTX-M-27 (n = 12), blaCTX-M-65 (n = 10), and blaCTX-M-15 (n = 9). The clinical and community isolates did not cluster together based on the pan-genome comparison, suggesting isolates from the two settings were clonally unrelated. However, cluster analysis based on carried plasmids, resistance genes and phenotypic susceptibility profiles identified four distinct clusters, with similar patterns between the community and clinical isolates. ESBL-EC from the clinical and community settings shared similar resistome profiles, suggesting the frequent exchange of genetic materials through horizontal gene transfer.

Identification of Plasmid-Mediated Tigecycline-Resistant Gene tet(X4) in Enterobacter cloacae from Pigs in China.

ABSTRACTTwo tet(X4)-positive Enterobacter cloacae isolates TECL_1 and TECL_2 were isolated from pigs in China. S1-PFGE and Southern blotting showed that tet(X4) located on plasmids in the size of ∼290 kb and ∼190 kb in TECL_1 and TECL_2, respectively. Conjugation experiment demonstrated that the tet(X4)-harboring plasmid can transfer from the donor strain TECL_1 and TECL_2 to the recipient strain Escherichia coli J53, and the tigecycline resistance of transconjugants was increased by 128-fold and 64-fold compared with E. coli J53, respectively. We obtained the complete plasmid sequence of pTECL_2-190k-tetX4 (190,185 bp) from E. cloacae TECL_2 and found that the plasmid was a hybrid plasmid with replicon types of IncFIA, IncHI1A and IncHI1B. We further analyzed 85 tet(X4)-carrying plasmids in the public database and clarified that pTECL_2-190k-tetX4-like plasmid was widespread in multiple species of Enterobacteriaceae.IMPORTANCE We identified two tet(X4)-positive E. cloacae isolates, which has not been previously reported. We obtained the complete sequence of pTECL_2-190k-tetX4 and found that it was a hybrid plasmid with multiple replicon types, including IncFIA, IncHI1A and IncHI1B. By comparing all the known tet(X4)-carrying plasmids, we found that pTECL_2-190k-tetX4-like plasmid has been disseminated across various species in China. Our study expanded the identification of tet(X4)-positive species and emphasized that pTECL_2-190k-tetX4-like plasmid has spread widely in various species.