Genetic diversity of Leptospira isolates in Lao PDR and genome analysis of an outbreak strain

Background Although Southeast Asia is one of the most leptospirosis afflicted regions, little is known about the diversity and molecular epidemiology of the causative agents of this widespread and emerging zoonotic disease. Methodology/Principal findings We used whole genome sequencing to examine genetic variation in 75 Leptospira strains isolated from patients in the Lao PDR (Laos) between 2006 and 2017. Eleven serogroups from 4 Leptospira species and 43 cgMLST-defined clonal groups (CGs) were identified. The most prevalent CG was CG272 (n = 18, 26.8%), composed of L. interrogans serogroup Autumnalis isolates. This genotype was recovered throughout the 12-year period and was associated with deaths, and with a large outbreak in neighbouring Thailand. Genome analysis reveals that the CG272 strains form a highly clonal group of strains that have, for yet unknown reasons, recently spread in Laos and Thailand. Additionally, accessory genes clearly discriminate CG272 strains from the other Leptospira strains. Conclusions/Significance The present study reveals a high diversity of Leptospira genotypes in Laos, thus extending our current knowledge of the pan- and core-genomes of these life-threatening pathogens. Our results demonstrate that the CG272 strains belong to a unique clonal group, which probably evolved through clonal expansion following niche adaptation. Additional epidemiological studies are required to better evaluate the spread of this genotype in Southeast Asia. To further investigate the key factors driving the virulence and spread of these pathogens, more intense genomic surveillance is needed, combining detailed clinical and epidemiological data.

Genomic evolution of the globally disseminated multidrug-resistant Klebsiella pneumoniae clonal group 147

Background: The rapid emergence of multidrug-resistant Klebsiella pneumoniae (Kp) is largely driven by the spread of specific clonal groups (CG). Of these, CG147 includes 7-gene MLST sequence types ST147, ST273 and ST392. CG147 has caused nosocomial outbreaks across the world, but its global population dynamics remain unknown. Here, we report a pandrug-resistant ST147 clinical isolate from India (strain DJ) and define the evolution and global emergence of CG147. Methods: Antimicrobial susceptibility testing (EUCAST guidelines) and genome sequencing (Illumina and Oxford Nanopore technologies, Unicycler assembly) were performed on strain DJ. Additionally, we collated 217 publicly available CG147 genomes (NCBI, May 2019). CG147 evolution was inferred within a temporal phylogenetic framework (BEAST) based on a recombination-free sequence alignment (Roary/Gubbins). Comparative genomic analyses focused on resistance and virulence genes and other genetic elements (BIGSdb, Kleborate, PlasmidFinder, PHASTER, ICEFinder and CRISPRCasFinder). Results: Strain DJ had a pandrug resistance phenotype. Its genome comprised 7 plasmids and 1 linear phage-plasmid. Four carbapenemase genes were detected: blaNDM-5 and 2 copies of blaOXA-181 in the chromosome, and a second copy of blaNDM-5 on an 84 kb IncFII plasmid. CG147 genomes carried a mean of 13 acquired resistance genes or mutations; 63% carried a carbapenemase gene and 83% harbored blaCTX-M. All CG147 genomes presented GyrA and ParC mutations and a common subtype IV-E CRISPR-Cas system. ST392 and ST273 emerged in 2005 and 1995, respectively. ST147, the most represented phylogenetic branch, was itself divided into two main clades with distinct capsular loci: KL64 (74%, DJ included, emerged in 1994 and disseminated worldwide, with carbapenemases varying among world regions) and KL10 (20%, 2002, predominantly found in Asian countries, associated with carbapenemases NDM and OXA-48-like). Further, subclades within ST147-KL64 differed in the yersiniabactin locus, OmpK35/K36 mutations, plasmid replicons and prophages. The absence of IncF plasmids in some subclades was associated with a possible activity of a CRISPR-Cas system. Conclusions: K. pneumoniae clonal group CG147 comprises pandrug- or extensively-resistant isolates and carries multiple and diverse resistance genes and mobile genetic elements, including chromosomal blaNDM-5. Its emergence is driven by the spread of several phylogenetic clades marked by their own genomic features and specific temporo-spatial dynamics. These findings highlight the need for precision surveillance strategies to limit the spread of particularly concerning CG147 subsets.

Molecular epidemiology of hypervirulent Klebsiella pneumoniae: a systematic review and meta-analysis

Classical (CKp) and hypervirulent (hvKp) Klebsiella pneumoniae are two different circulating pathotypes. The aim of this study was to assess the prevalence, epidemiology and molecular relatedness of hvKps using a systemic review and meta-analysis. The data extracted from Medline, Embase, and Web of Science and finally 14 studies met the eligible criteria. To combine prevalence proportions of all studies, we performed the metaprop command embedded in the Meta package software. Totally, of 1814 K. pneumoniae isolates, 21.7% (394/1814) were hvKp. The molecular typing showed that all hvKp isolates were grouped into 50 different sequence types (STs) of them ST23, ST11, ST65 and ST86 were common. K1, K2 and K64 were dominant capsule serotypes that strongly related to ST23, ST65 and ST11, respectively. It seems that clonal group 23 (CG23) is associated with liver abscess and CG11 related to various clinical sources.

Re-Emergence of Salmonellosis in Hog Farms: Outbreak and Bacteriological Characterization

Clinical salmonellosis has been increasing significantly in Brazil in recent years. A total of 130 outbreaks distributed among 10 swine-producing states were investigated. One representative Salmonella isolate from each outbreak was characterized through serotyping, antimicrobial resistance profiles, PFGE, and WGS. From 130 outbreaks: 50 were enteric, 48 were septicemic, 17 cases were characterized as hepato-biliary invasive, 13 as nodal and two were not classified. The most prevalent serovars were a monophasic variant of S. typhimurium (55/130), Choleraesuis (46/130), and Typhimurium (14/130). Most of the strains (86.92%) demonstrated a high rate of multi-drug resistance. The identification of a major Choleraesuis clonal group in several Brazilian states sharing the same resistance genes suggested that these strains were closely related. Six strains from this clonal group were sequenced, revealing the same ST-145 and 11 to 47 different SNPs. The detected plasmid type showed multiple marker genes as RepA_1_pKPC-CAV1321, the first to be reported in Salmonella. All AMR genes detected in the genomes were likely present on plasmids, and their phenotype was related to genotypic resistance genes. These findings reveal that salmonellosis is endemic in the most important pig-producing states in Brazil, emphasizing the need to make data available to aid in reducing its occurrence.

Usefulness of MALDI-TOF and REP-PCR against PFGE for the epidemiological study of Acinetobacter baumannii

Objective. To evaluate the ability of MALDI-TOF MS and rep-PCR to discriminate Acinetobacter baumannii clones. Material and methods. A total of 21 strains of A. baumannii with different epidemiological and phenotipycal characteristics were included in the study. All isolates were analyzed in parallel by MALDI-TOF MS and rep-PCR and the spectra obtained were compared with each other and with the results obtained by pulsed field gel electrophoresis (PFGE). Isolates with a similarity equal to or greater than 87% were considered to be part of the same clonal group. Results. The analysis of the 21 isolates included in the study, resulted in 8 clonal groups using PFGE, 3 groups by MALDI-TOF MS and 7 groups by rep-PCR analysis. The isolates that formed the different groups by the 3 techniques used were totally different, so it can be concluded that there is no equivalence between the results obtained with the three typing methods used. Conclusions. Despite its simplicity, neither MALDI-TOF MS nor rep-PCR can at this time replace PFGE for the epidemiological study of A. baumannii.

Hospital-diagnosed infections with Escherichia coli clonal group ST131 are mostly acquired in the community

The worldwide spread of E. coli ST131 has significantly contributed to the dissemination of E. coli producing extended-spectrum β-lactamases (ESBL). In a French University hospital, we assessed the molecular features of ESBL-producing E. coli and identified risk factors in patients for colonization or infection with E. coli ST131. Over a 2-year period (2015–2017), each patient with at least one clinical isolate or one screening isolate positive with ESBL-producing E. coli were included (n = 491). The ST131 clonal group accounted for 17.5% (n = 86) of all ESBL-producing E. coli and represented 57.3% isolates of phylogroup B2. FimH-based sub-typing showed that 79.1% (68/86) of ST131 isolates were fimH30, among which 67.6% (n = 46), 20.6% (n = 14) and 11.8% (n = 8) isolates harbored genes encoding the ESBL CTX-M-15, CTX-M-27, and CTX-M-14, respectively. The multivariate analysis identified two factors independently associated with ST131 ESBL-producing E. coli isolates: infection (Odds ratio [OR] = 1.887, 95% confidence interval [CI]: 1.143–3.115; p = 0.013) and community acquisition (OR = 2.220, 95% CI: 1.335–3.693; p = 0.002). In conclusion, our study confirmed the predominance of ST131 clonal group among ESBL-producing E. coli and the difficulty to identify common risk factors associated with carriage of this pandemic clonal group.

Predominance of Non-carbapenemase Producing Carbapenem-Resistant Enterobacterales in South Texas

BackgroundCarbapenem-resistant Enterobacterales (CRE) pose a significant global public health threat. Resistance among CRE is particularly complex, owing to numerous possible resistance mechanisms and broad definitions. We aimed to characterize the clinical and molecular profiles of CRE in the South Texas region.Materials and methodsWe compared the clinical, genotypic, and phenotypic profiles of carbapenemase producing Enterobacterales (CPE) with those of non-carbapenemase producers (NCPE) isolated from South Texas, United States between 2011 and 2019. Molecular characteristics and resistance mechanisms were analyzed using whole-genome sequences.ResultsThe majority (59%) of the CRE isolates were NCPE while 41% of isolates harbored carbapenemases, predmonantly blaKPC-type. The most common CPE was Klebsiella pneumoniae while majority of Enterobacter cloacae and Escherichia coli were NCPE Among K. pneumoniae, the clonal group 307 has emerged as a predmoninant group and was associated with as many CRE infections as the previous common clonal group 258. Patients with NCPE compared to CPE infections were associated with higher antimicrobial exposure prior to culture collection (days of therapy, 795 vs. 242; p < 0.001) and emergency department visits within past 90 days (22% vs. 4%; p = 0.011). The all cause 30-day mortality was 21%.ConclusionsThis study highlights the diversity of resistance mechanisms underlying CRE in South Texas, with 59% not harboring a carbapenemase. Individuals with NCPE infections were more likely to have had prior antimicrobial therapy and emergency department visits compared to those with CPE. Identification and distinction of these mechanisms by rapid identification of species and carbapenemase would allow for optimal treatment and infection control efforts.

Longitudinal Characterization and Transmission Dynamics of Antibiotic-Resistant Organisms in an ICU (LOCATE AROs)

Background: Healthcare-associated infections caused by antibiotic-resistant organisms (AROs) are a major cause of significant morbidity and mortality. To create and optimize infection prevention strategies, it is crucial to delineate the role of the environment and clinical infections. Methods: Over a 14-month period, we collected environmental samples, patient feces, and patient bloodstream infection (BSI) isolates in a newly built bone marrow transplant (BMT) intensive care unit (ICU). Samples were collected from 13 high-touch areas in the patient room and 4 communal areas. Samples were collected from the old BMT ICU, in the new BMT ICU before patients moved in, and for 1 year after patients moved in. Selective microbiologic culture was used to isolate AROs, and whole-genome sequencing (WGS) was used to determine clonality. Antibiotic susceptibility testing was performed using Kirby-Bauer disk diffusion assays. Using linear mixed modeling, we compared ARO recovery across time and sample area. Results: AROs were collected and cultured from environmental samples, patient feces, and BSI isolates (Fig. 1a). AROs were found both before and after a patient entered the ICU (Fig. 1b). Sink drains had significantly more AROs recovered per sample than any other surface area (P < .001) (Fig. 1c). The most common ARO isolates were Pseudomonas aeruginosa and Stenotrophomonas maltophila (Fig. 1d). The new BMT ICU had fewer AROs recovered per sample than the old BMT ICU (P < .001) and no increase in AROs recovered over the first year of opening (P > .05). Furthermore, there was no difference before versus after patients moved into the hospital (P > .05). Antibiotic susceptibility testing reveal that P. aeruginosa isolates recovered from the old ICU were resistant to more antibiotics than isolates recovered from the new ICU (Fig. 2a). ANI and clonal analyses of P. aeruginosa revealed a large cluster of clonal isolates (34 of 76) (Fig. 2b). This clonal group included isolates found before patients moved into the BMT ICU and patient blood isolates. Furthermore, this clonal group was initially found in only 1 room in the BMT ICU, and over 26 weeks, it was found in sink drains in all 6 rooms sampled (Fig. 2b). Conclusions: AROs are present before patients move into a new BMT ICU, and sink drains act as a reservoir for AROs over time. Furthermore, sink-drain P. aeruginosa isolates are clonally related to isolates found in patient BSIs. Overall, these results provide insight into ARO transmission dynamics in the hospital environment.Funding: Research reported in this publication was supported by the Washington University Institute of Clinical and Translational Sciences grant UL1TR002345 from the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH.Disclosures: None