wgMLST Analysis Research Articles

Genomic typing and virulence gene profile analysis of Salmonella Derby from different sources

Salmonella enterica serovar Derby (S. Derby) is one of the most common Salmonella serovars which can infect poultry, swine, and humans. With the reduction of the sequencing cost and the improvement of sequencing technology, whole genome sequencing (WGS) has become an important method for bacterial determination, molecular investigation, and pathogenic tracing analysis. In this study, we investigated S. Derby isolates from different sources in China using in-silico multilocus sequence typing (MLST), core genome MLST (cgMLST) and whole genome MLST (wgMLST) analysis based on WGS. The results showed that 21 S. Derby strains were divided into 3 STs using MLST analysis, including ST40 (n = 19, accounting for 90.48%), ST71 (n = 1, accounting for 4.76%) and ST8016 (n = 1, accounting for 4.76%). cgMLST and wgMLST analysis categorized the tested strains into 13 cgSTs and 21 wgSTs, respectively. The minimum spanning trees of cgMLST and wgMLST both divided these strains into 3 clusters and 4 singletons. In addition, virulence gene profiles of S. Derby isolates were also analyzed, and a total of 174 virulence genes belonged to 8 categories were identified. In summary, we studied genomic typing, phylogenetic relationship and virulence gene profiles of S. Derby strains from different sources in China. These findings were beneficial for the epidemiology and pathogenesis of Salmonella.

Genomes-based MLST, cgMLST, wgMLST and SNP analysis of Salmonella Typhimurium from animals and humans

Salmonella Typhimurium (S. Typhimurium) is an important food-borne and zoonotic pathogen that causes salmonellosis. With the development of whole genome sequencing (WGS), genome-based typing has been widely applied to bacteriology. In this study, we investigated genotyping and phylogenetic clusters of S. Typhimurium isolates from humans and animals in different provinces (including Beijing, Shandong, Guangxi, Shaanxi, Henan, and Shanghai) of China during 2009–2018 using multi locus sequence typing (MLST), core genome MLST (cgMLST), whole genome MLST (wgMLST) and single nucleotide polymorphism (SNP) based on WGS. 29 S. Typhimurium isolates from chicken (n = 22), sick pigeon (n = 2), patients (n = 4) and sick swine (n = 1) were tested. MLST analysis showed S. Typhimurium strains were divided into four STs, namely ST19 (n = 14), ST34 (n = 12), ST128 (n = 2) and ST1544 (n = 1). cgMLST and wgMLST divided 29 strains into 27 cgSTs and 29 wgST, respectively. Phylogenetic clustering showed that all isolates were divided into 4 clusters and 4 singletons. SNP analysis was used to examine MLST, cgMLST, wgMLST analysis. Finally, comparisons of MLST, cgMLST, wgMLST, and SNP were analyzed and the results showed their precision increased in order. In summary, genomic typing and phylogenetic relationships of 29 S. Typhimurium strains from different sources in China were analyzed. These findings were beneficial to investigate molecular pathogenesis, bacterial diversity, and traceability analysis of Salmonella.

Comparative analysis unravels genetic recombination events of Vibrio parahaemolyticus recA gene.

Vibrio parahaemolyticus is a gram-negative bacterium capable of causing diseases in humans and aquatic animals. The global relationships among V. parahaemolyticus genomes have been studied using multilocus sequence typing (MLST). Recently, the MLST gene recA has shown difficulties in amplification and/or a larger PCR fragment for some V. parahaemolyticus genomes due to genetic recombination. We aimed to investigate these recombination events of recA gene by analyzing 500 publicly available whole genomes from the NCBI database. The genomes with untypable recA genes were separated using BIGSdb and CGEMLST 2.0 servers, followed by annotation with RAST and NCBI pipelines. Moreover, the variable nature of V. parahaemolyticus was investigated by wgMLST analysis. The hypothetical proteins in recombinant regions were analyzed with VCIMPred tool. In the results, 3 genomes were detected with recA gene recombination, in which 2 were associated with phages and 1 to an AHPND causing strain. All 3 recombinant regions had a G+C content of 39%-40% with 15-30 ORFs, including a newly incorporated recA gene. These acquired recA genes were closely related to 3 different genera namely Aliivibrio, Photobacterium, and Vibrio. The wgMLST analysis indicated genetic recombination events occur independently among V. parahaemolyticus on a global scale. The in silico analysis revealed 4 hypothetical proteins associated with virulence factors in recombinant regions. The present study confirms, recombination events of V. parahaemolyticus recA gene, are diverse and may have an impact on the evolutionary process. Moreover, understanding these genetic recombination events of the recA gene is necessary to determine their STs and, therefore assessing epidemiological relationships.

1189. Clonal Recovery Pattern of Staphylococcus aureus during Sham-controlled, Interventional, Crossover Trial on the Effectiveness of Pulsed Xenon Ultraviolet Light (PX-UV) in Reducing Healthcare-associated Infections

Abstract Background PX-UV has shown great effectiveness at reducing Staphylococcus aureus (S. aureus) on healthcare surfaces. S. aureus is one of the most prevalent causes of healthcare-associated infections (HAIs). Here we assess the clonal recovery pattern and the disruption caused by PX-UV on S. aureus collected from patients, using Whole Genome Multilocus Sequence Type (wgMLST) analysis. Methods During a 3-year period, 2017 to 2020, a prospective sham-controlled double-blinded interventional crossover trial designed to assess the effectiveness of post-discharge PX-UV room disinfection on HAIs in 2 Detroit hospitals. The interventional group (Q) and the control group (W) was treated with PX-UV and sham UV light after terminal cleaning respectively, both treatments lasting 12 months. Crossover occurred after a 6-month washout period (R) during which no UV light was used. Whole Genome Sequencing (WGS) of the 291 S. aureus samples collected was performed using the Nextseq 550 (Illumina). Following de novo assembly, construction of minimum spanning tree (MST) and wgMLST analysis was completed using BioNumerics calculation engine (v7.6). Results A total of 20 sequence types (STs) were found throughout the trial, the most common being ST5 and ST8 with roughly 20 to 50 each found in all 3 treatment groups (Table 1. The other 18 STs were only found 7 times or less in each treatment group. ). During intervention (Q) 9 STs were found, however 11 were not recovered after PX-UV (ST1, ST1508, ST22, ST231, ST3287, ST3462, ST398, ST59, ST6790, ST840, ST97). During the sham treatment (W) 15 STs were found while 7 were found during the washout period (R). The minimum spanning tree (MST) shows clusters of related ST groups color coded by treatment type (Figure 1). Fig. 1:Minimum spanning tree (MST) for S. aureusTable 1:Total number of each sequence type (ST) per treatment group Conclusion The difference between the STs recovered during intervention and during sham UV treatment was inconsequential because many factors influence the amount of STs recovered (treatment, number of patients, sampling bias, and terminal cleaning methods). There was no clonality noted among any of the intervention group (Fig. 1). Both dominant strains (ST5, ST8) have been characterized as hypervirulent and predominant previously, consistent with our data. Our data does not demonstrate any notable change in the clonal prevalence of S. aureus after PX-UV use. Disclosures Piyali Chatterjee, PhD, AHRQ Grant # 1R03HS027667-01: Grant/Research Support|AHRQ Grant # 1R03HS027667-01: Central Texas Veterans Health Care System Keith S. Kaye, MD, MPH, Allecra: Advisor/Consultant|GlaxoSmithKline plc.: Receiving symposia honoraria|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502|Merck: Advisor/Consultant|qpex: Advisor/Consultant|Shionogi: Grant/Research Support|Spero: Advisor/Consultant Chetan Jinadatha, MD, MPH, AHRQ R01 Grant-5R01HS025598: Grant/Research Support|EOS Surfaces: Copper Coupons and materials for testing.

2048. Molecular Characterization and Resistance Factors of Circulating Acinetobacter baumannii isolates in South-East Michigan

Abstract Background Carbapenem-resistant Acinetobacter baumannii (CRAb) is increasing due to widespread use of antibiotics. Multidrug resistant (MDR) CRAb is a major threat to public health as treatment options are limited. The objective of this study is to elucidate the molecular epidemiology of circulating antibiotic resistance genes causing MDR CRAb infections by using a combination of whole-genome Multi-Locus Sequence Typing (wgMLST) and antibiotic susceptibility phenotyping. Table 1.Molecular characterization of MDR CRAb isolates*The numbers indicate % of sequence identity match for each Beta-lactamase gene. Methods Bacterial isolates were derived from cultures taken from subjects 48 hours following admission as part of routine clinical care for patients between 2017-2020. Isolates were obtained from 16 hospital units (both ICU and non-ICU) across two hospitals in the Detroit area. Whole Genome Sequencing (WGS) was performed using Illumina MiniSeq or Nextseq. WgMLST analysis was performed using BioNumerics software v7.6. ResFinder software was used for analysis of antibiotic resistance genes. Isolates underwent antibiotic susceptibility testing using a broth microdilution method (VITEK2) and Clinical & Laboratory Standards Institute (CLSI) minimum inhibitory concentration (MIC) cut offs. Results Out of the 95 total isolates, 51(54%) were CRAb isolates and of the CRAb isolates, 21(41%) were MDR CRAb. WgMLST identified that majority of the circulating MDR CRAb isolates belonged to ST2Pas (ST195Ox and ST208Ox) based on CDC definitions (Table 1). MDR CRAb isolates were resistant to 3 different classes of antibiotics including aminoglycosides, fluroquinolones and β-lactams. β-lactamase genes present include (blaADC-25, blaOXA-23, blaOXA-66 and blaTEM1D) for both ST195Ox and ST208Ox and (blaADC-25, blaOXA-23 and blaOXA-223) for ST406Pas (ST310Ox). Among the patients with MDR CRAb infections, most were males with respiratory infections in a non-ICU setting. Conclusion The study demonstrated a high proportion of isolates belonged to ST2 Pas carrying multiple beta-lactamase genes including blaOXA-23 gene. ST406Pas might be an emerging lineage carrying the blaOXA-23 gene. In addition to stringent infection control measures, continuous surveillance is recommended in limiting the spread of MDR CRAb isolates in the healthcare settings. Disclosures Chetan Jinadatha, MD, MPH, AHRQ R01 Grant-5R01HS025598: Grant/Research Support|EOS Surfaces: Copper Coupons and materials for testing Keith S. Kaye, MD, MPH, Allecra: Advisor/Consultant|GlaxoSmithKline plc.: Receiving symposia honoraria|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502|Merck: Advisor/Consultant|qpex: Advisor/Consultant|Shionogi: Grant/Research Support|Spero: Advisor/Consultant Piyali Chatterjee, PhD, AHRQ Grant # 1R03HS027667-01: Grant/Research Support|AHRQ Grant # 1R03HS027667-01: Central Texas Veterans Health Care System.

Campylobacter jejuni in Different Canine Populations: Characteristics and Zoonotic Potential

With most epidemiological studies focused on poultry, dogs are often overlooked as a reservoir of Campylobacter, even though these animals maintain close daily contact with humans. The present study aimed to obtain a first insight into the presence and characteristics of Campylobacter spp. in different canine populations in Portugal, and to evaluate its zoonotic potential through genomic analysis. From a total of 125 rectal swabs collected from companion (n = 71) and hunting dogs (n = 54) living in two different settings, rural (n = 75) and urban (n = 50), 32 Campylobacter spp. isolates were obtained. Four different Campylobacter species were identified by Multiplex PCR and MALDI-TOF mass spectrometry, of which Campylobacter jejuni (n = 14, 44%) was overall the most frequently found species. Relevant resistance phenotypes were detected in C. jejuni, with 93% of the isolates being resistant to ciprofloxacin, 64% to tetracycline, and 57% to ampicillin, and three isolates being multi-drug-resistant. Comparison of the phenotypic and genotypic traits with human isolates from Portuguese patients revealed great similarity between both groups. Particularly relevant, the wgMLST analysis allowed the identification of isolates from human and dogs without any apparent epidemiological relationship, sharing high genetic proximity. Notwithstanding the limited sample size, considering the high genomic diversity of C. jejuni, the genetic overlap between human and dog strains observed in this study confirmed that the occurrence of this species in dogs is of public health concern, reinforcing the call for a One Health approach.

679. Predominance of Acinetobacter baumannii Sequence Type 2 (ST2) among Clinical Isolates from Two Detroit Hospitals using Whole Genome Sequencing

BackgroundHealthcare-associated infections (HAIs) caused by Acinetobacter baumannii, an opportunistic gram-negative pathogen, often contribute to morbidity and mortality among hospitalized patients. To determine molecular epidemiology of circulating hospital Acinetobacter strains in Detroit area in a non-outbreak situation, we performed whole genome sequencing (WGS) and whole genome multilocus sequence typing (wgMLST) analysis to characterize genomic diversity.MethodsWe performed WGS on patient isolates from two disparate, geographically distinct tertiary care Detroit hospitals admitted to 16 intensive care units (ICU) and non-ICU wards between 2017-2019. The samples were obtained 48 hours following admission and WGS was performed using the Illumina NextSeq instrument. The contigs were de novo assembled using SPAdes and WgMLST analysis was performed using BioNumerics software v7.6. Minimum spanning tree (MST) was constructed to demonstrate the clusters in each hospital and their wards.ResultsWgMLST analysis was performed on a total of 62 Acinetobacter baumannii patient isolates, 33 of which were from hospital # 1 (H1) and 29 were from hospital # 2 (H2). ST2 (52%) was the predominant sequence type for both hospitals. In addition to ST2, ST93 (8%) and ST406 (8%) clusters were also found in H1. Most of the isolates were predominantly clustered in the intensive care unit (ICU) setting. Several identical sequence types of ST93 (2 identical in H1), ST36 (2 identical in H1) and ST2 (5 identical for H1 and 2 identical for H2) were also observed from different patients in both hospitals. Unlike H1, H2 only has ST2 as a predominant cluster. Other sporadic sequence types such as ST119, ST36, ST49, ST212, ST667 for H1 and ST395, ST214, ST427 and ST268 for H2 were also observed.Figure 1. MInimum Spanning Tree of Acinetobacter strains in hospitals (H1 and H2) and different wards (U1-U12). For identical strains circles are marked with dividing lines. Different sequence types (STs) are marked om the side bar. ConclusionOur data suggests that ST2 is the predominant sequence type circulating in both the geographically distinct hospitals and is endemic to the ICU. There was in hospital spread of this distinct Acinetobacter sequence type from one patient to the other, raising concerns about infection control practices and the role of environment in the spread of these infections. Early detection of these endemic strains within the hospital may be critical in preventing healthcare-associated infections in the future.Disclosures Chetan Jinadatha, MD, MPH, AHRQ (Research Grant or Support)Department of Veterans Affairs (Other Financial or Material Support, Owner: Department of Veterans Affairs. Licensed to: Xenex Disinfection System, San Antonio, TX)Inventor (Other Financial or Material Support, Methods for organizing the disinfection of one or more items contaminated with biological agents)NiH/NINR (Research Grant or Support)NSF (Research Grant or Support)Xenex Healthcare Services (Research Grant or Support) Mark Stibich, PhD MHS, Xenex Disinfection Services, Inc (Board Member, Employee)

1451. Genetic Analysis of Antibiotic Resistance Profiles of Acinetobacter baumannii Using Whole Genome Sequencing

Background Acinetobacter is known to quickly develop resistance to commonly used antibiotics. Previously we performed whole genome sequencing (WGS) and whole genome multilocus sequence typing (wgMLST) analysis in clinical Acinetobacter isolates to determine sequence types (ST) of these isolates and mapped their distribution. In this study, we sought to characterize the genetic antibiotic resistance patterns in these isolates.MethodsSixty-two clinical Acinetobacter isolates collected in two distinct large tertiary care hospitals in Detroit were analyzed. The samples were subjected to WGS using the NextSeq instrument (Illumina). The contigs were de novo assembled using SPAdes (v3.7.1) and wgMLST analysis was performed using BioNumerics software v7.6. The genomic sequence for each isolate was uploaded in ResFinder 3.2 and known antibiotic resistance genes were analyzed.ResultsThe most common resistance gene found is blaADC-25 conferring resistance to beta-lactams across all STs. Sulfonamide (sul1) and macrolide resistance (mphE/msrE) among STs were also common. ST2 (52%) was predominant for both hospitals (H1 and H2). ST2 in H2 exhibited the presence of the maximum number of resistance genes including resistance to aminoglycosides, macrolides (2), tetracyclines (tetB), beta-lactams, fluoroquinolones (aac(6’)-Ib-cr), sulphonamides (sul1, sul2). ST2 had a slightly different resistance profile of beta-lactams in H1 when compared to H2. ST406 and ST15 exhibited similar antibiotic profiles in both hospitals and a single isolate of ST20 from H2 is highly antibiotic resistant.Table 1. Antimicrobial Drug Resistance Profiles of all Acinetobacter Sequence Types (STs) in H1 and H2. Table 2. Antibiotic Resistance Gene Profiles of Sequence Type 2 (ST2) isolates in H1 and H2. ConclusionThis study provides us with a snapshot of antibiotic resistant genes among circulating Acinetobacter isolates prevalent in an area. All hospital isolates demonstrated resistance to beta-lactams. Multidrug resistant ST2 isolates from both hospitals demonstrate similar antibiotic resistance gene profiles suggesting a common circulating strain in the area. No colistin resistance genes were detected in any isolates. Because Acinetobacter infections are predominantly hospital acquired, it is important to continually monitor resistance profiles to determine the trends that may better serve both the patients and infection control practices.Disclosures Chetan Jinadatha, MD, MPH, AHRQ (Research Grant or Support)Department of Veterans Affairs (Other Financial or Material Support, Owner: Department of Veterans Affairs. Licensed to: Xenex Disinfection System, San Antonio, TX)Inventor (Other Financial or Material Support, Methods for organizing the disinfection of one or more items contaminated with biological agents)NiH/NINR (Research Grant or Support)NSF (Research Grant or Support)Xenex Healthcare Services (Research Grant or Support)

836. Complete genome sequencing reveals a melting pot of diverse Klebsiella pneumoniae pathogens in two Detroit hospitals

BackgroundKlebsiella pneumoniae is one of the leading causes of healthcare-associated infections. Treatment of Klebsiella pneumonia is difficult due to the antibiotic resistance and high survival on environmental surfaces. Whole genome sequencing analysis of Klebsiella pneumoniae clinical isolates were performed to study the transmission of Klebsiella pneumonia in hospital settings.Figure 1. Minimum spanning tree (MST) of wgMLST profiles of Klebsiella pneumoniae sequence types found in the two hospitals. Branch lengths reflect the number of allele differences between the isolates in the connected nodes Figure 2. Minimum spanning tree (MST) of wgMLST profiles of Klebsiella pneumoniae isolates. Node labels indicate patients’ hospital unit. MethodsClinical bacterial isolates from patients admitted to two disparate, geographically distinct tertiary care hospitals (H1, H2) in Detroit, Michigan, after 48 hours of admission from 2017- 2019 were collected and sequenced. Whole genome multilocus sequence typing (wgMLST) analysis was performed using Illumina NextSeq platform. De novo assembly of the contigs was performed using SPAdes assembler. WgMLST (assembly-free and assembly-based calls) was performed using calculation engine on the Bionumerics v7.6 platform. Minimum spanning tree with the isolates was constructed and arranged by their MLST Pasteur serotype and hospital/ward of the patient isolate collection.ResultsTotal 17 different MLST Pasteur serotypes were observed from WgMLST analyzation of forty-five Klebsiella pneumoniae clinical patient isolates. All the Klebsiella pneumoniae isolates of HAI obtained from two hospitals were genetically distinct. As shown in Figure 1, there were three distinct clusters on the minimum spanning tree. Out of 17 STs, 4 were present in both hospitals. Though there was no predominant ST type, ST15 and ST11 were the most frequent isotypes (18% each). Both ST15 and ST11 were evenly spread across both hospitals, but the pattern was different. While ST15 was predominantly found in two units (H1U3 and H2U4), ST11 was found in multiple units. ST348 and ST29 were predominantly found in H1, whereas ST16 was found in H2.ConclusionMajority of Klebsiella pneumoniae infection is sporadic and there was no evidence of hospital spread. The WgMLST analysis showed the isolates distributed across the phylogeny of Klebsiella with diverse serotypes from the three main diverse evolutionary origin. Our study showed that the global spread of various serotypes of Klebsiella pneumoniae has already reached a significant level in these two Detroit hospitals possibly the catchment area.Disclosures Chetan Jinadatha, MD, MPH, AHRQ (Research Grant or Support)Department of Veterans Affairs (Other Financial or Material Support, Owner: Department of Veterans Affairs. Licensed to: Xenex Disinfection System, San Antonio, TX)Inventor (Other Financial or Material Support, Methods for organizing the disinfection of one or more items contaminated with biological agents)NiH/NINR (Research Grant or Support)NSF (Research Grant or Support)Xenex Healthcare Services (Research Grant or Support) Mark Stibich, PhD MHS, Xenex Disinfection Services, Inc (Board Member, Employee)

Evaluation of the bioMérieux EPISEQ-CS Software for wgMLST-Based Bacterial Strain Typing

Background: Whole-genome sequencing (WGS) is becoming the method of choice for outbreak analysis of microbial pathogens. However, the main challenge with WGS for microbial strain typing is the conversion of raw sequencing data to actionable results for epidemiology and surveillance analysis. We evaluated the bioMrieux EPISEQ-CS, a cloud-based WGS data analysis software for outbreak detection to compare the results for 4 groups of different species previously characterized by strain typing and commonly isolated in hospital-acquired infections. Methods: In total, 30 methicillin-resistant Staphylococcus aureus (MRSA), 15 Clostridioides difficile (CDIFF), 17 Pseudomonas aeruginosa (PSA), and 10 Acinetobacter baumannii (ACB) isolates were included in this study. All isolates had been previously characterized by rep-PCR using the DiversiLab system (bioMrieux, France) and saved at 70C. Before testing, samples were thawed and plated, and DNA extraction was performed on the QIAcube (Qiagen, Hilden, Germany) using the DNEasy Ultra Clean Microbial kit extraction protocol. DNA libraries were prepared using the Nextera DNA Flex Kit and sequenced on the Illumina iSeq100 platform according to manufacturer’s recommendations (Illumina, San Diego, CA). Generated sequences were uploaded into EPISEQ-CS, and wgMLST-based analysis was performed. We compared clusters generated by the DiversiLab system and EPISEQ-CS. Results: DiversiLab identified 9 MRSA clusters among 30 isolates. EPISEQ-CS reclassified 14 of 30 isolates into 5 MRSA clusters and the remaining 16 isolates were unrelated. DiversiLab identified 2 CDIFF clusters among 15 isolates. EPISEQ-CS reclassified 3 isolates into 1 CDIFF cluster and determined the remaining 12 to be unrelated. DiversiLab identified 5 PSA clusters among 17 isolates, whereas EPISEQ-CS reclassified all 17 isolates as unrelated. DiversiLab identified 2 ACB clusters among 10 isolates, whereas EPISEQ-CS reclassified 2 ACB isolates into 1 cluster and determined 8 to be unrelated. Analysis using Simpson’s diversity index (D) suggested that the EPISEQ-CS showed increased diversity when compared to DiversiLab clustering across all bacterial species analyzed in this study. Conclusions: EPISEQ-CS enabled a comprehensive wgMLST analysis, including quality control and comparative epidemiological analysis, thereby providing a more reliable method for bacterial strain typing. As WGS becomes more affordable and applicable to routine epidemiological surveillance, EPISEQ-CS provides an informative tool in the monitoring of hospital-acquired infections.Funding: NoneDisclosures: None

Characterization of Clostridioides difficile strains isolated from manure and digestate in five agricultural biogas plants

Clostridioides difficile strains were isolated from manure and digestate samples from five biogas plants in France. The objective of this study was to characterize these isolates using PCR ribotyping, wgMLST, a multiplex PCR targeting genes encoding for the main virulence factors, i.e. tcdA, tcdB, cdtA and cdtB, and antimicrobial susceptibility assays. The 54 strains characterized were all positive for tcdA and tcdB and 83% (45/54) were positive for the binary toxin genes. PCR ribotypes 126 (59%) and 078 (37%) were predominant, and wgMLST analysis of 18 isolates showed close proximity of strains within a single biogas plant. Samples from the biogas plant supplied with cattle and poultry manure displayed the largest variety in PCR ribotypes. The in vitro activities of nine antimicrobial agents were determined. All the strains were susceptible to vancomycin and metronidazole, which are currently considered first-line treatments for C. difficile infection in humans. All the strains were resistant to clindamycin. The results of this study show that a high percentage of C. difficile strains present in the French biogas plants investigated are toxigenic strains from PCR ribotypes also commonly found in humans.

ISAba1-dependent overexpression of eptA in clinical strains of Acinetobacter baumannii resistant to colistin.

Colistin resistance in Acinetobacter baumannii often results from mutational activation of the two-component system PmrAB and subsequent addition of phospho-ethanolamine (pEtN) to lipooligosaccharide by up-regulated pEtN transferase PmrC. To characterize mechanisms of colistin resistance independent of PmrCAB in A. baumannii. Twenty-seven colistin-resistant A. baumannii were collected from 2012 to 2018. Analysis of operon pmrCAB was performed by PCR and sequencing. Seven strains were investigated further by WGS and whole-genome MLST (wgMLST). Seven out of the 27 selected isolates were found to overexpress eptA, a gene homologous to pmrC, likely as a consequence of upstream insertion of an ISAba1 element. Insertion sites of ISAba1 were mapped 13, 18 and 156 bp ahead of the start codon of eptA in five strains, one strain and one strain, respectively. The finding that the isolates did not cluster together when compared by wgMLST analysis supports the notion that distinct insertion events occurred in close, but different, genetic backgrounds. Activation of eptA and subsequent addition of pEtN to the cell surface represents a novel mechanism of resistance to colistin in A. baumannii.

Culture of Clinical Specimens Reveals Extensive Diversity of Legionella pneumophila Strains in Arizona.

Between 2000 and 2017, a total of 236 Legionella species isolates from Arizona were submitted to the CDC for reference testing. Most of these isolates were recovered from bronchoalveolar lavage specimens. Although the incidence of legionellosis in Arizona is less than the overall U.S. incidence, Arizona submits the largest number of isolates to the CDC for testing compared to those from other states. In addition to a higher proportion of culture confirmation of legionellosis cases in Arizona than in other states, all Legionellapneumophila isolates are forwarded to the CDC for confirmatory testing. Compared to that from other states, a higher proportion of isolates from Arizona were identified as belonging to L. pneumophila serogroups 6 (28.2%) and 8 (8.9%). Genome sequencing was conducted on 113 L. pneumophila clinical isolates not known to be associated with outbreaks in order to understand the genomic diversity of strains causing legionellosis in Arizona. Whole-genome multilocus sequence typing (wgMLST) revealed 17 clusters of isolates sharing at least 99% identical allele content. Only two of these clusters contained isolates from more than one individual with exposure at the same facility. Additionally, wgMLST analysis revealed a group of 31 isolates predominantly belonging to serogroup 6 and containing isolates from three separate clusters. Single nucleotide polymorphism (SNP) and pangenome analysis were used to further resolve genome sequences belonging to a subset of isolates. This study demonstrates that culture of clinical specimens for Legionella spp. reveals a highly diverse population of strains causing legionellosis in Arizona which could be underappreciated using other diagnostic approaches.IMPORTANCE Culture of clinical specimens from patients with Legionnaires' disease is rarely performed, restricting our understanding of the diversity and ecology of Legionella Culture of Legionella from patient specimens in Arizona revealed a greater proportion of non-serogroup 1 Legionellapneumophila isolates than in other U.S. isolates examined. Disease caused by such isolates may go undetected using other diagnostic methods. Moreover, genome sequence analysis revealed that these isolates were genetically diverse, and understanding these populations may help in future environmental source attribution studies.

Genomic Epidemiology of Campylobacter jejuni Transmission in Israel.

Objectives: Campylobacter jejuni is responsible for 80% of Campylobacter infections in Israel, a country with a high incidence reaching 91/100,000 population. We studied the phylogeny, diversity and prevalence of virulence factors using whole genome sequencing (WGS) of a national sample of C. jejuni clinical, food, and animal isolates collected over a 10-year period (2003–2012).Methods: C. jejuni isolates (n = 263) were subject to WGS using Illumina sequencing (PE 250bpx2). Raw reads and de novo assemblies were analyzed with the BioNumerics whole genome MLST (wgMLST) pipeline. Reads were screened for 71 virulence genes by the SRST2 script. Allelic profiles were analyzed to create minimum spanning trees and allelic core distances were investigated to determine a reliable cutoff for strain determination.Results: wgMLST analysis of 263 C. jejuni isolates indicated significant diversity among the prevalent clonal complexes (CCs) with CC-21 and CC-353 being the most diverse, and CC-574 the most clonal. Within CC-21, sequence type (ST)-1359 created a separate clade. Human, poultry and bovine isolates clustered together across the different STs. Forty four percent of studied isolates were assigned to 29 genetic clusters. Temporal and geographical relatedness were found among the minority of clusters, while most phylogenetically associated cases appeared diffuse and unassociated epidemiologically. The majority of virulence factors were highly prevalent across the dataset and not associated with genotype, source of isolation or invasiveness. Conversely, all 13 genes associated with type VI secretion system (T6SS) were lineage-related and identified in only 18% of the isolates. T6SS was detected in 95.2% of ST-1359, a common type in Israel.Conclusions: wgMLST supported the assessment that poultry and cattle are likely food sources of infection in Israel. Substantial genetic clustering among C. jejuni isolates suggested multiple point source and diffuse outbreaks that were previously unreported in Israel. The high prevalence of T6SS among ST-1359 isolates is unique to Israel, and requires further investigation. This study exemplifies the importance of studying foodborne pathogens using advanced genomic approaches across the entire spectrum of One Health.

Large-scale genomic analyses reveal the population structure and evolutionary trends of Streptococcus agalactiae strains in Brazilian fish farms

Streptococcus agalactiae is a major pathogen and a hindrance on tilapia farming worldwide. The aims of this work were to analyze the genomic evolution of Brazilian strains of S. agalactiae and to establish spatial and temporal relations between strains isolated from different outbreaks of streptococcosis. A total of 39 strains were obtained from outbreaks and their whole genomes were sequenced and annotated for comparative analysis of multilocus sequence typing, genomic similarity and whole genome multilocus sequence typing (wgMLST). The Brazilian strains presented two sequence types, including a newly described ST, and a non-typeable lineage. The use of wgMLST could differentiate each strain in a single clone and was used to establish temporal and geographical correlations among strains. Bayesian phylogenomic analysis suggests that the studied Brazilian population was co-introduced in the country with their host, approximately 60 years ago. Brazilian strains of S. agalactiae were shown to be heterogeneous in their genome sequences and were distributed in different regions of the country according to their genotype, which allowed the use of wgMLST analysis to track each outbreak event individually.

Genomic analyses of Neisseria gonorrhoeae reveal an association of the gonococcal genetic island with antimicrobial resistance

SummaryObjectivesAntimicrobial resistance (AMR) threatens our ability to treat the sexually transmitted bacterial infection gonorrhoea. The increasing availability of whole genome sequence (WGS) data from Neisseria gonorrhoeae isolates, however, provides us with an opportunity in which WGS can be mined for AMR determinants.MethodsChromosomal and plasmid genes implicated in AMR were catalogued on the PubMLST Neisseria database (http://pubmlst.org/neisseria). AMR genotypes were identified in WGS from 289 gonococci for which MICs against several antimicrobial compounds had been determined. Whole genome comparisons were undertaken using whole genome MLST (wgMLST).ResultsClusters of isolates with distinct AMR genotypes were apparent following wgMLST analysis consistent with the occurrence of genome wide genetic variation. This included the presence of the gonococcal genetic island (GGI), a type 4 secretion system shown to increase recombination and for which possession was significantly associated with AMR to multiple antimicrobials.ConclusionsEvolution of the gonococcal genome occurs in response to antimicrobial selective pressure resulting in the formation of distinct N. gonorrhoeae populations evidenced by the wgMLST clusters seen here. Genomic islands offer selective advantages to host bacteria and possession of the GGI may, not only facilitate the spread of AMR in gonococcal populations, but may also confer fitness advantages.

Tracing isolates from domestic human Campylobacter jejuni infections to chicken slaughter batches and swimming water using whole-genome multilocus sequence typing

Campylobacter jejuni is the leading cause of bacterial gastroenteritis and chicken is considered a major reservoir and source of human campylobacteriosis. In this study, we investigated temporally related Finnish human (n=95), chicken (n=83) and swimming water (n=20) C. jejuni isolates collected during the seasonal peak in 2012 using multilocus sequence typing (MLST) and whole-genome MLST (wgMLST). Our objective was to trace domestic human C. jejuni infections to C. jejuni isolates from chicken slaughter batches and swimming water. At MLST level, 79% of the sequence types (STs) of the human isolates overlapped with chicken STs suggesting chicken as an important reservoir. Four STs, the ST-45, ST-230, ST-267 and ST-677, covered 75% of the human and 64% of the chicken isolates. In addition, 50% of the swimming water isolates comprised ST-45, ST-230 and ST-677. Further wgMLST analysis of the isolates within STs, accounting their temporal relationship, revealed that 22 of the human isolates (24%) were traceable back to C. jejuni positive chicken slaughter batches. None of the human isolates were traced back to swimming water, which was rather sporadically sampled. The highly discriminatory wgMLST, together with the patient background information and temporal relationship data with possible sources, offers a new, accurate approach to trace back the origin of domestic campylobacteriosis. Our results suggest that potentially a substantial proportion of campylobacteriosis cases during the seasonal peak most probably are due to other sources than chicken meat consumption. These findings warrant further wgMLST-based studies to reassess the role of other reservoirs in the Campylobacter epidemiology both in Finland and elsewhere.