Multilocus Sequence Typing Loci Research Articles

Epidemiological and molecular investigations of Salmonella isolated from duck farms in southwest and around area of Shandong, China

Salmonella is a zoonotic pathogen posing a serious risk to the farming industry and public health due to food animals serving as reservoirs for future contamination and spread of Salmonella. The present study is designed to monitor the contamination status of Salmonella in duck farms and the main control points during breeding. 160 strains of duck-derived Salmonella were isolated from the 736 samples (cloacal swabs, feces, water, feed, soil, and air and dead duck embryos) collected in southwest Shandong Province and the province's surrounding area. The percentage of Salmonella-positive samples collected was 21.74% (160/736), and the greatest prevalence from duck embryo samples (40.00%, 36/90). These Salmonella were classified into 23 serotypes depending on their O and H antigens, in which S. Typhimurium (30.15%), S. Kottbus (13.97%) and S. Enteritidis (10.29%) were the prevailing serotypes. Subsequently, the molecular subtyping was done. Clustered regularly interspaced short palindromic repeats (CRISPR) analysis showed that 41 strains of S. Typhimurium and 14 strains of S. Enteritidis were classified into 13 and 3 genotypes, respectively. 19 S. Kottbus isolates from different sources featured ST1546, ST198, ST321, and ST1690 by multilocus sequence typing (MLST) analysis, among which ST1546 belongs to S. Kottbus was a new ST. The minimum spanning tree analysis based on the two CRISPR loci and seven MLST loci from all S. Typhimurium, S. Enteritidis and S. Kottbus isolates revealed that duck embryos, feed and water were key control points to the spread of Salmonella along the breeding chain. Meanwhile, the emergence of S. Kottbus in duck flocks was considered a potential public health hazard.

Emerging novel sequence types of Staphylococcus aureus in Pakistan

BackgroundDespite an increasing incidence of Staphylococcus aureus infection and dissemination in Pakistan, the epidemiology of different Staphylococcus aureus research clones has been the subject of only a small number of investigations. By analyzing the collected data sequence, this study was designed to study the epidemiology of Staphylococcus aureus in the area using multilocus sequence typing (MLST). MethodsA total of 1015 staphylococcus strains collected from the city's tertiary care facilities were biochemically screened, followed by antimicrobial susceptibility testing against a panel of 13 antibiotics. Analyzed methicillin-resistant Staphylococcus aureus (MRSA) was subjected to molecular characterization using multilocus sequence typing (MLST), clonal complex analysis, recombination testing, and phylogenetic analysis. ResultsApproximately 421 bacteria were verified as Staphylococcus aureus by biochemical analysis. 57% of the isolates exhibited multidrug resistance, of which 89% were found to be methicillin-resistant Staphylococcus aureus (MRSA). MLST results in a total of 39 sequence types (ST) and 5 clonal complexes (CC), out of which twenty-two STs were newly documented worldwide. The most common CC identified was CC8. The direct sequencing data also revealed significant shifts at MLST loci, with point mutations resulting in the aroE-343 and tpi-278 alleles. ConclusionsThis study concludes that there is high diversity in the locally circulating clones of Staphylococcus aureus present in nature and that they are defined by their geographic epidemiology. These findings have practical implications for public health, including the need for tailored infection control strategies, antibiotic stewardship, global surveillance, and a deeper understanding of bacterial evolution.

Assessment of genetic markers for multilocus sequence typing (MLST) of Fasciola isolates from Iran.

Several markers have been described to characterise the population structure and genetic diversity of Fasciola species (Fasciola hepatica (F. hepatica) and Fasciola gigantica (F. gigantica). However, sequence analysis of a single genomic locus cannot provide sufficient resolution for the genetic diversity of the Fasciola parasite whose genomes are ∼1.3 GB in size. To gain a better understanding of the gene diversity of Fasciola isolates from western Iran and to identify the most informative markers as candidates for epidemiological studies, five housekeeping genes were evaluated using a multilocus sequence typing (MLST) approach. MLST analysis was developed based on five genes (ND1, Pepck, Pold, Cyt b and HSP70) after genomic DNA extraction, amplification and sequencing. Nucleotide diversity and phylogeny analysis were conducted on both concatenated MLST loci and each individual locus. A median joining haplotype network was created to examine the haplotypes relationship among Fasciola isolates. Thirty-three Fasciola isolates (19 F. hepatica and 14 F. gigantica) were included in the study. A total of 2971bp was analysed for each isolate and 31 sequence types (STs) were identified among the 33 isolates (19 for F. hepatica and 14 for F. gigantica isolates). The STs produced 44 and 42 polymorphic sites and 17 and 14 haplotypes for F. hepatica and F. gigantica, respectively. Haplotype diversity was 0.982 ± 0.026 and 1.000 ± 0.027 and nucleotide diversity was 0.00200 and 0.00353 ± 0.00088 for F. hepatica and F. gigantica, respectively. There was a high degree of genetic diversity with a Simpson's index of diversity of 0.98 and 1 for F. hepatica and F. gigantica, respectively. While HSP70 and Pold haplotypes from Fasciola species were separated by one to three mutational steps, the haplotype networks of ND1 and Cyt b were more complex and numerous mutational steps were found, likely due to recombination. Although HSP70 and Pold genes from F. gigantica were invariant over the entire region of sequence coverage, MLST was useful for investigating the phylogenetic relationship of Fasciola species. The present study also provided insight into markers more suitable for phylogenetic studies and the genetic structure of Fasciola parasites.

S8.5c MLST genotyping and phylogenetics of AD-hybrids

S8.5 Genotyping of Cryptococcus neoformans and C. gattii, September 23, 2022, 3:00 PM - 4:30 PMObjectivesIn a previous study a set of new molecular-type specific primers were designed to apply the standard ISHAM consensus multi-locus sequence typing (MLST) scheme to Cryptococcus neoformans AD hybrids. In the present study, we report the preliminary results of the investigation by MLST of a large number of AD hybrids with the aim to identify the circulating genotypes, their phylogenesis, and population genetics.MethodsA total of 50 AD-hybrid isolates from different parts of the world and from different sources were genotyped by MLST. Minimum spanning trees using GoeBurst algorithm were generated by comparing hybrid genotypes and by comparing separately either allele-A and allele-D portions of the hybrid genotypes to the haplotypes recorded in the MLST global database.ResultsAnalysis identified 32 hybrid genotypes grouped in three distinct main clusters (CC12, CC21, and CC30) including 12 isolates each. Both CC12 and CC21 clusters included isolates from different countries and continents but the former grouped only isolates with mating type aADalpha whereas the latter those with mating type alphaADa. Cluster CC30 included only isolates from Ivory Coasts. Heterozygous allelic combinations in each of the seven MLST loci presented two or three combinations more frequent than the other ones. In some isolates, one or more alleles were not amplified after multiple attempts, and therefore, they were considered as lacking. A total of 22 MLST profiles were identified by analyzing separately the allele-A combinations of the hybrids. Comparison with all MLST profiles of VNI, VNII, and VNB included in the MLST global database showed that the allele-A portion of the hybrid genotypes was grouped in few VNI or VNB clusters. In none of the investigated hybrids, the allele-A portion originated from VNII genotypes. Similarly, when the MLST profile of allele-D portion of hybrids was compared to all VNIV genotypes present in the global MLST database, few clusters were identified but, in this case, mostly originated from genotypes not yet found among VNIV haplotypes.ConclusionsThese preliminary results suggest that the AD hybrids here investigated originated from the mating of A haploids very common in both clinical and environmental isolates and D haploids that are not circulating at present or are very rare. Therefore, it is likely that hybrids originated in the environment where VNIV genotypic diversity is higher and suitable AD combinations can occur. Sequencing of further AD hybrids is in progress to confirm these results.

Analysis of bacterial diversity and genetic evolution of Lacticaseibacillus paracasei isolates in fermentation pit mud.

Since little is known about the genetic diversity of lactic acid bacteria (LAB) isolates from the fermentation pit mud (FPM), we sought to evaluate the bacterial structure, identify the LAB isolates and investigate the genotype and genetic diversity of the LAB isolates. Using high-throughput MiSeq sequencing, we identified seven dominant bacterial genera in FPM. Lactobacillus had the highest abundance. We isolated 55 LAB strains. These isolates were all identified as Lacticaseibacillus paracasei. Using an extant multilocus sequence typing (MLST) scheme, isolates were assigned to 18 sequence types (STs) and three clonal complexes. ST1, the largest group, mainly comprised FPM isolates. Niche-specific ST2 to ST18 only contained FPM isolates. Isolates could be divided into four lineages, with most assigned to Lineage 1. Only one FPM isolate was classified as L. paracasei subsp. paracasei. Other isolates could not be classified at the subspecies level using the seven MLST loci. Lactobacilli account for a high proportion of bacteria in pit mud. Based on the traditional culture method, L. paracasei was the dominant species, and these isolates exhibit a high ethanol tolerance, high intraspecific diversity and specific genetic profiles. The study described the characterization of FPM bacterial diversity, giving an insight into the genetic diversity of L. paracasei strains present in FPM.

Phenotypic and genotypic characterisation of Lactobacillus and yeast isolates from a traditional New Zealand Māori potato starter culture

Parāroa Rēwena is a traditional Māori sourdough produced by fermentation using a potato starter culture. The microbial composition of the starter culture is not well characterised, despite the long history of this product. The morphological, physiological, biochemical and genetic tests were conducted to characterise 26 lactic acid bacteria (LAB) and 15 yeast isolates from a Parāroa Rēwena potato starter culture. The results of sugar fermentation tests, API 50 CHL tests, and API ID 32 C tests suggest the presence of four different LAB phenotypes and five different yeast phenotypes. 16S rRNA and 26S rRNA sequencing identified the LAB as Lacticaseibacillus paracasei and the yeast isolates as Saccharomyces cerevisiae, respectively. Multilocus sequence typing (MLST) of the L. paracasei isolates indicated that they had identical genotypes at the MLST loci, to L. paracasei subsp. paracasei IBB 3423 or L. paracasei subsp. paracasei F19. This study provides new insights into the microbial composition of the traditional sourdough Parāroa Rēwena starter culture.

Prevalence and molecular characterization of Wolbachia in field-collected Aedes albopictus, Anopheles sinensis, Armigeres subalbatus, Culex pipiens and Cx. tritaeniorhynchus in China.

Wolbachia are maternally transmitted intracellular bacteria that can naturally and artificially infect arthropods and nematodes. Recently, they were applied to control the spread of mosquito-borne pathogens by causing cytoplasmic incompatibility (CI) between germ cells of females and males. The ability of Wolbachia to induce CI is based on the prevalence and polymorphism of Wolbachia in natural populations of mosquitoes. In this study, we screened the natural infection level and diversity of Wolbachia in field-collected mosquitoes from 25 provinces of China based on partial sequence of Wolbachia surface protein (wsp) gene and multilocus sequence typing (MLST). Among the samples, 2489 mosquitoes were captured from 24 provinces between July and September, 2014 and the remaining 1025 mosquitoes were collected month-by-month in Yangzhou, Jiangsu province between September 2013 and August 2014. Our results showed that the presence of Wolbachia was observed in mosquitoes of Aedes albopictus (97.1%, 331/341), Armigeres subalbatus (95.8%, 481/502), Culex pipiens (87.0%, 1525/1752), Cx. tritaeniorhynchus (17.1%, 14/82), but not Anopheles sinensis (n = 88). Phylogenetic analysis indicated that high polymorphism of wsp and MLST loci was observed in Ae. albopictus mosquitoes, while no or low polymorphisms were in Ar. subalbatus and Cx. pipiens mosquitoes. A total of 12 unique mutations of deduced amino acid were identified in the wsp sequences obtained in this study, including four mutations in Wolbachia supergroup A and eight mutations in supergroup B. This study revealed the prevalence and polymorphism of Wolbachia in mosquitoes in large-scale regions of China and will provide some useful information when performing Wolbachia-based mosquito biocontrol strategies in China.

Centre-specific bacterial pathogen typing affects infection-control decision making.

Whole-genome sequencing is becoming the de facto standard for bacterial outbreak surveillance and infection prevention. This is accompanied by a variety of bioinformatic tools and needs bioinformatics expertise for implementation. However, little is known about the concordance of reported outbreaks when using different bioinformatic workflows. In this multi-centre proficiency testing among 13 major Dutch healthcare-affiliated centres, bacterial whole-genome outbreak analysis was assessed. Centres who participated obtained two randomized bacterial datasets of Illumina sequences, a Klebsiella pneumoniae and a Vancomycin-resistant Enterococcus faecium, and were asked to apply their bioinformatic workflows. Centres reported back on antimicrobial resistance, multi-locus sequence typing (MLST), and outbreak clusters. The reported clusters were analysed using a method to compare landscapes of phylogenetic trees and calculating Kendall–Colijn distances. Furthermore, fasta files were analysed by state-of-the-art single nucleotide polymorphism (SNP) analysis to mitigate the differences introduced by each centre and determine standardized SNP cut-offs. Thirteen centres participated in this study. The reported outbreak clusters revealed discrepancies between centres, even when almost identical bioinformatic workflows were used. Due to stringent filtering, some centres failed to detect extended-spectrum beta-lactamase genes and MLST loci. Applying a standardized method to determine outbreak clusters on the reported de novo assemblies, did not result in uniformity of outbreak-cluster composition among centres.

Phylogeny and Strain Typing of Wolbachia from Yamatotettix flavovittatus Matsumura Leafhoppers.

Wolbachia is a maternally inherited bacterium of insects that can alter the reproduction, biology, and fitness of the hosts. It was detected in natural populations of the Yamatotettix flavovittatus Matsumura leafhoppers, the vector of phytoplasma, which is responsible for sugarcane white leaf disease. Wolbachia infection prolongs the longevity of female leafhoppers and promotes a strong reproductive incompatibility; importantly, highly maternal transmission rate was observed. However, limited data on the diversity or strain typing of Wolbachia in Y. flavovittatus are available. We aimed here to detect the presence of Wolbachia in different populations by amplification of the wsp gene, which was then sequenced. Multilocus sequence typing (MLST) was also performed to explore the diversity of the Wolbachia strains. Based on the wsp sequences, Wolbachia in the Y. flavovittatus leafhoppers belonged to supergroup B, and formed a distinct evolutionary lineage; therefore, we designated this new specific strain as wYfla. The MLST profiles revealed ten potential new sequence types (STs) in different leafhopper populations. Multiple STs were detected in individual leafhoppers, among which the ST-wYfla1 strain was predominant. Furthermore, we obtained congruent results for the phylogenetic analyses using the wsp gene and MLST loci. To the best of our knowledge, this is the first study characterizing Wolbachia strains in Y. flavovittatus. Our results reveal a novel strain and multiple STs of Wolbachia, and these data may prove useful in the exploitation of Wolbachia as a biological Y. flavovittatus control agent.

1199. Phylogenomic analysis of Campylobacter jejuni isolated from gastroenteritis cases in Michigan

Background C. jejuni is the leading cause of bacterial gastroenteritis worldwide. It has been classified as a serious antibiotic resistant threat, causing 13,000 hospitalizations and 120 deaths annually. Our goal was to describe the diversity of clinical C. jejuni using phylogenomics and classify resistance mechanisms.MethodsIsolates were collected via sentinel surveillance at four hospitals, and demographic and clinical data were obtained. DNA was extracted and sequenced. Raw reads were processed with Trimmomatic and quality checked with FastQC. De novo genome assembly was performed in Spades. Assembled genomes were filtered for quality and completeness; samples of 1.4-2.1MB were annotated in Prokka followed by pangenome and phylogenetic analyses. Multilocus sequence typing loci and virulence and antibiotic resistance genes were extracted from each genome.ResultsAmong the 214 C. jejuni isolates recovered, 86 unique sequence types (STs) were identified; five were novel STs with unique allele combinations. ST353 (8.3%: n=18), ST982 (7.4%: n=16), ST50 (5.1 %: n=11) and ST48 (5.1%: n=11) were the most prevalent STs identified, while the majority (50.1%: n=50) of STs were singletons. The pangenome analysis identified 8781, 615, and 1169 total, core, and shell core genes, respectively, which grouped the isolates into three major clades. Most isolates belonged to clade 1. A neighbor-net analysis detected significant recombination among all 86 STs (pairwise homoplasy index p=< 0.00001) and evidence of horizontal gene transfer across clades. The beta-lactamase gene, blaOXA-605, was the most common resistance gene identified (58.8%: n=125) followed by tet(O) (56.0%: n=121), which mediate resistance to beta-lactams and tetracyclines, respectively. Resistance phenotypes were confirmed using microbroth dilution.Conclusion: Together, these data demonstrate that the C. jejuni population is highly diverse and carries important resistance determinants. The phylogenomic analyses also provide insight into the evolution of this major foodborne pathogen. Future work will focus on identifying molecular and epidemiological factors associated with specific strain types and resistance and virulence profiles circulating in Michigan.Disclosures All Authors: No reported disclosures

New multilocus sequence typing primers to enable genotyping of AD hybrids within the Cryptococcus neoformans species complex.

We designed and tested new specific primers to amplify the two alleles of each of the seven MLST loci in C. neoformans species complex hybrids. The sequences obtained from hybrids can be compared with those present in the Cryptococcus global MLST database for future molecular epidemiology studies.

A multilocus sequence typing scheme of Pseudomonas putida for clinical and environmental isolates

Pseudomonas putida is a bacterium commonly found in soils, water and plants. Although P. putida group strains are considered to have low virulence, several nosocomial isolates with carbapenem- or multidrug-resistance have recently been reported. In the present study, we developed a multilocus sequence typing (MLST) scheme for P. putida. MLST loci and primers were selected and designed using the genomic information of 86 clinical isolates sequenced in this study as well as the sequences of 20 isolates previously reported. The genomes were categorised into 68 sequence types (STs). Significant linkage disequilibrium was detected for the 68 STs, indicating that the P. putida isolates are clonal. The MLST tree was similar to the haplotype network tree based on single nucleotide morphisms, demonstrating that our MLST scheme reflects the genetic diversity of P. putida group isolated from both clinical and environmental sites.

Identification of Haemophilus influenzae serotype e strains missing the fucK gene in clinical isolates from Japan

Introduction. Certain nontypeable Haemophilus influenzae cannot be assigned a sequence type (ST) by Multilocus Sequence Typing (MLST) due to the lack of the fucK gene, one of seven MLST loci in H. influenzae, which encodes a fucose-operon enzyme.Aims. To confirm whether the loss of fucK is also found in the encapsulated strains, we analysed clinical isolates of H. influenzae serotype e (Hie).Methodology. We conducted MLST, PFGE, and antimicrobial susceptibility tests of 45 Hie strains; the majority (n=43) were derived from respiratory samples of pediatric patients at Chiba Children's Hospital between 2000 and 2016. The two remaining strains were obtained from the blood of elderly patients with invasive H. influenzae diseases (IHiDs) between 2015 and 2016 at general hospitals. For the fucK-negative strains, PCR analysis for fucose operon was also performed.Results. Four STs (ST18, 122, 621 and 1758) were assigned to 13 strains, and remaining 32 (including one associated with IHiD) were fucK-negative, completely missing the fucose operon. The allelic profiles of six other loci were identical among 31 strains and to that of ST18, 122 and 621, and these strains were genetically closely related. Forty of 45 isolates were ampicillin-sensitive.Conclusions. The loss of fucK was frequently observed in clinical isolates of Hie from children. Moreover, fucK-negative Hie may be the cause of IHiD in adult patients. The majority of Hie, including fucK-negative strains, were shown to be clonally related and were ampicillin sensitive. This represents the first report examining fucK losses in encapsulated H. influenzae.

Tracing the environmental footprint of the Burkholderia pseudomallei lipopolysaccharide genotypes in the tropical "Top End" of the Northern Territory, Australia.

The Tier 1 select agent Burkholderia pseudomallei is an environmental bacterium that causes melioidosis, a high mortality disease. Variably present genetic markers used to elucidate strain origin, relatedness and virulence in B. pseudomallei include the Burkholderia intracellular motility factor A (bimA) and filamentous hemagglutinin 3 (fhaB3) gene variants. Three lipopolysaccharide (LPS) O-antigen types in B. pseudomallei have been described, which vary in proportion between Australian and Asian isolates. However, it remains unknown if these LPS types can be used as genetic markers for geospatial analysis within a contiguous melioidosis-endemic region. Using a combination of whole-genome sequencing (WGS), statistical analysis and geographical mapping, we examined if the LPS types can be used as geographical markers in the Northern Territory, Australia. The clinical isolates revealed that LPS A prevalence was highest in the Darwin and surrounds (n = 660; 96% being LPS A and 4% LPS B) and LPS B in the Katherine and Katherine remote and East Arnhem regions (n = 79; 60% being LPS A and 40% LPS B). Bivariate logistics regression of 999 clinical B. pseudomallei isolates revealed that the odds of getting a clinical isolate with LPS B was highest in East Arnhem in comparison to Darwin and surrounds (OR 19.5, 95% CI 9.1–42.0; p<0.001). This geospatial correlation was subsequently confirmed by geographically mapping the LPS type from 340 environmental Top End strains. We also found that in the Top End, the minority bimA genotype bimABm has a similar remote region geographical footprint to that of LPS B. In addition, correlation of LPS type with multi-locus sequence typing (MLST) was strong, and where multiple LPS types were identified within a single sequence type, WGS confirmed homoplasy of the MLST loci. The clinical, sero-diagnostic and vaccine implications of geographically-based B. pseudomallei LPS types, and their relationships to regional and global dispersal of melioidosis, require global collaborations with further analysis of larger clinically and geospatially-linked datasets.

Multilocus sequence typing analysis of Candida africana from vulvovaginal candidiasis

BackgroundCandida africana is distributed worldwide and colonized in human genitalia and cause mainly vulvovaginal candidiasis (VVC). We report the multilocus sequence typing (MLST) analysis of C. africana from VVC.MethodsMLST analysis of 43 strains of C. africana, which were isolated from vaginal specimens of patients with VVC, was performed. The enzymatic activity of phospholipase, esterase and haemolysis enzyme production was evaluated.The level of virulent genes and resistant genes mRNA expression was determined by using real-time PCR. Antifungal susceptibilities of the isolates were assayed by using the broth microdilution method. The statistical of the results was determined by the T test and Pearson chi-squared test.ResultsThe MLST analysis revealed a substantial degree of genetic homogeneity. The DST782 and DST182 were the main MLST genotypes in C. africana. All the patients were symptomatic and with a high mycological cure rate when treated with commonly used antifungal agents.There were statistically significant differences in biofilm formation and phospholipase activity between C. africana and C.albicans. The level of virulent genes and resistant genes mRNA expression was higher in fluconazole-resistant strains. All C. africana isolates were susceptible to fluconazole, itraconazole, voriconazole, caspofungin, and micafungin. These isolates also exhibited low MICs to amphotericin B, flucytosine, and posaconazole.ConclusionsCandida africana appear to be with a low level of sequence variation in MLST loci. Candida africana, a lower virulence candida, is susceptible to commonly used antifungal agents.This paper was presented at the conference of 8th Trend in Medical Mycology (6–9 October 2017, Belgrade, Serbia) and was published on conference abstract.

A six-member SNP assay on the iPlex MassARRAY platform provides a rapid and affordable alternative for typing major African Staphylococcus aureus types.

Purpose Data on the clonal distribution of Staphylococcus aureus in Africa are scanty, partly due to the high costs and long turnaround times imposed by conventional genotyping methods such as spa and multilocus sequence typing (MLST), which means there is a need for alternative typing approaches. This study evaluated the discriminatory power, cost of and time required for genotyping Kenyan staphylococcal isolates using iPlex MassARRAY compared to conventional methods.Methodology Fifty-four clinical S. aureus isolates from three counties were characterized using iPlex MassARRAY, spa and MLST typing methods. Ten single-nucleotide polymorphisms (SNPs) from the S. aureus MLST loci were assessed by MassARRAY.>Results The MassARRAY assay identified 14 unique SNP genotypes, while spa typing and MLST revealed 22 spa types and 21 sequence types (STs) that displayed unique regional distribution. spa type t355 (ST152) was the dominant type overall while t037/t2029 (ST 241) dominated among the methicillin-resistant S. aureus (MRSA) isolates. MassARRAY showed 83 % and 82 % accuracy against spa typing and MLST, respectively, in isolate classification. Moreover, MassARRAY identified all MRSA strains and a novel spa type. MassARRAY had a reduced turnaround time (<12 h) compared to spa typing (4 days) and MLST (20 days). The MassARRAY reagent and consumable costs per isolate were approximately $18 USD compared to spa typing ($30 USD) and MLST ($126 USD).Conclusion This study demonstrated that iPlex MassARRAY can be adapted as a useful surveillance tool to provide a faster, more affordable and fairly accurate method for genotyping African S. aureus isolates to identify clinically significant genotypes, MRSA strains and emerging strain types.

Genetic differences in Chlamydia pecorum between neighbouring sub-populations of koalas (Phascolarctos cinereus)

Chlamydiosis, caused by Chlamydia pecorum, is regarded as an important threat to koala populations. Across the koala’s geographical range, disease severity associated with C. pecorum infection varies, with pathogen diversity and strain pathogenicity being likely important factors. To examine C. pecorum diversity on a sub-population level a Multi-Locus Sequence Typing (MLST) scheme, containing the housekeeping genes; gatA, oppA_3, hflX, gidA, enoA, hemN and fumC, was used to type strains from two sub-populations of koalas from the Liverpool Plains, NSW, Australia, with different disease expressions. Typing of samples from 2015 to 2017, revealed a significant association between sequence type ST 69 and clinical disease and a significant difference in sequence type frequencies between sub-populations. Sequence type ST 69 has previously been identified in both subclinical and clinically diseased koalas indicating that these markers alone are not illustrative of pathogenicity. However, recent emergence of this sequence type in a naïve population may explain the differing disease expressions. Sequence types ST 73 and ST 69 have been described in koalas across a broad geographic range, indicating multiple introduction events and/or a limited veracity of the MLST loci to explore fine scale epidemiological investigations, particularly those examining the interface between pathogenic strain and disease outcome.

Intensive targeting of regulatory competence genes by transposable elements in streptococci.

Competence for natural transformation is a widespread developmental process of streptococci. By allowing the uptake and recombination of exogenous naked DNA into the genome, natural transformation, as transposable elements, plays a key role in the plasticity of bacterial genomes. We previously analysed the insertion sites of IS1548, an insertion sequence present in Streptococcus agalactiae and S. pyogenes, and showed that some targeted loci are involved in competence induction. In this work, we investigated on a large scale if loci coding for early competence factors (ComX and the two pheromone-dependent signalling systems ComCDE and ComRS) of streptococci are especially targeted by transposable elements. The transposable elements inserted in regions surrounding these genes and housekeeping genes used for Multilocus Sequence Typing (MLST) were systematically searched for. We found numerous insertion events in the close vicinity of early competence genes, but only very few into the MLST loci. The incidence of transposable elements, mainly insertion sequences, is particularly high in the intergenic regions surrounding comX alleles in numerous species belonging to most streptococcal groups. The identification of scarce disruptive insertions inside early competence genes indicates that the maintenance of competence is essential for streptococci. The specific association of transposable elements with intergenic regions bordering the main regulatory genes of competence may impact on the induction of transformability and so, on the genome plasticity and adaptive evolution of streptococci. This widespread phenomenon brings new perspectives on our understanding of competence regulation and its role in the bacterial life cycle.

Ruminant and chicken: important sources of campylobacteriosis in France despite a variation of source attribution in 2009 and 2015

Pathogen source attribution studies are a useful tool for identifying reservoirs of human infection. Based on Multilocus Sequence Typing (MLST) data, such studies have identified chicken as a major source of C. jejuni human infection. The use of whole genome sequence-based typing methods offers potential to improve the precision of attribution beyond that which is possible from 7 MLST loci. Using published data and 156 novel C. jejuni genomes sequenced in this study, we performed probabilistic host source attribution of clinical C. jejuni isolates from France using three types of genotype data: comparative genomic fingerprints; MLST genes; 15 host segregating genes previously identified by whole genome sequencing. Consistent with previous studies, chicken was an important source of campylobacteriosis in France (31–63% of clinical isolates assigned). There was also evidence that ruminants are a source (22–55% of clinical isolates assigned), suggesting that further investigation of potential transmission routes from ruminants to human would be useful. Additionally, we found evidence of environmental and pet sources. However, the relative importance as sources varied according to the year of isolation and the genotyping technique used. Annual variations in attribution emphasize the dynamic nature of zoonotic transmission and the need to perform source attribution regularly.

Phenotypic and molecular characterization of Klebsiella spp. isolates causing community-acquired infections.

Klebsiella spp. isolates from community-acquired infections were characterized. A total of 39 Klebsiella spp. isolates were obtained from outpatients at four rural hospitals in Mexico (2013–2014). The biochemical tests identified all as being K. pneumoniae. The molecular multiplex-PCR test identified 36 (92.4%) K. pneumoniae isolates and one (2.5%) K. variicola isolate, and phylogenetic analysis of the rpoB gene identified two isolates (5.1%) belonging to K. quasipneumoniae subsp. quasipneumoniae and K. quasivariicola. The last one was confirmed by phylogenetic analysis of six-loci concatenated genes. Mostly the isolates were multidrug resistant; however, a minority were extended-spectrum β-lactamase producing (10.2%). The extended-spectrum β-lactamase CTX-M-15 gene was identified in these isolates. Analysis of biofilm production and the hypermucoviscosity phenotype showed a total of 35 (92.3%) and seven (17.9%) of the isolates were positive for these phenotypes respectively. The K2 (4/39, 10.2%), K5 (2/39, 5.1%) and K54 (1/39, 2.5%) serotypes were identified in seven (17.9%) of the isolates, and only 28.5% (2/7) hypermucoviscous isolates were positive for the K2 and K5 serotypes. In general, the sequence type (ST) analysis and phylogenetic analysis of seven multilocus sequence typing loci were heterogeneous; however, ST29 was the most prevalent ST in the analysed isolates, accounting for 19% (4/21) of the total isolates. Two of the four ST29 isolates had the hypermucoviscosity phenotype. The virulence factors for fimbriae were the most prevalent, followed by siderophores. Community-acquired infections are caused by various species from Klebsiella genus, with different profiles of antibiotic resistance and heterogeneous virulence factors.