Multilocus Sequence Typing Scheme Research Articles

RIPS (rapid intuitive pathogen surveillance): a tool for surveillance of genome sequence data from foodborne bacterial pathogens.

Monitoring data submitted to the National Center for Biotechnology Information's Pathogen Detection whole-genome sequence database, which includes the foodborne bacterial pathogens Listeria monocytogenes, Salmonella enterica, and Escherichia coli, has proven effective for detecting emerging outbreaks. As part of the submission process, new sequence data are typed using a whole-genome multi-locus sequence typing scheme and clustered with sequences already in the database. Publicly available text files contain the results of these analyses. However, contextualizing and interpreting this information is complex. We present the Rapid Intuitive Pathogen Surveillance (RIPS) tool, which shows the results of the NCBI Rapid Reports, along with appropriate metadata, in a graphical, interactive dashboard. RIPS makes the information in the Rapid Reports useful for real-time surveillance of genome sequence databases.

Multi-locus sequencing typing reveals geographically related intraspecies variability of Sporothrix brasiliensis

Sporotrichosis is a subcutaneous mycosis caused by pathogenic Sporothrix species. Among them, Sporothrix brasiliensis is the main species associated with endemic regions in South America, especially Brazil. It is highly virulent and can be spread through zoonotic transmission. Molecular epidemiological surveys are needed to determine the extent of genetic variation, to investigate outbreaks, and to identify genotypes associated with antifungal resistance and susceptibility. This study investigated the sequence variation of different constitutive genes and established a novel multilocus sequence typing (MLST) scheme for S. brasiliensis. Specific primers were designed for 16 genes using Primer-BLAST software based on the genome sequences of three S. brasiliensis strains (ATCC MYA-4823, A001 and A005). Ninety-one human, animal, and environmental S. brasiliensis isolates from different Brazilian geographic regions (South, Southeast, Midwest and Northeast) andtwo isolates from Paraguay were sequenced. The loci that presented the highest nucleotide diversity (π) were selected for the MLST scheme. Among the 16 studied genetic loci, four presented increased π value and were able to distinguish all S. brasiliensis isolates into seven distinct haplotypes. The PCR conditions were standardized for four loci. Some of the obtained haplotypes were associated with the geographic origin of the strains. This study presents an important advance in the understanding of this important agent of sporotrichosis in Brazil. It significantly increased the discriminatory power for genotyping of S. brasiliensis isolates, and enabled new contributions to the epidemiological studies of this human and animal pathogen in Brazil and in other countries.

1932. Molecular Epidemiology of NDM-producing Acinetobacter baumannii in the US—October 2013—March 2022

Abstract Background In the United States, most carbapenem-resistant Acinetobacter baumannii (CRAB) produce carbapenem-hydrolyzing class D β-lactamases (OXA); other carbapenemase classes have historically been uncommon. We describe the epidemiology and molecular characteristics of New Delhi Metallo-β-lactamase (NDM)-producing CRAB reported to CDC since October 2013.Figure 1:A core genome multilocus sequence typing (cgMLST) scheme phylogenetic tree of 208 the New Delhi Metallo-β-lactamase (NDM), sequenced by US public health laboratories and with publicly available sequences, with dates of collection between November 1, 2018 – March 31, 2022. Four regional clusters were identified in Mountain, Mid-Atlantic, Midwest and West regions. Isolates within each cluster were closely related to each other. Methods We included a patient’s first NDM-CRAB isolate reported to CDC through reference antimicrobial susceptibility testing, outbreak response, or Antimicrobial Resistance Laboratory Network, with specimen collection dates during 10/1/2013–3/31/2022. Two or more NDM-CRAB linked in space and time were classified as cluster-associated; other NDM-CRAB as sporadic. We described patient demographics and clinical characteristics and analyzed whole genome sequence (WGS) data for relatedness using both traditional multilocus sequence typing (MLST) with the Oxford scheme (STOX) and core genome (cg) MLST. We compared publicly available sequences of US NDM-CRAB to NDM-CRAB from non-US locations. Results CDC received reports of 263 NDM-CRAB from unique patients in 21 states. Patients had a median age of 65 years (IQR: 55-74) (Table 1). NDM-CRAB were from clinical cultures of respiratory (60), wound (44), urine (32), blood (11), and other sites (11) and from rectal or axilla/groin cultures (96). Eleven of 103 patients with information available were hospitalized outside the US ≤12 months prior to NDM-CRAB specimen collection, 82% were sporadic cases and 19% were a part of clusters. Overall, 191 were from cluster-associated cases in four distinct geographic regions that reported multifacility outbreaks; all cluster-associated cases were from cultures collected after June 15, 2019. WGS data were available for 208 (79%) isolates. The four regional outbreaks identified in the epidemiologic investigation were different STOX (Fig. 1); three (ST218, ST281, and ST1697) were closely related to NDM-CRAB isolates from outside the US. Conclusion NDM-CRAB reported to CDC appears to reflect domestic acquisition rather than importation. ST differences across the regional outbreaks indicate they arose from unique introductions rather than inter-regional spread or dissemination of a successful clone. Disclosures All Authors: No reported disclosures

2180. Pseudomonas aeruginosa epidemic high-risk clones and their association with multidrug-resistant

Abstract Background Pseudomonas aeruginosa (PAE) is an opportunistic pathogen associated with a variety of hospital infections. Treatment of infections caused by PAE is challenging due to the emergence of multidrug-resistant (MDR), which is associated with increased morbidity, mortality, and healthcare costs. Clonal spread and the presence of resistance genes probably explain the successful propagation of MDR. In Ecuador, data on the molecular epidemiology, as well as circulating clones, are limited. Therefore, the objective of this study is to know the population structure of PAE by identifying clones in clinical samples in Quito-Ecuador. Methods A significant set (45) of randomly selected clinical PAE isolates, including multidrug and non-multidrug resistant isolates, were assigned to sequence types (STs) and compared them with their antibiotic susceptibility profile. The genetic diversity was assessed by applying the multilocus sequence typing (MLST) scheme (Figure 1) and the genetic relationships between different STs were corroborated by the phylogenetic neighbor-net network (Figure 2).Figure 1.Analysis of the allelic profiles using eBURST algorithm.Figure 2.Splits Tree showing the distribution of all the sequence types obtained for the clinical Pseudomonas aeruginosa isolates studied. The Splits Tree was based on the analysis of the allelic profiles of the acsA, aroE, guaA, mutL, nuoD, ppsA and trpE genes. Asterisk mark (*) indicates the MDR isolates Results The analysis of seven loci, acsA, aroE, guaA, mutL, nuoD, ppsA and trpE, identified 24 different ST and the most prevalent STs were ST-3750 and ST-253. A SplitsTree was constructed with all the isolates analyzed. The majority of the MDR isolates were included in ST-3750 and ST-253, also 3 singleton ST was identified in to MDR isolates. The 20 different ST was found to non-MDR isolates, and only 3 ST were found in more the one isolates. Conclusion The population structure of clinical PAE presents in these isolates indicate a significant association between MDR isolates and the clonal types: all ST-3750 and ST-253 isolates were MDR. ST-3750 is closely related strains to the ST111 (CC111), and ST-253 and ST111 are a group of successful high-risk clones and widely distributed worldwide. The multiresistant-isolates studied are grouped in the most prevalent sequence types found, and the susceptible isolates correspond mainly to singleton sequence types. Therefore, these high-risk clones and their association with multidrug resistance phenotypes are contributing to the spread of MDR in Quito-Ecuador. Disclosures Jeannete Zurita, n/a, Pfizer: Grant/Research Support

Genetic markers of Chlamydia pecorum virulence in ruminants support short term host-pathogen evolutionary relationships in the koala, Phascolarctos cinereus

In ruminants infected with Chlamydia pecorum, shorter lengths of coding tandem repeats (CTR) within two genes, the inclusion membrane protein (incA) and Type III secretor protein (ORF663), have been previously associated with pathogenic outcomes. In other chlamydial species, the presence of a chlamydial plasmid has been linked to heightened virulence, and the plasmid is not ubiquitous in C. pecorum across the koala's range. We therefore investigated these three markers: incA, ORF663 and C. pecorum plasmid, as potential indicators of virulence in two koala populations in New South Wales with differing expression of urogenital chlamydiosis; the Liverpool Plains and one across the Southern Highlands and South-west Sydney (SHSWS). We also investigated the diversity of these loci within strains characterised by the national multi-locus sequence typing (MLST) scheme. Although CTR lengths of incA and ORF663 varied across the populations, they occurred only within previously described pathogenic ranges for ruminants. This suggests a relatively short-term host-pathogen co-evolution within koalas and limits the utility of CTR lengths for incA and ORF663 as virulence markers in the species. However, in contrast to reports of evolution of C. pecorum towards lower virulence, as indicated by longer CTR lengths in ruminants and swine, CTR lengths for ORF663 appeared to be diverging towards less common shorter CTR lengths within strains recently introduced to koalas in the Liverpool Plains. We detected the plasmid across 90% and 92% of samples in the Liverpool Plains and SHSWS respectively, limiting its utility as an indicator of virulence. It would be valuable to examine the CTR lengths of these loci across koala populations nationally. Investigation of other hypervariable loci may elucidate the evolutionary trajectory of virulence in C. pecorum induced disease in koalas. Profiling of virulent strains will be important in risk assessments for strain movement to naïve or susceptible populations through translocations and wildlife corridor construction.

Core-genome multilocus sequence typing and core-SNP analysis of Clostridium neonatale strains isolated in different spatio-temporal settings.

Clostridium neonatale was described as a new species within the Clostridium genus cluster I sensu stricto. It was recovered from the gut microbiota of infants and has been associated with necrotizing enterocolitis (NEC), a severe gastrointestinal disease affecting preterm neonates. In the absence of molecular typing methods, we developed a core (cg)-genome multilocus sequence typing (MLST) scheme based on 48 newly sequenced and 12 publicly available genomes of C. neonatale. Using the ChewBBACA algorithm, a stable MLST scheme consisting of 2,350 target genes (with genomes having ≥95% cgMLST targets) is proposed. The strains are distributed among five clades, and while some are clonality related within clades, the C. neonatale strain distribution is independent of geographic or temporal clustering. When considering strains isolated from patients with and without NEC, there were no observable differences in clustering. When compared to a core-single nucleotide polymorphism (SNP) analysis approach (31,248 positions), we showed that both cgMLST and cgSNP have comparable genetic discrimination of strains. In this study, we validated the use of cgMLST and cgSNP-based typing methods for the genetic comparison of clinical isolates of C. neonatale, which will allow for future surveillance and epidemiological clinical investigations of this potential opportunistic pathogen.IMPORTANCEClostridium neonatale has been isolated from the fecal samples of asymptomatic neonates and cases of necrotizing enterocolitis (NEC). Taking advantage of a large collection of independent strains isolated from different spatio-temporal settings, we developed and established a cgMLST scheme for the molecular typing of C. neonatale. Both the cgMLST and cgSNP methods demonstrate comparable discrimination power. Results indicate geographic- and temporal- independent clustering of C. neonatale NEC-associated strains. No specific cgMLST clade of C. neonatale was genetically associated with NEC.

Phylogenomics of Acinetobacter species and analysis of antimicrobial resistance genes.

Non-baumannii Acinetobacter species are increasingly isolated in the clinical setting and the environment. The aim of the present study was to analyze a genome database of 837 Acinetobacter spp. isolates, which included 798 non-baumannii Acinetobacter genomes, in order to define the concordance of classification and discriminatory power of 7-gene MLST, 53-gene MLST, and single-nucleotide polymorphism (SNPs) phylogenies. Phylogenies were performed on Pasteur Multilocus Sequence Typing (MLST) or ribosomal Multilocus Sequence Typing (rMLST) concatenated alleles, or SNPs extracted from core genome alignment. The Pasteur MLST scheme was able to identify and genotype 72 species in the Acinetobacter genus, with classification results concordant with the ribosomal MLST scheme. The discriminatory power and genotyping reliability of the Pasteur MLST scheme were assessed in comparison to genome-wide SNP phylogeny on 535 non-baumannii Acinetobacter genomes assigned to Acinetobacter pittii, Acinetobacter nosocomialis, Acinetobacter seifertii, and Acinetobacter lactucae (heterotypic synonym of Acinetobacter dijkshoorniae), which were the most clinically relevant non-baumannii species of the A. baumannii group. The Pasteur MLST and SNP phylogenies were congruent at Robinson-Fould and Matching cluster tests and grouped genomes into four and three clusters in A. pittii, respectively, and one each in A. seifertii. Furthermore, A. lactucae genomes were grouped into one cluster within A. pittii genomes. The SNP phylogeny of A. nosocomialis genomes showed a heterogeneous population and did not correspond to the Pasteur MLST phylogeny, which identified two recombinant clusters. The antimicrobial resistance genes belonging to at least three different antimicrobial classes were identified in 91 isolates assigned to 17 distinct species in the Acinetobacter genus. Moreover, the presence of a class D oxacillinase, which is a naturally occurring enzyme in several Acinetobacter species, was found in 503 isolates assigned to 35 Acinetobacter species. In conclusion, Pasteur MLST phylogeny of non-baumannii Acinetobacter isolates coupled with in silico detection of antimicrobial resistance makes it important to study the population structure and epidemiology of Acinetobacter spp. isolates.

The Molecular Genetic Epidemiology and Antimicrobial Resistance of Neisseria gonorrhoeae Strains Obtained from Clinical Isolates in Central Panama.

We aim to analyze Neisseria gonorrhoeae isolates in central Panama, characterize the associated gonococcal antimicrobial resistance (AMR) and conduct molecular epidemiology and genetic typing. We conducted a retrospective study based on N. gonorrhoeae hospital isolates collected between 2013 and 2018. AMR was determined using dilution agar and Etest®. Molecular typing was conducted using the Multilocus Sequence Typing (ST) scheme. The isolates analyzed (n = 30) showed resistance to penicillin (38%), tetracycline (40%), and ciprofloxacin (30%), and sensitivity to extended-spectrum cephalosporins and azithromycin. We identified 11 STs, the most frequent of which was ST1901 among the strains with decreased sensitivity and resistance to three types of antibiotics. We identified eight variations for the penA gene, all non-mosaic, with type II LVG as the most frequent (50%). To the best of our knowledge, we conducted the first Central American genomic study that analyzes a collection of gonococcal isolates, which represents a benchmark for future epidemiological and molecular genetic studies. The high prevalence of ciprofloxacin, tetracycline, and penicillin resistance, in addition to the identification of the worldwide spread of multidrug-resistant clone ST1901, should prompt the continuous and reinforced surveillance of AMR, including the molecular epidemiology of N. gonorrhoeae in Panama.

A core-genome multilocus sequence typing scheme for the detection of genetically related Streptococcus pyogenes clusters.

The recently observed increase in invasive Streptococcus pyogenes infections causes concern in Europe. However, conventional molecular typing methods lack discriminatory power to aid investigations of outbreaks caused by S. pyogenes. Therefore, there is an urgent need for high-resolution molecular typing methods to assess genetic relatedness between S. pyogenes isolates. In the current study, we aimed to develop a novel high-resolution core-genome multilocus sequence typing (cgMLST) scheme for S. pyogenes and compared its discriminatory power to conventional molecular typing methods. The cgMLST scheme was designed with the commercial Ridom SeqSphere+ software package. To define a cluster threshold, the scheme was evaluated using publicly available data from nine defined S. pyogenes outbreaks in the United Kingdom. The cgMLST scheme was then applied to 23 isolates from a suspected S. pyogenes outbreak and 117 S. pyogenes surveillance isolates both from the Netherlands. MLST and emm-typing results were used for comparison to cgMLST results. The allelic differences between isolates from defined outbreaks ranged between 6 and 31 for isolates with the same emm-type, resulting in a proposed cluster threshold of <5 allelic differences out of 1,095 target loci. Seven out of twenty-three (30%) isolates from the suspected outbreak had an allelic difference of <2, thereby identifying a potential cluster that could not be linked to other isolates. The proposed cgMLST scheme shows a higher discriminatory ability when compared to conventional typing methods. The rapid and simple analysis workflow allows for extended detection of clusters of potential outbreak isolates and surveillance and may facilitate the sharing of sequencing results between (inter)national laboratories.

Classification and prediction of Klebsiella pneumoniae strains with different MLST allelic profiles via SERS spectral analysis.

The Gram-negative non-motile Klebsiella pneuomoniae is currently a major cause of hospital-acquired (HA) and community-acquired (CA) infections, leading to great public health concern globally, while rapid identification and accurate tracing of the pathogenic bacterium is essential in facilitating monitoring and controlling of K. pneumoniae outbreak and dissemination. Multi-locus sequence typing (MLST) is a commonly used typing approach with low cost that is able to distinguish bacterial isolates based on the allelic profiles of several housekeeping genes, despite low resolution and labor intensity of the method. Core-genome MLST scheme (cgMLST) is recently proposed to sub-type and monitor outbreaks of bacterial strains with high resolution and reliability, which uses hundreds or thousands of genes conserved in all or most members of the species. However, the method is complex and requires whole genome sequencing of bacterial strains with high costs. Therefore, it is urgently needed to develop novel methods with high resolution and low cost for bacterial typing. Surface enhanced Raman spectroscopy (SERS) is a rapid, sensitive and cheap method for bacterial identification. Previous studies confirmed that classification and prediction of bacterial strains via SERS spectral analysis correlated well with MLST typing results. However, there is currently no similar comparative analysis in K. pneumoniae strains. In this pilot study, 16 K. pneumoniae strains with different sequencing typings (STs) were selected and a phylogenetic tree was constructed based on core genome analysis. SERS spectra (N = 45/each strain) were generated for all the K. pneumoniae strains, which were then comparatively classified and predicted via six representative machine learning (ML) algorithms. According to the results, SERS technique coupled with the ML algorithm support vector machine (SVM) could achieve the highest accuracy (5-Fold Cross Validation = 100%) in terms of differentiating and predicting all the K. pneumoniae strains that were consistent to corresponding MLSTs. In sum, we show in this pilot study that the SERS-SVM based method is able to accurately predict K. pneumoniae MLST types, which has the application potential in clinical settings for tracing dissemination and controlling outbreak of K. pneumoniae in hospitals and communities with low costs and high rapidity.

Phenotypic and genotypic characterization of Neisseria gonorrhoeae isolates from Yaoundé, Cameroon, 2019 to 2020.

This study investigated antimicrobial resistance (AMR) phenotypes and genotypes exhibited by Neisseria gonorrhoeae from Yaoundé, Cameroon. AMR to tetracycline, penicillin and ciprofloxacin was observed although none of the isolates had reduced susceptibility to azithromycin, cefixime or ceftriaxone. Whole genome sequence (WGS) data were obtained and, using a threshold of 300 or fewer locus differences in the N. gonorrhoeae core gene multilocus sequence typing (cgMLST) scheme, four distinct core genome lineages were identified. Publicly available WGS data from 1355 gonococci belonging to these four lineages were retrieved from the PubMLST database, allowing the Cameroonian isolates to be examined in the context of existing data and compared with related gonococci. Examination of AMR genotypes in this dataset found an association between the core genome and AMR with, for example, isolates belonging to the core genome group, Ng_cgc_300 : 21, possessing GyrA and ParC alleles with amino acid substitutions conferring high-level resistance to ciprofloxacin while lineages Ng_cgc_300 : 41 and Ng_cgc_300 : 243 were predicted to be susceptible to several antimicrobials. A core genome lineage, Ng_cgc_300 : 498, was observed which largely consisted of gonococci originating from Africa. Analyses from this study demonstrate the advantages of using the N. gonorrhoeae cgMLST scheme to find related gonococci to carry out genomic analyses that enhance our understanding of the population biology of this important pathogen.

Characterization Integrons and Gene Cassettes in Trimethoprim/Sulfamethoxazole-resistant Stenotrophomonas maltophilia

Background: Integrons in Stenotrophomonas maltophilia are the major mechanism for trimethoprim/sulfamethoxazole (TMP/SMX) resistance. Molecular epidemiology of S. maltophilia with integrons has not been studied till now, and gene cassettes of the integron in S. maltophilia may change with time. Aim: Molecular typing and interrelatedness between TMP/SMX-resistant S. maltophilia (TSRSM) isolates in a hospital in 2017 were analyzed and gene context of cassettes in integrons was studied. Methods: Molecular typing was determined through a multilocus sequence typing (MLST) scheme, while pulsed-field gel electrophoresis (PFGE) was used for relatedness analysis of TSRSM with integrons. Mapping of gene cassettes in the integron was also performed through sequencing. Results: From 214 S. maltophilia isolates collected in 2017, 34 of them (15.9%) were TSRSM. A total of 20 (58.8%) from 34 TSRSM isolates harboring the class 1 integron were analyzed. The MLST analysis revealed 11 different sequence types, 5 out of which were novel STs (ST 830, ST 833, ST 836, ST 837, and ST 839), suggesting a wide genetic diversity. There were two clones with intrahospital dissemination between different hospital settings, according to PFGE. Mapping of gene cassettes of the integron revealed four novel combinations of multiresistance genes (aacA4-aadA5, aacA7-catB, cmlA10-aadA2, and aacA4-aphA15-catB3), indicating the continued evolutionary change of the gene cassettes. Conclusion: Evidence of clonal transmission within the hospital and continuous change of multiresistant gene combinations in the cassettes of the integron showed that S. maltophilia with resistance integrons may play a role in the spread of antimicrobial resistance.

Genomic analysis of the MLST population structure and antimicrobial resistance genes associated with Salmonella enterica in Mexico.

Salmonella enterica is one of the most commonly reported foodborne pathogens by public health agencies worldwide. In this study, the multilocus sequence typing (MLST) population structure and frequency of antimicrobial resistance (AMR) genes were evaluated in S. enterica strains from Mexico (n=2561). The most common sources of isolation were food (44.28%), environment (27.41%), animal-related (24.83%), and human (3.48%). The most prevalent serovars were Newport (8.51%), Oranienburg (7.03%), Anatum (5.78%), Typhimurium (5.12%), and Infantis (4.57%). As determined by the 7-gene MLST scheme, the most frequent sequence types were ST23, ST64, and ST32. The core genome MLST scheme identified 132 HC2000 and 195 HC900 hierarchical clusters, with the HC2000_2 cluster being the most prevalent in Mexico (n=256). A total of 78 different AMR genes belonging to 13 antimicrobial classes were detected in 638 genomic assemblies of S. enterica. The most frequent class was aminoglycosides (31.76%), followed by tetracyclines (12.53%) and sulfonamides (11.91%). These results can help public health agencies in Mexico prioritize their efforts and resources to increase the genomic sequencing of circulating Salmonella strains. Additionally, they provide valuable information for local and global public health efforts to reduce the impact of foodborne diseases and AMR.

Acinetobacter baumannii from grass: novel but non-resistant clones.

Acinetobacter baumannii is one the most worrisome nosocomial pathogens, which has long been considered almost mainly as a hospital-associated bacterium. There have been some studies about animal and environmental isolates over the last decade. However, little effort has been made to determine if this pathogen dwells in the grass. Here, we aim to determine the evolutionary relationships and antibiotic resistance of clones of A. baumannii sampled from grass to the major human international clones and animal clones. Two hundred and forty genomes were considered in total from four different sources for this study. Our core and accessory genomic epidemiology analyses showed that grass isolates cluster in seven groups well differentiated from one another and from the major human and animal isolates. Furthermore, we found new sequence types under both multilocus sequence typing schemes: two under the Pasteur scheme and seven for the Oxford scheme. The grass isolates contained fewer antibiotic-resistance genes and were not resistant to the antibiotics tested. Our results demonstrate that these novel clones appear to have limited antibiotic resistance potential. Given our findings, we propose that genomic epidemiology and surveillance of A. baumannii should go beyond the hospital settings and consider the environment in an explicit One Health approach.

Household transmission of non-toxigenic diphtheria toxin gene-bearing Corynebacterium diphtheriae following a cluster of cutaneous cases in a specialist outpatient setting.

Introduction. Combination of PCR and Elek testing to identify toxigenic corynebacteria has revealed organisms described as non-toxigenic toxin-gene bearing (NTTB) Corynebacterium diphtheriae or C. ulcerans (i.e. PCR tox positive; Elek negative). These organisms carry part or all of tox, but are unable to express diphtheria toxin (DT) and present a challenge to clinical and public health case management.Gap analysis/Hypothesis. There are few data on the theoretical risk of NTTB reversion to toxigenicity. This unique cluster and subsequent epidemiologically linked isolates allowed the opportunity to determine any change in DT expression status.Aim. To characterize a cluster of infections due to NTTB in a skin clinic and subsequent cases in two household contacts.Methodology. Epidemiological and microbiological investigations were carried out according to existing national guidance at the time. Susceptibility testing used gradient strips. The tox operon analysis and multi-locus sequence typing (MLST) was derived from whole-genome sequencing. Alignment of the tox operon and phylogenetic analyses were performed using clustalW, mega, the public core-genome MLST (cgMLST) scheme and an in-house bioinformatic single nucleotide polymorphism (SNP) typing pipeline.Results. Isolates of NTTB C. diphtheriae were recovered from four cases (cases 1 to 4) with epidermolysis bullosa attending the clinic. Two further isolates were subsequently recovered from case 4, >18 months later, and from two household contacts (cases 5 and 6) after a further 18 months and 3.5 years, respectively. All eight strains were NTTB C. diphtheriae biovar mitis, belonged to the same sequence type (ST-336) with the same deletion in tox. Phylogenetic analysis showed relatively high diversity between the eight strains with 7-199 SNP and 3-109 cgMLST loci differences between them. The number of SNPs between the three isolates from case 4 and two household contacts (cases 5 and 6) was 44-70 with 28-38 cgMLST loci differences.Conclusions. We report a cluster of NTTB C. diphtheriae cases in a skin clinic and evidence of onward household transmission. We conclude the deletion in the tox was responsible for the non-expression of DT. There was no evidence of reversion to DT expression over the 6.5 year period studied. These data informed revision to guidance in the management of NTTB cases and their contacts in the UK.

Clonal Diversity of Candida auris, Candida blankii, and Kodamaea ohmeri Isolated from Septicemia and Otomycosis in Bangladesh as Determined by Multilocus Sequence Typing.

Candida auris, Candida blankii, and Kodamaea ohmeri have been regarded as emerging fungal pathogens that can cause infections with high mortality. For genotyping of C. auris, a multilocus sequence typing (MLST) scheme based on four locus sequences has been reported, while there is no typing scheme for C. blankii and K. ohmeri. In the present study, the existing MLST scheme of C. auris was modified by adding more locus types deduced from sequence data available in the GenBank database. Furthermore, MLST schemes of C. blankii and K. ohmeri were developed using the four cognate loci (ITS, RPB1, RPB2, D1/D2) and similar sequence regions to those of C. auris. These MLST schemes were applied to identify the ST (sequence type) of clinical isolates of C. auris (n = 7), C. blankii (n = 9), and K. ohmeri (n = 6), derived from septicemia or otomycosis in Bangladesh in 2021. All the C. auris isolates were classified into a single ST (ST5) and clade I, having a Y132F substitution in ERG11p, which is associated with azole resistance. Similarly, all the C. blankii isolates belonged to a single type (ST1). In contrast, six K. ohmeri isolates were assigned to five types (ST1-ST5), suggesting its higher genetic diversity. These findings revealed the availability of MLST schemes for these three fungal species for understanding their clonal diversity among clinical isolates.

Core-Genome Multilocus Sequence Typing for Epidemiological and Evolutionary Analyses of Phytopathogenic Xanthomonas citri.

Xanthomonas citri subsp. citri is the cause of bacterial citrus canker, responsible for major economic losses to the citrus industry. X. citri subspecies and pathovars are responsible for diseases in soybean, common bean, mango, pomegranate, and cashew. X. citri disease has been tracked using several typing methods, but recent studies using genomic sequencing have been key to understanding the evolutionary relationships within the species, including fundamental differences among X. citri subsp. citri pathotypes. Here, we describe a core-genome multilocus sequence typing (cgMLST) scheme for X. citri based on 250 genomes comprising multiple examples of X. citri subsp. citri pathotypes A, A*, and Aw; X. citri subsp. malvacearum; X. citri pv. aurantifolii, pv. fuscans, pv. glycines, pv. mangiferaeindicae, pv. viticola, and pv. vignicola; and single isolates of X. citri pv. dieffenbachiae and pv. punicae. This data set included genomic sequencing of 100 novel X. citri subsp. citri isolates. cgMLST, based on 1,618 core genes across 250 genomes, is implemented at PubMLST (https://pubmlst.org/organisms/xanthomonas-citri/). GrapeTree minimum-spanning tree and Interactive Tree of Life (iTOL) neighbor-joining phylogenies generated from the cgMLST data resolved almost identical groupings of isolates to a core-genome single nucleotide polymorphism (SNP)-based neighbor-joining phylogeny. These resolved identical groupings of X. citri subsp. citri pathotypes and X. citri subspecies and pathovars. X. citri cgMLST should prove to be an increasingly valuable resource for the study of this key species of plant-pathogenic bacteria. Users can submit genomic data and associated metadata for comparison with previously characterized isolates at PubMLST to allow the rapid characterization of the local, national, and global epidemiology of these pathogens and examine evolutionary relationships. IMPORTANCE Xanthomonas citri is a plant pathogen that causes major economic losses to the citrus industry and sweet orange production in particular. Several subspecies and pathogens are recognized, with host ranges including soybean, common bean, mango, pomegranate, and cashew, among others. Recent genomic studies have shown that host-adapted X. citri subspecies and pathovars and X. citri subsp. citri pathotypes form distinct clades. In this study, we describe a core-genome multilocus sequence typing (cgMLST) scheme for this species that can rapidly and robustly discriminate among these ecologically distinct, host-adapted clades. We have established this scheme and associated databases containing genomic sequences and metadata at PubMLST, which users can interrogate with their own genome sequences to determine X. citri subspecies, pathovars, and pathotypes. X. citri cgMLST should prove to be an invaluable tool for the study of the epidemiology and evolution of this major plant pathogen.

The role of plasmids in carbapenem resistant E. coli in Alameda County, California

BackgroundAntimicrobial resistant infections continue to be a leading global public health crisis. Mobile genetic elements, such as plasmids, have been shown to play a major role in the dissemination of antimicrobial resistance (AMR) genes. Despite its ongoing threat to human health, surveillance of AMR in the United States is often limited to phenotypic resistance. Genomic analyses are important to better understand the underlying resistance mechanisms, assess risk, and implement appropriate prevention strategies. This study aimed to investigate the extent of plasmid mediated antimicrobial resistance that can be inferred from short read sequences of carbapenem resistant E. coli (CR-Ec) in Alameda County, California. E. coli isolates from healthcare locations in Alameda County were sequenced using an Illumina MiSeq and assembled with Unicycler. Genomes were categorized according to predefined multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) schemes. Resistance genes were identified and corresponding contigs were predicted to be plasmid-borne or chromosome-borne using two bioinformatic tools (MOB-suite and mlplasmids).ResultsAmong 82 of CR-Ec identified between 2017 and 2019, twenty-five sequence types (STs) were detected. ST131 was the most prominent (n = 17) followed closely by ST405 (n = 12). blaCTX−M were the most common ESBL genes and just over half (18/30) of these genes were predicted to be plasmid-borne by both MOB-suite and mlplasmids. Three genetically related groups of E. coli isolates were identified with cgMLST. One of the groups contained an isolate with a chromosome-borne blaCTX−M−15 gene and an isolate with a plasmid-borne blaCTX−M−15 gene.ConclusionsThis study provides insights into the dominant clonal groups driving carbapenem resistant E. coli infections in Alameda County, CA, USA clinical sites and highlights the relevance of whole-genome sequencing in routine local genomic surveillance. The finding of multi-drug resistant plasmids harboring high-risk resistance genes is of concern as it indicates a risk of dissemination to previously susceptible clonal groups, potentially complicating clinical and public health intervention.

A Core Genome Multilocus Sequence Typing Scheme for Proteus mirabilis

ObjectiveA core genome multilocus sequence typing (cgMLST) scheme to genotype and identify potential risk clonal groups (CGs) in Proteus mirabilis. MethodsIn this work, we propose a publicly available cgMLST scheme for P. mirabilis using chewBBACA. In total 72 complete P. mirabilis genomes, representing the diversity of this species, were used to set up a cgMLST scheme targeting 1,842 genes, 635 unfinished (contig, chromosome, and scaffold) genomes were used for its validation. ResultsWe identified a total of 205 CGs from 695 P. mirabilis strains with regional distribution characteristics. Of these, 159 unique CGs were distributed in 16 countries. CG20 and CG3 carried large numbers of shared and unique antibiotic resistance genes. Nine virulence genes (papC, papD, papE, papF, papG, papH, papI, papJ, and papK) related to the P fimbrial operon that cause severe urinary tract infections were only found in CG20. These CGs require attention due to potential risks. ConclusionThis research innovatively performs high-resolution molecular typing of P. mirabilis using whole-genome sequencing technology combined with a bioinformatics pipeline (chewBBACA). We found that the CGs of P. mirabilis showed regional distribution differences. We expect that our research will contribute to the establishment of cgMLST for P. mirabilis.

Evaluation of core genome and whole genome multilocus sequence typing schemes for Campylobacter jejuni and Campylobacter coli outbreak detection in the USA.

Campylobacter is a leading causing of bacterial foodborne and zoonotic illnesses in the USA. Pulsed-field gene electrophoresis (PFGE) and 7-gene multilocus sequence typing (MLST) have been historically used to differentiate sporadic from outbreak Campylobacter isolates. Whole genome sequencing (WGS) has been shown to provide superior resolution and concordance with epidemiological data when compared with PFGE and 7-gene MLST during outbreak investigations. In this study, we evaluated epidemiological concordance for high-quality SNP (hqSNP), core genome (cg)MLST and whole genome (wg)MLST to cluster or differentiate outbreak-associated and sporadic Campylobacter jejuni and Campylobacter coli isolates. Phylogenetic hqSNP, cgMLST and wgMLST analyses were also compared using Baker's gamma index (BGI) and cophenetic correlation coefficients. Pairwise distances comparing all three analysis methods were compared using linear regression models. Our results showed that 68/73 sporadic C. jejuni and C. coli isolates were differentiated from outbreak-associated isolates using all three methods. There was a high correlation between cgMLST and wgMLST analyses of the isolates; the BGI, cophenetic correlation coefficient, linear regression model R 2 and Pearson correlation coefficients were >0.90. The correlation was sometimes lower comparing hqSNP analysis to the MLST-based methods; the linear regression model R 2 and Pearson correlation coefficients were between 0.60 and 0.86, and the BGI and cophenetic correlation coefficient were between 0.63 and 0.86 for some outbreak isolates. We demonstrated that C. jejuni and C. coli isolates clustered in concordance with epidemiological data using WGS-based analysis methods. Discrepancies between allele and SNP-based approaches may reflect the differences between how genomic variation (SNPs and indels) are captured between the two methods. Since cgMLST examines allele differences in genes that are common in most isolates being compared, it is well suited to surveillance: searching large genomic databases for similar isolates is easily and efficiently done using allelic profiles. On the other hand, use of an hqSNP approach is much more computer intensive and not scalable to large sets of genomes. If further resolution between potential outbreak isolates is needed, wgMLST or hqSNP analysis can be used.