Multilocus Sequence Typing Scheme Research Articles

Establishment of a Multilocus Sequence Typing Scheme for the Characterization of Avibacterium paragallinarum

Infectious coryza is an acute respiratory infection caused by Avibacterium paragallinarum, which is widely distributed throughout the world. However, there is no effective molecular typing scheme to obtain basic knowledge about the Av. paragallinarum population structure. This study aimed to develop a multilocus sequence typing (MLST) scheme for Av. paragallinarum that allows for the worldwide comparison of sequence data. For this purpose, the genetic variability of 59 Av. paragallinarum strains from different geographical origins and serovars was analyzed to identify correlations. The MLST scheme was developed using seven conserved housekeeping genes, which identified eight STs that clustered all of the strains into three evolutionary branches. The analytical evaluation of the clone group relationship between the STs revealed two clone complexes (CC1 and CC2) and three singletons (ST2, ST5, and ST6). Most of the isolates from China belonged to ST1 and ST3 in CC1. ST8 from Peru and ST7 from North America together formed CC2. Our results showed that the Av. paragallinarum strains isolated from China had a distant genetic relationship with CC2, indicating strong regional specificity. The MLST scheme established in this study can monitor the dynamics and genetic differences of Av. paragallinarum transmission.

A Multilocus Sequence Tying Scheme for Rapid Identification of Xanthomonas citri Based on Whole Genome Sequencing Data.

Xanthomonas citri is a plant-pathogenic bacterium associated with a diverse range of plant host species. It has undergone substantial reclassification and currently consists of fourteen different subspecies or pathovars that are responsible for a wide range of plant diseases. Whole genome sequencing (WGS) provides a cutting-edge advantage over other diagnostic techniques in epidemiological and evolutionary studies of X. citri because it has a higher discriminatory power and is replicable across laboratories. Also, WGS allows the improvement of multi-locus sequence typing (MLST) schemes. In this study, we used genome sequences of Xanthomonas isolates from the NCBI RefSeq database to develop a seven-gene MLST scheme that yielded 19 sequence types (STs) that correlated with phylogenetic clades of X. citri subspecies/pathovars. Using this MLST scheme, we examined 2,911 assemblies from NCBI GenBank and identified 15 novel STs from 37 isolates that were misclassified in the NCBI. In total, we identified 545 X. citri assemblies from GenBank with 95% average nucleotide identity to the X. citri type strain and all were classified as one of the 34 STs. All MLST classifications correlated with phylogenetic position inferred from alignments using 92 conserved genes. We observed several instances where strains from different pathovars formed closely related monophyletic clades and shared the same ST, indicating that further investigation of the validity of these pathovars is required. Our MLST scheme described here is a robust tool for rapid classification of X. citri pathovars using WGS and a powerful method for further comprehensive taxonomic revision of X. citri pathovars.

A core genome multi-locus sequence typing scheme for Streptococcus uberis: an evolution in typing a genetically diverse pathogen.

Streptococcus uberis is a globally endemic and poorly controlled cause of bovine mastitis impacting the sustainability of the modern dairy industry. A core genome was derived from 579 newly sequenced S. uberis isolates, along with 305 publicly available genome sequences of S. uberis isolated from 11 countries around the world and used to develop a core genome multi-locus sequence typing (cgMLST) scheme. The S. uberis core genome comprised 1475 genes, and these were used to identify 1447 curated loci that were indexed into the cgMLST scheme. This was able to type 1012 of 1037 (>97  %) isolates used and differentiated the associated sequences into 932 discrete core genome sequence types (cgSTs). Analysis of the phylogenetic relationships of cgSTs revealed no clear clustering of isolates based on metadata such as disease status or year of isolation. Geographical clustering of cgSTs was limited to identification of a UK-centric clade, but cgSTs from UK isolates were also dispersed with those originating from other geographical regions across the entire phylogenetic topology. The cgMLST scheme offers a new tool for the detailed analysis of this globally important pathogen of dairy cattle. Initial analysis has re-emphasized and exemplified the genetically diverse nature of the global population of this opportunistic pathogen.

Characterization of Gallibacterium anatis Isolated from Pathological Processes in Domestic Mammals and Birds in the Czech Republic.

Gallibacterium anatis, recognized as a resident and opportunistic pathogen primarily in poultry, underwent investigation in unwell domestic mammals and birds. The study encompassed the mapping and comparison of G. anatis isolates, evaluation of their genetic diversity, and determination of their susceptibility to antimicrobials. A total of 11,908 clinical samples were analyzed using cultivation methods and MALDI-TOF. Whole-genome sequencing was performed on seven calf isolates and six hen isolates. Among mammals, G. anatis was exclusively detected in 22 young dairy calves, while among domestic birds, it was found in 35 individuals belonging to four species. Pathological observations in calves were predominantly localized in the digestive tract, whereas in birds, multi-organ infections and respiratory system infections were most prevalent. Distinct groups of genes were identified solely in calf isolates, and conversely, those unique to hen isolates were also recognized. Novel alleles in the multilocus sequence typing scheme genes and previously unidentified sequence types were observed in both calf and hen isolates. Antimicrobial susceptibility exhibited variation between bird and calf isolates. Notably, G. anatis isolates from calves exhibited disparities in genotype and phenotype compared to those from hens. Despite these distinctions, G. anatis isolates demonstrated the capability to induce septicemia in both species.

Multilocus sequence typing schemes for the emerging swine pathogen Mycoplasma hyosynoviae

Mycoplasma (M.) hyosynoviae is a commensal of the upper respiratory tract in swine, which has the potential to spread systemically, usually resulting in arthritis in fattening pigs and gilts. To date, very little is known about the epidemiology of M. hyosynoviae, mainly due to a lack of suitable typing methods. Therefore, this study aimed to develop both a conventional multi locus sequence typing (MLST) and a core genome (cg) MLST scheme. The development of the cgMLST was based on whole genome sequences of 64 strains isolated from pigs and wild boars during routine diagnostics as well as nine publicly available genomes. A cgMLST scheme containing 390 target genes was established using the Ridom© SeqSphere+ software. Using this scheme as a foundation, seven housekeeping genes were selected for conventional MLST based on their capability to reflect genome wide relatedness and subsequently, all 73 strains were typed by applying both methods. Core genome MLST results revealed a high diversity of the studied strain population and less than 100 allele differences between epidemiologically unrelated strains were only detected for four isolates from the US. On the other hand, seven clonal clusters (≤ 12 allele differences) comprising 20 isolates were identified. Comparison of the two typing methods resulted in highly congruent phylogenetic trees and an Adjusted Rand Coefficient of 0.893, while cgMLST showed marginally higher resolution when comparing closely related isolates, indicated by a slightly higher Simpson’s ID (0.992) than conventional MLST (Simpson’s ID = 0.990). Overall, both methods seem well suited for epidemiological analyses for scientific as well as diagnostic purposes. While MLST is faster and cheaper, cgMLST can be used to further differentiate closely related isolates.

Investigating environmental transmission to resolve a Bacillus cereus group outbreak in a neonatal intensive care unit using core genome multilocus sequence typing

BackgroundWe analyzed an outbreak of Bacillus cereus group (Bcg) at a single-center neonatal intensive care unit level IV by conducting comprehensive sampling of both patients and the environment.MethodsBetween 06/2020 and 10/2021, all Bcg isolates identified by both regular colonization screening and additional sampling of the environment were subjected to whole-genome sequencing, followed by in vitro extraction of MLST ST, resistance genes and virulence factors. Using publicly available genome sequences, we defined an ad hoc core genome multilocus sequence typing (cgMLST) scheme comprising 2759 target genes for Bcg typing, which we applied to the detected isolates. We have compared the results with a stable cgMLST that was published in the meantime and completed the investigation with a SNP analysis.ResultsWe analyzed 28 Bcg isolates from patient and environmental samples using MLST and cgMLST. This revealed multiple sequence types, with ST127 being the most common (n = 13). Both cgMLST schemes grouped ten of the 13 ST127 isolates into a cluster, including two invasive isolates from two different patients and several environmental samples. SNP analysis postulated a screen from a ventilation machine as a possible reservoir.ConclusionIn sensitive settings such as neonatal intensive care units, considering the environment in outbreak analyses is crucial, especially when investigating potential transmission routes through shared devices. When dealing with widespread bacteria such as Bcg, high-resolution typing techniques are necessary. In this study, we successfully resolved an outbreak of Bcg infections using a custom cgMLST scheme combined with a SNP analysis.

Low genetic diversity of Treponema pallidum ssp. pertenue (TPE) isolated from patients' ulcers in Namatanai District of Papua New Guinea: Local human population is infected by three TPE genotypes.

Yaws is an endemic disease caused by Treponema pallidum subsp. pertenue (TPE) that primarily affects children in rural regions of the tropics. The endemic character of yaws infections and the expected exclusive reservoir of TPE in humans opened a new opportunity to start a yaws eradication campaign. We have developed a multi-locus sequence typing (MLST) scheme for TPE isolates combining the previously published (TP0548, TP0488) and new (TP0858) chromosomal loci, and we compared this typing scheme to the two previously published MLST schemes. We applied this scheme to TPE-containing clinical isolates obtained during a mass drug administration study performed in the Namatanai District of Papua New Guinea between June 2018 and December 2019. Of 1081 samples collected, 302 (28.5%) tested positive for TPE DNA, from which 255 (84.4%) were fully typed. The TPE PCR-positivity in swab samples was higher in younger patients, patients with single ulcers, first ulcer episodes, and with ulcer duration less than six months. Non-treponemal serological test positivity correlated better with PCR positivity compared to treponema-specific serological tests. The MLST revealed a low level of genetic diversity among infecting TPE isolates, represented by just three distinct genotypes (JE11, SE22, and TE13). Two previously used typing schemes revealed similar typing resolutions. Two new alleles (one in TP0858 and one in TP0136) were shown to arise by intragenomic recombination/deletion events. Compared to samples genotyped as JE11, the minor genotypes (TE13 and SE22) were more frequently detected in samples from patients with two or more ulcers and patients with higher values of specific TP serological tests. Moreover, the A2058G mutation in the 23S rRNA genes of three JE11 isolates was found, resulting in azithromycin resistance.

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.