MLST Scheme Research Articles

Genome sequence of a ST65 hypervirulent Klebsiella pneumoniae isolate carrying mcr-8 from China

ObjectivesIn this study, we report the complete genome sequence of a ST65 hypervirulent Klebsiella pneumoniae isolate carrying mcr-8 from China. The aim was to investigate its molecular characteristics and resistant mechanism. MethodsA colistin-resistant hvKP was isolated from an inpatient in China. The whole genome was sequenced on Illumina NovaSeq 6000 and long-read ONT platforms. de novo assembly was conducted using SPAdes and Unicycler. S1 nuclease pulsed-field gel electrophoresis, Southern-blot, and antimicrobial susceptibility testing were performed. Sequence type, antimicrobial resistance and virulence-related genes were predicted from the sequence. The circular maps of multiple plasmids comparisons were drawn by the BLAST Ring Image. ResultsThe complete genome sequence of K. pneumoniae ACESH00926 consists of one chromosome and two plasmids. ACESH00926 belongs to K2 ST65 according to the MLST scheme. ACESH00926 showed high resistance to colistin (MIC > 8 µg/mL). Several ARGs were identified, including mobile colistin-resistant gene mcr-8 which was located in an IncFIl(K)/IncFIA(HI1) type plasmid. The bigger plasmid was a pK244-like virulence plasmid. It carrying a series of virulence genes, such as the regulator of the mucoid phenotype (rmpA and rmpA2), salmochelin siderophore biosynthesis (iroB), ABC transporter (iroC), ferric aerobactin receptor (iutA), aerobactin siderophore biosynthesis protein (iucC), and aerobactin synthetase (iucA) encoded genes. And another plasmid carrying mcr-8 with a conserved genetic context (dgkA-sasA-copR-mcr-8-ccdA-ccdB-xerD-repE-parM-umuC-lexA-klcA). ConclusionsIt is necessary to emphasize the necessity of monitoring a combination of colistin-resistant and hypervirulent Klebsiella pneumoniae strains in the future.

Sequential use of capsular typing and whole-genome sequencing-based analysis for transmission of carbapenem-resistant Acinetobacter baumannii in a tertiary medical center

BackgroundDuring the COVID-19 pandemic, there has been an increasing trend in healthcare-associated infections (HAIs) caused by carbapenem-resistant Acinetobacter baumannii (CRAB), posting a global public health concern. The heightened sensitivity of whole-genome sequencing (WGS) renders it an optimal and potent tool for monitoring outbreaks and tracing the transmission routes of nosocomial pathogens. MethodWe collected CRAB isolates from March 1, 2023, to April 6, 2023 in Chang Gung Memorial Hospital Lin Kou branch, a tertiary medical center in northern Taiwan. Any two or more isolates with the same identifiable capsular K-locus (KL) types were selected, and analyzed via WGS to identify putative transmission clusters, combined with epidemiologic and retrospective analysis on medical records to confirm risk factors and hidden transmission chains. ResultA total of 48 non-redundant CRAB isolates were collected, belonging to ST2 of Pasteur MLST scheme and identifiable KL types of KL2, KL3, KL9, KL10, KL22, KL52. Excluding the KL types that was only found in 1 case, KL2 (n = 9, 22.5 %), KL3 (n = 24, 60 %), KL9 (n = 3, 7.5 %), and KL10 (n = 4, 10 %) were selected for further WGS analysis. Four distinct transmission clusters comprised of 2, 3, 10, and 23 cases were identified on a basis of phylogenetic status. 12 probable transmission chains were revealed, and 2 hidden transmission routes can be speculated. ConclusionThis study referred to some hidden transmission chains that may be missed from traditional surveillance measures. Despite its low prevalence and high cost currently, implementing WGS could be a efficient, prompt, and unequivocal option for future MDRO infection control.

Whole genome sequencing insight into carbapenem-resistant and multidrug-resistant Acinetobacter baumannii harboring chromosome-borne blaOXA-23.

Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a significant threat to hospitalized patients as effective therapeutic options are scarce. Based on the genomic characteristics of the CRAB strain AB2877 harboring chromosome-borne blaOXA-23, which was isolated from the bronchoalveolar lavage fluid (BALF) of a patient in a respiratory intensive care unit (RICU), we systematically analyzed antibiotic resistance genes (ARGs) and the genetic context associated with ARGs carried by CRAB strains harboring chromosome-borne blaOXA-23 worldwide. Besides blaOXA-23, other ARGs were detected on the chromosome of the CRAB strain AB2877 belonging to ST208/1806 (Oxford MLST scheme). Several key genetic contexts associated with the ARGs were identified on the chromosome of the CRAB strain AB2877, including (1) the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph(6)-Id (2); the resistance island AbGRI3 harboring armA and mph(E)-msr(E) (3); the Tn3-like composite transposon containing blaTEM-1D and aph(3')-Ia; and (4) the structure "ISAba1-blaADC-25." The first two genetic contexts were most common in ST195/1816, followed by ST208/1806. The last two genetic contexts were found most frequently in ST208/1806, followed by ST195/1816.IMPORTANCEThe blaOXA-23 gene can be carried by plasmid or chromosome, facilitating horizontal genetic transfer and increasing carbapenem resistance in healthcare settings. In this study, we focused on the genomic characteristics of CRAB strains harboring the chromosome-borne blaOXA-23 gene, and the important genetic contexts associated with blaOXA-23 and other ARGs were identified, and their prevalent clones worldwide were determined. Notably, although the predominant clonal CRAB lineages worldwide containing the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph (6)-Id was ST195/1816, followed by ST208/1806, the CRAB strain AB2877 in our study belonged to ST208/1806. Our findings contribute to the knowledge regarding the dissemination of CRAB strains and the control of nosocomial infection.

A Case of Food-Borne Salmonellosis in a Corn Snake (Pantherophis guttatus) after a Feeder Mouse Meal

Reptiles are usually asymptomatic carriers of Salmonella, with the manifestation of typical clinical signs of acute forms in adult and non-immunocompromised animals being considered exceptions. In the present case, an adult male corn snake (Pantherophis guttatus) was found dead due to septic shock 48 h after consuming a feeder mouse purchased online. The snake’s tissue samples and faeces were cultured for bacteria isolation. Microbiological examinations of the snake and mouse livers revealed the presence of Salmonella enterica subsp. enterica serovar Midway. A whole-genome analysis of these two isolates showed a high correlation between them: they belonged to the strain type ST-357 for the classic MLST scheme and to the strain type ST 171322 for the cgMLST scheme. Also, a virulence gene analysis revealed the presence of stdB and STM3026 genes. This report conveys a case of food-borne salmonellosis in a pet snake, transmitted from a feeder mouse, likely responsible for the snake’s death due to septic shock. It highlights the relevance of feeder mice as a source of Salmonella infections in snakes and the associated risks to human health.

Streptococcus equi subsp. zooepidemicus: High molecular diversity of Argentinian strains isolated from mares with endometritis

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a mucosal commensal of the lower genital tract in horses and is the most isolated bacterium causing endometritis in mares. The aim of this study was to determine the molecular diversity of S. zooepidemicus obtained from endometritis in mares in Buenos Aires province, Argentina. Thirty isolates obtained from the uterus of mares in 2005 and 2017 were studied. The MLST scheme was applied to identify the Argentinian genotypes and the clonal relationships and patterns of evolutionary descent were identified using the eBURST algorithm – goeBURST. Twenty six different Sequence types (STs) were identified, being only 11 of them previously reported in horses and also, from several host species and tissues. The other 15 STs were reported in Argentinian reproductive strains of mares in our study for the first time. The genotypes obtained from uterus in Argentina were not evenly distributed when all the published S. zooepidemicus STs were analysed, thus, it was not possible to establish that the same lineage circulates in our equine population. The fact that the identified genotypes were also reported in other countries, diverse samples and host species suggest that there is not a host, and an anatomical niche adaptation. Finally, the isolation of the same genotype in the vagina/clitoris and the uterus of the same mare highlights the versatility of S. zooepidemicus and its role as an opportunistic pathogen.

Genomic insights into a blaNDM-5-carrying Escherichia coli ST167 isolate recovered from faecal sample of a healthy individual in China

ObjectivesSince its discovery, blaNDM-5 has spread widely amongst Escherichia coli strains in clinical patients, causing carbapenem resistance. Here we report the complete genome sequence of an NDM-5-producing E. coli strain isolated from the faecal sample of a healthy individual in Hangzhou, China. MethodsThe whole-genome sequence of E. coli CREC8 was obtained utilising both the Nanopore sequencer and the Illumina NovaSeq 6000 platform. Antimicrobial resistance genes, multilocus sequence typing, and plasmid replicons were identified using the BacWGSTdb server. The phylogenetic relationship between CREC8 and other E. coli strains was investigated using the core genome multilocus sequence typing (cgMLST) strategy. ResultsThe complete genome sequence of E. coli CREC8 consists of one chromosome and 7 plasmids. CREC8 belongs to ST167 according to the MLST scheme. Seven ARGs were identified, including carbapenem resistance gene blaNDM-5 which was located in an IncFIA/IncFII type plasmid. A total of 164 E. coli ST167 strains related to 25 countries across four continents can be retrieved from the NCBI database, 95 of them carrying the blaNDM gene with blaNDM-5 the most (N = 79). Phylogenetic analysis revealed a worldwide distribution of E. coli ST167 strains, with China having the highest prevalence (37%, 61/165). ConclusionIn summary, we reported a blaNDM-5-carrying E. coli ST167 strain isolated from a healthy individual in China. Such strains are more commonly isolated from hospitalised patients but are rarely isolated from healthy individuals. This indicates a further epidemic of carbapenem-resistant E. coli strains in the healthy population which needs our attention.

Modern Features of Pathogenic Leptospira Isolation and Identification in Siberia and the Far East

Recently, pathogenic Leptospira culture isolation is an extremely rare phenomenon in Russia.The aim of our work was to synthesize the lessons learned at the Irkutsk Anti-Plague Institute from Leptospira culture isolation and identification since 2011.Materials and methods. Material from eight individuals with suspected leptospirosis and from 942 small mammals (SM) was examined using PCR and microscopic agglutination test (MAT), from humans and 260 SM, applying bacteriological method. Bacteriological temperature test, MAT, PCR, MLST and MALDI-TOF mass spectrometry with the original Leptospira protein profiles base were used to identify cultures. Six complete genomes were generated at the Central Research Institute of Epidemiology of the Rospotrebnadzor.Results and discussion. Leptospira have not been isolated from humans against the background of taking antibiotics, despite the positive PCR and MAT results. Four cultures of Leptospira borgpetersenii of the Javanica serogroup and three L. kirschneri (Grippotyphosa) have been isolated from SM. The results of identification using MLST scheme No. 1 and MALDI-TOF MS are identical. MLST in silico has shown the uniformity of two Grippotyphosa serogroup strains from Primorie and Khabarovsk with a sequence-type (ST) profile 110:100:94. ST146 is determined in four Javanica serogroup strains according to scheme No. 1, and unique single nucleotide polymorphisms are detected according to schemes No. 2–3. Thus, in Siberia and the Far East, between 2012 and 2016, seven pathogenic Leptospira cultures were isolated from carriers in natural foci; carrier infection rate being12.0–48.9 %. Javanica serogroup strains differ in the MLST profile characteristics and adapt to nutrient media for a longer time than Grippotyphosa serogroup strains.

Genomic characterization of a carbapenem-resistant Citrobacter freundii clinical isolate from China carrying blaNDM-5 on a novel IncC-IncFIB-IncX3 plasmid

Citrobacter freundii is one of the important pathogens that can cause nosocomial infections. The advent of carbapenem-resistant C. freundii complicates clinical treatment. Here, we reported the genome sequence of a carbapenem-resistant C. freundii strain carrying a novel IncC-IncFIB-IncX3 plasmid in China. The genome sequence of C. freundii CRNMS1 was obtained using the Illumina NovaSeq 6000 platform and the long-read Nanopore sequencer. Multilocus sequence typing was identified using MLST (v.2.23.0). The identification of antimicrobial resistance genes (ARGs) and plasmid replicons was performed using the resfinder and plasmidfinder of ABRicate (v.1.0.1). Circular comparisons of plasmids were performed using the BLAST Ring Image Generator (BRIG). CRNMS1 belongs to ST116 in the C. freundii MLST scheme. Thirteen ARGs were predicted in all, including blaNDM-5, which was located in a plasmid. The plasmid pblaNDM5-S1, which carried the blaNDM-5 gene, was discovered to be a novel plasmid including three plasmid replicons (IncC, IncFIB, and IncX3) as well as seven ARGs (sul1, sul2, floR, dfrA17, aadA5, qnrA1, and blaNDM-5). A total of 38 blaNDM-5-bearing C. freundii strains can be retrieved from the NCBI database. Phylogenetic analysis revealed a worldwide distribution of C. freundii strains carrying the blaNDM-5 gene, with China having the highest prevalence (39%, 15/38). However, they were distantly related to CRNMS1 with SNP differences >2545. In summary, we reported a novel IncC-IncFIB-IncX3 plasmid carrying blaNDM-5 in a carbapenem-resistant C. freundii strain in China. The development of such hybrid plasmids facilitates the transmission of ARGs.

Genotypic characterization of Staphylococcus chromogenes and Staphylococcus simulans from Swedish cases of bovine subclinical mastitis

Staphylococcus chromogenes and Staphylococcus simulans are commonly found in intramammary infections (IMI) associated with bovine subclinical mastitis, but little is known about genotypic variation and relatedness within species. This includes knowledge about genes encoding antimicrobial resistance (AMR) and potential virulence factors (pVF). The aim of this study was therefore to investigate these aspects by whole-genome sequencing of milk isolates from Swedish dairy cows with subclinical mastitis in an observational study. We also wanted to study if specific genotypes were associated with persistent IMI and the inflammatory response at udder quarter level. In total, 105 and 118 isolates of S. chromogenes and S. simulans, respectively, were included. Isolates were characterized using a 7-locus multilocus sequence typing (7-MLST), core genome analysis and in-silico analysis of AMR and pVF genes. Forty-seven sequence types (ST) and 7 core genome clusters of S. chromogenes were identified, and the most common ST were ST-6 and ST-109, both belonging to cluster VII. A 7-locus MLST scheme for S. simulans was not available, but 3 core genome clusters and 5 subclusters were described. Overall, substantial variation in ST and clusters among cows and herds were found in both species. Some ST of S. chromogenes were found in several herds, indicating spread between herds. Moreover, within-herd spread of the same genotype was observed for both species. Only a few AMR genes (blaZ, strpS194, vga(A)) were detected in a limited number of isolates, with the exception of blaZ coding for β-lactamase, which was identified in 22% of the isolates of S. chromogenes with ST-19, ST-102, and ST-103 more commonly carrying this gene compared with other STs. However, the blaZ gene was not identified in S. simulans. The average total number of pVF detected per isolate was similar in S. chromogenes (n = 30) and S. simulans (n = 33), but some variation in total numbers and presence of specific pVF or functional groups of pVF, was shown between ST/clusters within species. Differences in inflammatory response and potentially in persistent IMI at udder quarter level were found between S. chromogenes subtypes but not between S. simulans subtypes. In conclusion, the results from the present study generates new insight into the epidemiology of bovine S. chromogenes and S. simulans IMI, which can have implications for future prevention and antimicrobial treatment of infections related to these species.

New Multilocus Sequence Typing Scheme for Enterococcus faecium Based on Whole Genome Sequencing Data.

The MLST scheme currently used for Enterococcus faecium typing was designed in 2002 and is based on putative gene functions and Enterococcus faecalis gene sequences available at that time. As a result, the original MLST scheme does not correspond to the real genetic relatedness of E. faecium strains and often clusters genetically distant strains to the same sequence types (ST). Nevertheless, typing has a significant impact on the subsequent epidemiological conclusions and introduction of appropriate epidemiological measures, thus it is crucial to use a more accurate MLST scheme. Based on the genome analysis of 1,843 E. faecium isolates, a new scheme, consisting of 8 highly discriminative loci, was created in this study. These strains were divided into 421 STs using the new MLST scheme, as opposed to 223 STs assigned by the original MLST scheme. The proposed MLST has a discriminatory power of D = 0.983 (CI95% 0.981 to 0.984), compared to the original scheme's D = 0.919 (CI95% 0.911 to 0.927). Moreover, we identified new clonal complexes with our newly designed MLST scheme. The scheme proposed here is available within the PubMLST database. Although whole genome sequencing availability has increased rapidly, MLST remains an integral part of clinical epidemiology, mainly due to its high standardization and excellent robustness. In this study, we proposed and validated a new MLST scheme for E. faecium, which is based on genome-wide data and thus reflects the tested isolates' more accurate genetic similarity. IMPORTANCE Enterococcus faecium is one of the most important pathogens causing health care associated infections. One of the main reasons for its clinical importance is a rapidly spreading resistance to vancomycin and linezolid, which significantly complicates antibiotic treatment of infections caused by such resistant strains. Monitoring the spread and relationships between resistant strains causing severe conditions represents an important tool for implementing appropriate preventive measures. Therefore, there is an urgent need to establish a robust method enabling strain monitoring and comparison at the local, national, and global level. Unfortunately, the current, extensively used MLST scheme does not reflect the real genetic relatedness between individual strains and thus does not provide sufficient discriminatory power. This can lead directly to incorrect epidemiological measures due to insufficient accuracy and biased results.

Putative transmission of extended-spectrum β-lactamase-producing Escherichia coli among men who have sex with men in Amsterdam, the Netherlands

ObjectivesA previous study showed higher prevalence of Escherichia coli (E. coli) expressing extended-spectrum β-lactamases (ESBL-Ec) among men who have sex with men (MSM) in Amsterdam, the Netherlands, compared with the general Dutch population. This study genetically characterised the ESBL-Ec isolates and investigated whether the increased prevalence could be explained by transmission between participants. MethodsWhole-genome sequences were obtained from 93 unique ESBL-Ec isolates that were cultured from rectal swabs of 79 participants. Isolates were typed according to the Achtman MLST scheme and ESBL and virulence genes were identified. Pairwise SNP distances were determined between isolates. Isolate pairs with ≤ 25 SNPs were considered part of a putative transmission event, and events between multiple participants formed putative transmission clusters. To investigate whether putatively transmitted isolates belonged to globally expanded lineages, the level of hierarchical clustering with international isolates was assessed using core genome MLST (cgMLST) implemented on the Enterobase platform. ResultsThe most frequently detected E. coli types were ST131:blaCTX-M-15 (16 of 117, 13.5%), ST131:blaCTX-M-27, ST3075:blaCTX-M-15 and ST14:blaSHV-12 (all six of 117, 6.5%). Fourteen putative transmission events were identified, forming four putative transmission clusters. The largest putative transmission cluster contained ST131 isolates, which clustered with multiple international isolates in SNP and cgMLST analysis. One other transmission cluster (ST14:blaSHV-12) and two transmission events (ST14:blaSHV-12 and ST394:blaCTX-M-15) contained very rarely reported strains. ConclusionsThe identification of unique ESBL-Ec strains involved in putative transmission and carried by multiple participants demonstrated a high probability of ESBL-Ec transmission between MSM in Amsterdam; therefore, ESBL-Ec infection should be considered in cases of sexually active MSM having associated symptoms.

ShigaPass: an in silico tool predicting Shigella serotypes from whole-genome sequencing assemblies.

Shigella is one of the commonest causes of diarrhoea worldwide and a major public health problem. Shigella serotyping is based on a standardized scheme that splits Shigella strains into four serogroups and 60 serotypes on the basis of biochemical tests and O-antigen structures. This conventional serotyping method is laborious, time-consuming, impossible to automate, and requires a high level of expertise. Whole-genome sequencing (WGS) is becoming more affordable and is now used for routine surveillance, opening up possibilities for the development of much-needed accurate rapid typing methods. Here, we describe ShigaPass, a new in silico tool for predicting Shigella serotypes from WGS assemblies on the basis of rfb gene cluster DNA sequences, phage and plasmid-encoded O-antigen modification genes, seven housekeeping genes (EnteroBase's MLST scheme), fliC alleles and clustered regularly interspaced short palindromic repeats (CRISPR) spacers. Using 4879 genomes, including 4716 reference strains and clinical isolates of Shigella characterized with a panel of biochemical tests and serotyped by slide agglutination, we show here that ShigaPass outperforms all existing in silico tools, particularly for the identification of Shigella boydii and Shigella dysenteriae serotypes, with a correct serotype assignment rate of 98.5 % and a sensitivity rate (i.e. ability to make any prediction) of 100 %.

Development and Clinical Application of a Multilocus Sequence Typing Scheme for Bacteroides fragilis Based on Whole-Genome Sequencing Data

Bacteroides fragilis is among the most abundant and pathogenic bacterial species in the gut microbiota and is associated with diarrheal disease in children, inflammatory bowel disease, and the development of colorectal cancer. It is increasingly resistant to potent antimicrobial agents such as carbapenems and metronidazole, making it among the most resistant anaerobic bacteria. These factors combined call for increased monitoring of B. fragilis and its population structure on a worldwide scale. Here, we present a possible solution through the development of a multilocus sequence typing scheme (MLST). The scheme is based on seven core gene fragments: groL (hsp60), rpoB, recA, dnaJ, rprX, prfA, and fusA. These gene fragments possess high discriminatory power while retaining concordance with whole core genome-based phylogenetic analysis. The scheme proved capable of differentiating B. fragilis isolates at the strain level. It offers a standardized method for molecular typing and can be applied to isolates from various sampling backgrounds, such as patient isolates, environmental samples, and strains obtained from food and animal sources. In total, 567 B. fragilis genomes were sequence typed and their isolate data collected. The MLST scheme clearly divided the B. fragilis population into two divisions based on the presence of the cfiA and cepA resistance genes. However, no other specific subpopulations within the analyzed genomes were found to be associated with any specific diseases or geographical location. With this MLST scheme, we hope to provide a powerful tool for the study and monitoring of B. fragilis on an international scale. IMPORTANCE Here, we present the first MLST scheme for Bacteroides fragilis, one of the most abundant pathogenic bacteria in the human gut microbiota. The scheme enables standard classification and monitoring of B. fragilis on a worldwide scale and groups the majority of current isolate data in one place. A more unified approach to the collection and analysis of B. fragilis data could provide crucial insights into how the pathogen operates and develops as a species. Close monitoring of B. fragilis is especially relevant, as it is increasingly resistant to potent antimicrobial agents and engages in horizontal gene transfer with other bacteria. Hopefully, this approach will guide new discoveries into how B. fragilis evolves and interacts with its human host. Additionally, the scheme could potentially be applied to other species of the genus Bacteroides.

Class 1 integrons and multiple mobile genetic elements in clinical isolates of the Klebsiella pneumoniae complex from a tertiary hospital in eastern China.

The emergence of highly drug-resistant K. pneumoniae, has become a major public health challenge. In this work, we aim to investigate the diversity of species and sequence types (STs) of clinical Klebsiella isolates and to characterize the prevalence and structure of class 1 integrons. Based on the whole genome sequencing, species identification was performed by 16S rRNA gene homology and average nucleotide identity (ANI) analysis. STs were determined in accordance with the international MLST schemes for K. pneumoniae and K. variicola. Integron characterization and comparative genomic analysis were performed using various bioinformatic tools. Species identification showed that the 167 isolates belonged to four species: K. pneumoniae, K. variicola subsp. variicola, K. quasipneumoniae and K. aerogenes. Thirty-six known and 5 novel STs were identified in K. pneumoniae, and 10 novel STs were identified in K. variicola subsp. variicola. Class 1 integrons were found in 57.49% (96/167) of the isolates, and a total of 169 resistance gene cassettes encoding 19 types of resistance genes, including carbapenem resistance gene (bla IPM-4) and class D β-lactamases gene (bla OXA-1 and bla OXA-10), were identified. Among the 17 complete genomes, 29 class 1 integrons from 12 groups were found, only 1 group was encoded on chromosomes. Interestingly, one plasmid (pKP167-261) carrying two copies of approximately 19-kb IS26-Int1 complex resistance region that contains an integron and a multidrug resistance gene fragment. The results of this work demonstrated that the species and STs of the clinical Klebsiella isolates were more complex by the whole genome sequence analysis than by the traditional laboratory methods. Finding of the new structure of MGEs related to the resistance genes indicates the great importance of deeply exploring the molecular mechanisms of bacterial multidrug resistance.

Population Structure and Genomic Characteristics of Australian Erysipelothrix rhusiopathiae Reveals Unobserved Diversity in the Australian Pig Industry.

Erysipelothrix rhusiopathiae is a bacterial pathogen that is the causative agent of erysipelas in a variety of animals, including swine, emus, turkeys, muskox, caribou, moose, and humans. This study aims to investigate the population structure and genomic features of Australian isolates of E. rhusiopathiae in the Australian pig industry and compare them to the broader scope of isolates worldwide. A total of 178 isolates (154 Australian, seven vaccine isolates, six international isolates, and 11 of unknown origin) in this study were screened against an MLST scheme and publicly available reference isolates, identifying 59 new alleles, with isolates separating into two main single locus variant groups. Investigation with BLASTn revealed the presence of the spaA gene in 171 (96%) of the isolates, with three main groups of SpaA protein sequences observed amongst the isolates. Novel SpaA protein sequences, categorised here as group 3 sequences, consisted of two sequence types forming separate clades to groups 1 and 2, with amino acid variants at positions 195 (D/A), 303 (G/E) and 323(P/L). In addition to the newly identified groups, five new variant positions were identified, 124 (S/N), 307 (Q/R), 323 (P/L), 379 (M/I), and 400 (V/I). Resistance screening identified genes related to lincomycin, streptomycin, erythromycin, and tetracycline resistance. Of the 29 isolates carrying these resistance genes, 82% belonged to SpaA group 2-N101S (n = 22) or 2-N101S-I257L (n = 2). In addition, 79% (n = 23) of these 29 isolates belonged to MLST group ST 5. Our results illustrate that Australia appears to have a unique diversity of E. rhusiopathiae isolates in pig production industries within the wider global context of isolates.

Application of Multilocus Sequence Typing for the Characterization of Leptospira Strains in Malaysia.

Leptospirosis is a common zoonotic disease in tropical and subtropical countries. It is considered an emerging disease in Malaysia and is a notifiable disease. This study was conducted to characterize Malaysian isolates from human, animal and environmental samples via MLST and rrs2 sequencing in an attempt to develop a Malaysian genotypic database. An existing polymerase chain reaction (PCR)-based MLST scheme was performed to facilitate subsequent sequencing. Out of 46 extracted DNA, 36 had complete MLST profiles whereby all six genes were amplified and sequenced. Most of the pathogenic Leptospira genotypes with full MLST profiles were L. interrogans serogroup Bataviae (n = 17), followed by L. borgpetersenii serogroup Javanica (n = 9), L. interrogans serogroup Sejroe (n = 2), L. interrogans serogroup Australis (n = 2), L. kirschneri (n = 2), L. interrogans serogroup Grippotyphosa (n = 1) and L. interrogans serogroup Pyrogenes (n = 3). Two samples (R3_SER/17 and R4_SER/17) were not closely related with any of the reference strains. For the samples with incomplete MLST profiles, leptospiral speciation was conducted through rrs2 analysis, in which four samples were identified as L. borgpetersenii, five samples were closely related to L. kmetyi and one sample was known as L. yasudae. This study shows that molecular approaches that combine both MLST and rrs2 sequencing have great potential in the comprehensive characterization of pathogenic Leptospira because they can be performed directly from cultured and clinical samples.

Genotypic Diversity and Antifungal Susceptibility of Clinical Isolates of Cryptococcus Gattii Species Complex from Argentina.

The aim of this study was to determine the genotypic diversity of 22 Cryptococcus gattii species complex clinical isolates from Argentina and to place these genotypes within the diversity of clinical, veterinary and environmental isolates from Latin America. Mating type and antifungal susceptibility of the isolates were also determined. By URA5-RFLP, nine isolates were identified as molecular type VGI, 10 as VGII, one as VGIII and two as VGIV. Multilocus sequence typing (MSLT), following the International Society for Human and Animal Mycology (ISHAM) consensus MLST scheme, was used to determine the genotypic diversity. Our results suggest that, in Argentina, VGI isolates have low genetic diversity, while VGII isolates have high genetic diversity. Both isolates identified as VGIV by URA5-RFLP were genotyped by MLST as belonging to the currently named VGVI clade. From all isolates, eight sequence types (STs) were unique for Argentina, while five STs have been reported already in other countries, being of high interest the genotypes ST20 and ST7 since they belong to the subtypes VGIIa and VGIIb, respectively, which are associated with hypervirulent strains responsible for outbreaks in North America. To note, geographical analysis showed that some genotypes may be associated with some regions in Argentina. Most isolates were MATα, but we are reporting one isolate MATa for the first time in the country. Antifungal susceptibility tests showed that itraconazole, voriconazole and posaconazole had high activity against all isolates, while amphotericin B, fluconazole and 5-fluorocytosine were the least active drugs against all studied isolates.

Utility evaluation of two molecular methods for Leptospira spp. typing in human serum samples

Most of the available genotyping methods were applied and evaluated in Leptospira isolates and only few of them in a relevant sample size of blood specimens but not of sera. The objective of this study was to evaluate the utility of one partial 16S rRNA gene sequencing assay (16S rRNA) and an optimized. Multilocus sequence typing scheme (MLST) for Leptospira typing directly in serum samples. Confirmed leptospirosis patients (n = 228) from Argentina (2005–2016) were randomly included. Septicemic-phase serum samples (n = 228) were studied by two genotyping methods. Available immune-phase serum samples of the included patients (n = 159) were studied by MAT to compare serological and molecular results. In culture-proven cases (n = 8), genotyping results between clinical samples and isolates were compared. Typing success rate (TSR) was 21.9% for 16S rRNA and 11.4% for MLST (full allelic profile) and a positive trend in both TSR during the study period was observed. Two species (L. interrogans and L. borgpertesenii) were identified by both methods and MLST assigned 8 different STs. The probable serogroups identified by MLST were coincident with the presumptive infecting serogroups identified by MAT, but with different frequencies. The three serogroups (Canicola, Sejroe and Icterohaemorrhagiae) most frequently identified by MAT were also genotyped by MLST. Typing results via 16S rRNA and MLST in clinical samples and isolates of culture-proven cases, were consistent except for one case. Performance of partial 16S rRNA gene sequencing assay and the optimized MLST scheme directly in sera may increase and improve the knowledge about species and serogroups causing human leptospirosis, especially in countries with low rates of culture sample collection or Leptospira isolation.

Carbapenem resistance in Acinetobacter pittii isolates mediated by metallo-β-lactamases.

To characterize the genetic environment of metallo-β-lactamases (MBL) in carbapenem-resistant clinical Acinetobacter pittii isolates. Seventeen carbapenem-resistant A. pittii isolates harbouring an MBL were collected between 2010 and 2015 in Germany. Antimicrobial susceptibility testing was performed using agar dilution. Presence of MBLs was confirmed by PCR and their genetic location determined by S1-pulsed-field gel electrophoresis followed by Southern blot hybridization. Whole-genome sequencing was performed using the Miseq and MinION platforms. Isolates were typed using an ad hoc core genome MLST scheme. Conjugation into A. baumannii was tested by broth mating. In 10 isolates the MBL was plasmid-encoded and in seven isolates chromosomally encoded. blaGIM-1 and blaVIM-2 were plasmid-encoded, blaVIM-4 was chromosomally encoded, while blaNDM-1 was chromosomally encoded in four and plasmid-encoded in three isolates. Seven of ten plasmids were conjugative into A. baumannii. Although most isolates were unrelated, the backbones of the MBL-encoding plasmid showed >99% similarity and only differed in the MBL-encoding area. blaNDM-1-harbouring plasmids were highly similar to other plasmids from Acinetobacter isolates worldwide while the blaVIM-2- and blaGIM-1-encoding plasmids have not been described. These data show the existence of a promiscuous plasmid circulating in A. pittii isolates in Germany that differs only in the MBL-encoding region. Its plasmid backbone has been found globally among multiple Acinetobacter spp. These data should raise awareness of an epidemic conjugative plasmid that has independently acquired MBLs. We should also consider that future comparative plasmid analysis will look beyond solely the resistome and include the mobile elements carrying the resistance genes.

Characteristics of population structure, antimicrobial resistance, virulence factors, and morphology of methicillin-resistant Macrococcus caseolyticus in global clades

Macrococcus caseolyticus is an opportunistic pathogen that is frequently isolated from dairy products and veterinary infections. Recent studies have reported the possibility of methicillin resistance that be transferred among staphylococcal species in foods. The present study examined the population structure, antimicrobial resistance, virulence factors, and morphology of methicillin-resistant M. caseolyticus by investigation of 94 genomes derived from both isolates in beef (n = 7) and pork (n = 2) at Shanghai and those deposited in public domain (n = 85). Phylogenetically, M. caseolyticus were divided into four clades, which each consisted of genomes isolated from continent of European countries (82.4%, n = 78), Asian countries (11.3%, n = 10), United States (4.1%, n = 4), Australia (1%, n = 1), and Sudan (1%, n = 1). The M. caseolyticus isolated from present study formed a genetically distinguished clade, which was characterized by novel alleles in the traditional 7-gene MLST scheme. Furthermore, we identified 24 AMR genes that were associated with 10 classes of antimicrobial agents in M. caseolyticus. Most AMR genes were carried by dominant plasmids such as rep7a, rep22 and repUS56. The genomes in the global clades carried significantly less AMR genes (p < 0.05) and more virulence factors (p < 0.001) than present clade. Virulence factors were detected in methicillin resistant M. caseolyticus including genes coding hemolysin, adherence, biofilm formation, exotoxin, and capsule that associated to human health and infection. Finally, as the close relative of the genus Staphylococcus, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed for morphological comparison that M. caseolyticus has a larger diameter and thicker cell wall compared with S. aureus ATCC 25,923. Taken together, our study suggested that M. caseolyticus mediating divergent antimicrobial resistance and virulence factors could serve as the vector for methicillin resistance habitats in foodborne microorganisms.