Multilocus Sequence Typing Data Research Articles

Regulation of virulence and \u03b2-lactamase gene expression in Staphylococcus aureus isolates: cooperation of two-component systems in bloodstream superbugs

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA)-bloodstream infections (BSI) are predominantly seen in the hospital or healthcare-associated host. Nevertheless, the interactions of virulence factor (VFs) regulators and β-lactam resistance in MRSA-BSI are unclear. This study aims to characterize the molecular relationship of two-component systems of VFs and the expression of the β-lactamase gene in MRSA-BSI isolates. In this study, 639 samples were collected from BSI and identified by phenotypic methods. We performed extensive molecular characterization, including SCCmec type, agr type, VFs gene profiles determinations, and MLST on isolates. Also, a quantitative real-time PCR (q-RT PCR) assay was developed for identifying the gene expressions.ResultsNinety-one (91) S. aureus and 61 MRSA (67.0%) strains were detected in BSI samples. The presence of VFs and SCCmec genes in MRSA isolates were as follows: tst (31.4%), etA (18.0%), etB (8.19%), lukS-PVL (31.4%), lukF-PV (18.0%), lukE-lukD (16.3%), edin (3.2%), hla (16.3%), hlb (18.0%), hld (14.7%), hlg (22.9%), SCCmecI (16.3%), SCCmecII (22.9%), SCCmecIII (36.0%), SCCmecIV (21.3%), and SCCmecV (16.3%). Quantitative real-time PCR showed overexpression of mecRI and mecI in the toxigenic isolates. Moreover, RNAIII and sarA genes were the highest expressions of MRSA strains. The multi-locus sequence typing data confirmed a high prevalence of CC5, CC8, and CC30. However, ST30, ST22, and ST5 were the most prevalent in the resistant and toxigenic strains.ConclusionWe demonstrated that although regulation of β-lactamase gene expressions is a significant contributor to resistance development, two-component systems also influence antibiotic resistance development in MRSA-BSI isolates. This indicates that resistant strains might have pathogenic potential. We also confirmed that some MLST types are more successful colonizers with a potential for MRSA-BSI.

Investigation of the Domestic Reservoirs of Diarrheagenic Escherichia coli in Diarrhea Case Households of Urban Bangladesh.

This study collected rectal swabs from diarrheal patients and in-house environmental samples from low-income households in Dhaka City, Bangladesh, over a 4-month period and investigated these to determine the domestic transmission pathways of Escherichia coli-associated diarrhea. The environmental samples included swabs from four frequently touched surfaces, drinking water and food. Both the rectal swabs and environmental samples were examined for virulence genes characteristic of diarrheagenic E. coli pathotypes by PCR. In addition, each sample was cultured for E. coli, and the strains were analyzed for virulence profile and subjected to multilocus sequence typing (MLST). The results showed that 31% (73 of 233) of all samples including rectal swabs and household samples were positive for one or more of the diarrheagenic E. coli virulence factors. PCR analyses showed that 28% (10/36) of the rectal swabs, 43% (58/136) of household swabs, 9% (3/32) of the food, and 7% (2/29) of the water samples were positive for various virulence genes. 6 Out of the 36 rectal swab samples and associated household samples were shown to have similar E. coli pathotypic genes, and the drinking vessel surface was identified as the major source of contamination. EAEC and CTEC were the most commonly identified pathotypes in the cultured isolates. The phylogenetic tree constructed by MLST data showed that the diarrheagenic isolates were clustered in several diversified lineages. This study supports the hypothesis that there are high-risk hotspots, particularly those surfaces associated with food consumption, for diarrheagenic E. coli contamination within the household environments of Bangladesh.

Characterization of oxazolidinone and phenicol resistance genes in non-clinical enterococcal isolates from Korea

ObjectivesTo investigate the distribution and genetic characteristics of linezolid-resistant enterococci. MethodsEnterococcus faecalis and Enterococcus faecium strains were isolated from pigs, equipment, grounds, and employees of 19 Korean swine farms in 2017. Antimicrobial susceptibility testing was then performed and linezolid resistance genes were detected via PCR. For genetic epidemiological characterization, multilocus sequence typing and whole-genome sequencing data were analysed. ResultsTwenty-eightE. faecalis and five E. faecium strains were isolated from 1026 samples obtained from the 19 farms. Ten sequence types were identified among the E. faecalis strains, of which ST256 (42.9%) and ST86 (25%) were the most abundant. The oxazolidinone and phenicol resistance genes poxtA, optrA, and fexA were detected in isolates of E. faecalis (100%, 85.7%, and 67.9%, respectively) and E. faecium (100%, 60%, and 80%, respectively). The minimum inhibitory concentrations of linezolid in these isolates ranged from 2 mg/L to 12 mg/L. The whole-genome sequencing data indicated that fexA was located upstream of poxtA. ConclusionsThis is the first study to report the detection of poxtA in isolates that were both susceptible and resistant to linezolid in Korea. These results demonstrate the importance of antimicrobial resistance monitoring programmes, including regular antimicrobial susceptibility testing and resistance gene expression analysis, to facilitate the control of the spread of antibiotic resistance in non-clinical settings in Korea.

Comparison of Microbiological Characteristics and Genetic Diversity between Burkholderia cepacia Complex Isolates from Vascular Access and Other Clinical Infections.

Burkholderia cepacia complex (BCC) is a group of closely related bacteria with widespread environmental distribution. BCC bacteria are opportunistic pathogens that cause nosocomial infections in patients, especially cystic fibrosis (CF). Multilocus sequence typing (MLST) is used nowadays to differentiate species within the BCC complex. This study collected 41 BCC isolates from vascular access infections (VAIs) and other clinical infections between 2014 and 2020. We preliminarily identified bacterial isolates using standard biochemical procedures and further conducted recA gene sequencing and MLST for species identification. We determined genetic diversity indices using bioinformatics software. We studied 14 isolates retrieved from patients with VAIs and observed that Burkholderia cepacia was the predominant bacterial species, and B. contaminans followed by B. cenocepacia were mainly retrieved from patients with other infections. According to MLST data, we identified that all B. contaminans isolates belonged to ST102, while a wide variety of sequence types (STs) were found in B. cenocepacia isolates. In summary, the high diversity and easy transmission of BCC increase BCC infections, which provides insights into their potential clinical effects in non-CF infections.

Source attributed case-control study of campylobacteriosis in New Zealand

BackgroundFollowing an initial reduction in human campylobacteriosis in New Zealand after the implementation of poultry food chain-focused interventions during 2006–2008, further decline has been relatively small.We report a year-long study of notified campylobacteriosis cases, incorporating a case control study combined with a source attribution study. The purpose was to generate up-to-date evidence on the relative contributions of different sources of campylobacteriosis in New Zealand. MethodsThe study approach included:• A case-control study of notified cases (aged six months or more) sampled in a major urban centre (Auckland, every second case) and a mixed urban/rural area (Manawatū/Whanganui, every case), between 12 March 2018 and 11 March 2019.• Source attribution of human campylobacteriosis cases sampled from these two regions over the study period by modelling of multilocus sequence typing data of Campylobacter jejuni and C. coli isolates from faecal samples of notified human cases and relevant sources (poultry, cattle, sheep). ResultsMost cases (84%) were infected with strains attributed to a poultry source, while 14% were attributed to a cattle source. Approximately 90% of urban campylobacteriosis cases were attributed to poultry sources, compared to almost 75% of rural cases.Poultry consumption per se was not identified as a significant risk factor. However specific risk factors related to poultry meat preparation and consumption did result in statistically significantly elevated odds ratios. ConclusionsThe overall findings combining source attribution and analysis of specific risk factors indicate that poultry meat remains a dominant pathway for exposure and infection.

Chlamydia pecorum-Associated Sporadic Ovine Abortion.

Despite previous detection of Chlamydia pecorum in sporadic ovine abortions, published descriptions of naturally occurring infections with fetoplacental lesions are lacking. This report provides the first descriptions of severe necrosuppurative chorionitis with vasculitis, and fetal pyelonephritis and enteritis in late-term abortions of maiden ewes. Chlamydial infection was detected using a Chlamydia genus-specific qPCR (quantitative polymerase chain reaction) on tissue extracts from 3 fetuses. C. pecorum was identified using a targeted qPCR assay, which also determined infectious load within fetal tissues. The presence of viable C. pecorum in fetal samples was confirmed by cell culture. Multilocus sequence typing (MLST) data indicated that the C. pecorum strains from each fetus were identical and of sequence type (ST) 23. Chlamydia sp. immunohistochemistry showed strong positive immunolabeling of fetoplacental lesions. Other infectious abortigenic agents were excluded with specific testing. This report confirms C. pecorum as a likely cause of ovine abortion and provides the first descriptions of associated fetoplacental lesions in naturally infected sheep.

Molecular epidemiology and phylogenetic analyses of environmental and clinical isolates of Cryptococcus gattii sensu lato in Taiwan.

The rare occurrence of cryptococcosis caused by Cryptococcus gattii sensu lato (C. gattii s.l.) leads to the difficulties in studying the molecular epidemiology of this globally emerging disease. To establish the molecular epidemiological profile of C. gattii s.l. in Taiwan, and understand the genetic relationship between locally endemic and global isolates. A nationwide survey on environmental C. gattii s.l. in Taiwan was conducted from 2017 to 2019. The geographic distribution and molecular epidemiology based on multilocus sequence typing (MLST) data of the environmental isolates were compared with 18 previously collected clinical isolates. Phylogenetic analysis was performed to elucidate the genetic relationship between the global isolates and the isolates endemic to Taiwan. From a total of 622 environmental samples, 104 (16.7%) were positive for C. gattii s.l.. Seven sequence types were identified among the environmental isolates. The genetic population structure showed that the environmental and clinical isolates were closely linked by sequence types and geographical locations. Phylogenetic analysis revealed the association between the C. gattii s.l. isolates in Taiwan and those from South America and South Asia. The recombination test suggested that, in Taiwan, the C. gattii sensu stricto (C. gattii s.s). isolates undergo clonal reproduction and sexual recombination, whereas C. deuterogattii isolates were clonal. The molecular epidemiology of environmental C. gattii s.l. isolates is closely linked to the clinical isolates. Phylogenetic analysis of the environmental isolates provides an insight into the mechanisms underlying reproduction and dispersal of C. gattii s.l. in Taiwan.

Occurrence and molecular characteristics of Listeria monocytogenes isolated from ready-to-eat meats in Hanoi, Vietnam.

Listeria monocytogenes represents one of the most serious threats to food safety. Several studies have shown that Ready-To- Eat (RTE) meats are an important vehicle responsible for listeriosis in human. In Vietnam, little is known about the occurrence and molecular characteristics of L. monocytogenes in meat products, which are essential for developing monitoring plans and control measures. In the present study, we investigated the occurrence of L. monocytogenes in 258 sausage and sliced meat samples collected during the period of 2013-2015 and determined the genetic diversity of the isolates using multi-locus sequence typing (MLST). Overall, L. monocytogenes was present in 19/129 (14.7 %) and 40/129 (31.0 %) sausage and sliced meat samples respectively, with the peak of occurrence being in summer. Furthermore, a minimum spanning tree was constructed based on MLST data of 47 isolates. A total of 15 sequence types were found, with five being novel. Notably, the majority of the isolates (34/47) belonged to the hypervirulent clonal complexes 1, 2, and 3.

Molecular characterization of clinical isolates from vascular access infection: A single-institution study.

Hemodialysis requires repeated, reliable access to the systemic circulation; therefore, a well‐functioning vascular access (VA) procedure is crucial for stable hemodialysis. VA infections (VAIs) constitute the most challenging complication and cause considerable morbidity, loss of access, and even death. In this study, we investigated the molecular profiles of different bacterial isolates retrieved from various types of VA grafts. We collected clinical isolates from hemodialysis patients with VAIs in our institution for the period between 2013 and 2018. We identified the bacterial isolates using standard biochemical procedures; we used a polymerase chain reaction for coagulase‐negative staphylococci (CoNS) and Burkholderia cepacia complex (BCC) species identification. The antibiotic resistance and molecular profile were analyzed using the disk diffusion method and multilocus sequence typing, respectively. We studied 150 isolates retrieved from patients with VAI and observed that Staphylococcus aureus was the predominant bacterial species, followed by S. argenteus, BCC, and CoNS. According to multilocus sequence typing data, we identified a wide variety of sequence types (STs) in S. aureus isolates, with ST59, ST45, and ST239 being the predominant types. Burkholderia cepacia with two new ST types, namely ST1723 and ST1724, accounted for most of the BCC infections, along with ST102 B. contaminans, which were mainly isolated from infected tunneled‐cuffed catheters. In summary, the increased incidence of S. argenteus and BCC infections provides insights into their potential clinical effects in VAIs. The various STs identified in different bacterial species indicate the high genetic diversity of bacterial species isolated from VAIs in our institution.

No Assembly Required: Using BTyper3 to Assess the Congruency of a Proposed Taxonomic Framework for the Bacillus cereus Group With Historical Typing Methods.

The Bacillus cereus group, also known as B. cereus sensu lato (s.l.), is a species complex comprising numerous closely related lineages, which vary in their ability to cause illness in humans and animals. The classification of B. cereus s.l. isolates into species-level taxonomic units is essential for facilitating communication between and among microbiologists, clinicians, public health officials, and industry professionals, but is not always straightforward. A recently proposed genomospecies-subspecies-biovar taxonomic framework aims to provide a standardized nomenclature for this species complex but relies heavily on whole-genome sequencing (WGS). It thus is unclear whether popular, low-cost typing methods (e.g., single- and multi-locus sequence typing) remain congruent with the proposed taxonomy. Here, we characterize 2,231 B. cereus s.l. genomes using a combination of in silico (i) average-nucleotide identity (ANI)-based genomospecies assignment, (ii) ANI-based subspecies assignment, (iii) seven-gene multi-locus sequence typing (MLST), (iv) single-locus panC group assignment, (v) rpoB allelic typing, and (vi) virulence factor detection. We show that sequence types (STs) assigned using MLST can be used for genomospecies assignment, and we provide a comprehensive list of ST/genomospecies associations. For panC group assignment, we show that an adjusted, eight-group framework is largely, albeit not perfectly, congruent with the proposed eight-genomospecies taxonomy, as panC alone may not distinguish (i) B. luti from Group II B. mosaicus and (ii) B. paramycoides from Group VI B. mycoides. We additionally provide a list of loci that capture the topology of the whole-genome B. cereus s.l. phylogeny that may be used in future sequence typing efforts. For researchers with access to WGS, MLST, and/or panC data, we showcase how our recently released software, BTyper3 (https://github.com/lmc297/BTyper3), can be used to assign B. cereus s.l. isolates to taxonomic units within this proposed framework with little-to-no user intervention or domain-specific knowledge of B. cereus s.l. taxonomy. We additionally outline a novel method for assigning B. cereus s.l. genomes to pseudo-gene flow units within proposed genomospecies. The results presented here highlight the backward-compatibility and accessibility of the recently proposed genomospecies-subspecies-biovar taxonomic framework and illustrate that WGS is not a necessity for microbiologists who want to use the proposed nomenclature effectively.

Phylogeography of the marine pathogen, Vibrio vulnificus, revealed the ancestral scenarios of its evolution.

Vibrio vulnificus is the leading cause of seafood‐associated deaths worldwide. Despite the growing knowledge about the population structure of V. vulnificus, the evolutionary history and the ancestral relationships of strains isolated from various regions around the world have not been determined. Using the largest collection of sequence and isolate data of V. vulnificus to date, we applied ancestral character reconstruction to study the phylogeography of V. vulnificus. Multilocus sequence typing data from 10 housekeeping genes were used for the inference of ancestral states and reconstruction of the evolutionary history. The findings showed that the common ancestor of all V. vulnificus populations originated from East Asia, and later evolved into two main clusters that spread with time and eventually evolved into distinct populations in different parts of the world. While we found no meaningful insights concerning the evolution of V. vulnificus populations in the Middle East; however, we were able to reconstruct the ancestral scenarios of its evolution in East Asia, North America, and Western Europe.

Molecular characterisation of Trichomonas vaginalis isolates in Southwest Turkey with multilocus sequence typing and genetic structure analysis in relation to different countries

Trichomonas vaginalis, a flagellated protozoan parasite, is among the most common sexually transmitted pathogens in the world. The present study aimed to identify the genetic profiles of T. vaginalis in the southwest of Turkey with multilocus sequence typing (MLST) and to analyse the genetic structure of the parasite in a collection of isolates from different countries. The study included 27 T. vaginalis isolates from symptomatic females in Aydin, Turkey. Seven housekeeping genes of T. vaginalis were partially amplified and sequenced after genomic DNA extraction from in vitro cultures. The allele profiles and sequence types (STs) of the isolates were determined by using the MLST database (https://pubmlst.org/tvaginalis). The genetic structure and differentiation of the parasite were analysed in relation to findings from other countries by assembling the available MLST sequences. When referred to the database, a total of 22 STs, including 18 new STs were found; besides, there were two new allele types. The genetic analysis of MLST data demonstrated the presence of two main genetic structures: Type I and Type II. In addition, the neighbor-joining method also revealed that the isolates were clustered into two groups. The genetic types distributed almost equally in the Netherlands and the USA, however, the predominance of Type I was noted in Turkey and the UK. The genetic differentiation among four countries was significant (p < .05), the gene flow was relatively high between the Netherlands and the USA, in contrast to Turkey. Finally, genetic variations were originated within populations (93.8%) rather than among populations (6.2%). In conclusion, we studied the genetic diversity of T. vaginalis isolates with MLST in the southwest of Turkey and showed the origin of genetic differentiation of the parasite among different countries. The presentation of MLST profiles and genetic variance of T. vaginalis isolates will contribute to the development of new diagnostic and treatment options for the parasite.

Toxigenic Corynebacterium ulcerans: re-emergence of a zoonotic infection

Diphtheria is a potentially life-threatening infection in humans. The three species capable of causing diphtheria are: Corynebacterium diphtheriae, C. ulcerans and C. pseudotuberculosis. Although UK cases are rare, recently there has been an increase in reported toxigenic C. ulceransassociated with companion animals. Potentially toxigenic corynebacteria are sent to the National Reference Laboratory, Public Health England (PHE), London, UK for species confirmation, determination of presence of the diphtheria toxin gene by real-time PCR, and confirmation of toxin expression by the Elek test. We reviewed submissions of C. ulcerans between January 2006 and November 2019. Fifty-three isolates of toxigenic C. ulcerans were received, 29 from humans and 24 from animals. The most common animal hosts were dogs (15) and cats (7), but isolates were also received from a horse and a captive rhinoceros. Multi-locus sequence typing (MLST) data were derived from whole genome sequencing. In three human cases, C. ulcerans was isolated from companion animals and in all three, typing data supported an epidemiological link between human and animal hosts. The sequence types (STs) for these linked isolates were ST331 (human and dog, human and cat) and ST551 (human and cat). Although MLST data are limited, the finding of the same ST (ST331) from canine and feline sources indicates that strains can be carried by both hosts. Management of diphtheria cases is an important public health issue. Typing data can support epidemiological linkage and identify possible sources, allowing the potential intervention and treatment of animals thus avoiding onward transmission.

Machine learning-based analyses support the existence of species complexes for Strongyloides fuelleborni and Strongyloides stercoralis.

Human strongyloidiasis is a serious disease mostly attributable to Strongyloides stercoralis and to a lesser extent Strongyloides fuelleborni, a parasite mainly of non-human primates. The role of animals as reservoirs of human-infecting Strongyloides is ill-defined, and whether dogs are a source of human infection is debated. Published multi-locus sequence typing (MLST) studies attempt to elucidate relationships between Strongyloides genotypes, hosts, and distributions, but typically examine relatively few worms, making it difficult to identify population-level trends. Combining MLST data from multiple studies is often impractical because they examine different combinations of loci, eliminating phylogeny as a means of examining these data collectively unless hundreds of specimens are excluded. A recently-described machine learning approach that facilitates clustering of MLST data may offer a solution, even for datasets that include specimens sequenced at different combinations of loci. By clustering various MLST datasets as one using this procedure, we sought to uncover associations among genotype, geography, and hosts that remained elusive when examining datasets individually. Multiple datasets comprising hundreds of S. stercoralis and S. fuelleborni individuals were combined and clustered. Our results suggest that the commonly proposed 'two lineage' population structure of S. stercoralis (where lineage A infects humans and dogs, lineage B only dogs) is an over-simplification. Instead, S. stercoralis seemingly represents a species complex, including two distinct populations over-represented in dogs, and other populations vastly more common in humans. A distinction between African and Asian S. fuelleborni is also supported here, emphasizing the need for further resolving these taxonomic relationships through modern investigations.

Hospital-Associated Multidrug-Resistant MRSA Lineages Are Trophic to the Ocular Surface and Cause Severe Microbial Keratitis.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of severe and difficult to treat ocular infection. In this study, the population structure of 68 ocular MRSA isolates collected at Massachusetts Eye and Ear between January 2014 and June 2016 was assessed. By using a combination of multilocus sequence typing (MLST) analysis, SCCmec typing and detection of the panton-valentine leukocidin (PVL) gene, we found that the population structure of ocular MRSA is composed of lineages with community and hospital origins. As determined by eBURST analysis of MLST data, the ocular MRSA population consisted of 14 different sequence types (STs) that grouped within two predominant clonal complexes: CC8 (47.0%) and CC5 (41.2%). Most CC8 strains were ST8, harbored type IV SCCmec and were positive for the PVL-toxin (93.7%). The CC5 group was divided between strains carrying SCCmec type II (71.4%) and SCCmec type IV (28.6%). Remaining isolates grouped in 6 different clonal complexes with 3 isolates in CC6 and the other clonal complexes being represented by a single isolate. Interestingly, major MRSA CC5 and CC8 lineages were isolated from discrete ocular niches. Orbital and preseptal abscess/cellulitis were predominantly caused by CC8-SCCmec IV PVL-positive strains. In contrast, infections of the cornea, conjunctiva and lacrimal system were associated with the MDR CC5 lineage, particularly as causes of severe infectious keratitis. This niche specialization of MRSA is consistent with a model where CC8-SCCmec IV PVL-positive strains are better adapted to cause infections of the keratinized and soft adnexal eye tissues, whereas MDR CC5 appear to have greater ability in overcoming innate defense mechanisms of the wet epithelium of the ocular surface.

Genetic Diversity of Non-O157 Shiga Toxin-Producing Escherichia coli Recovered From Patients in Michigan and Connecticut.

Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens and non-O157 serotypes have been gradually increasing in frequency. The non-O157 STEC population is diverse and is often characterized using serotyping and/or multilocus sequence typing (MLST). Although spacers within clustered regularly interspaced repeat (CRISPR) regions were shown to comprise horizontally acquired DNA elements, this region does not actively acquire spacers in STEC. Hence, it is useful for further characterizing non-O157 STEC and examining relationships between strains. Our study goal was to evaluate the genetic relatedness of 41 clinical non-O157 isolates identified in Michigan between 2001 and 2005 while comparing to 114 isolates from Connecticut during an overlapping time period. Whole genome sequencing (WGS) was performed, and sequences were extracted for serotyping, MLST and CRISPR analysis. Phylogenetic analysis of MLST and CRISPR data was performed using the Neighbor joining and unweighted pair group method with arithmetic mean (UPGMA) algorithms, respectively. In all, 29 serogroups were identified; eight were unique to Michigan and 13 to Connecticut. “Big-six” serogroup frequencies were similar by state (Michigan: 73.2%, Connecticut: 81.6%), though STEC O121 was not found in Michigan. The distribution of sequence types (STs) and CRISPR profiles was also similar across states. Interestingly, big-six serogroups such as O103 and O26, grouped into different STs located on distinct branches of the phylogeny, further confirming that serotyping alone is not adequate for evaluating strain relatedness. Comparatively, the CRISPR analysis identified 361 unique spacers that grouped into 80 different CRISPR profiles. CRISPR spacers 231 and 317 were isolated from 79.2% (n = 118) and 59.1% (n = 88) of strains, respectively, regardless of serogroup and ST. Spacer profiles clustered according to the MLST analysis, though some discrepancies were noted. Indeed, use of both MLST and CRISPR typing enhanced the discriminatory power when compared to the use of each tool separately. These data highlight the genetic diversity of clinical STEC from different locations and show that CRISPR profiling can be used alongside MLST to discriminate related strains. Use of targeted sequencing approaches are particularly helpful for sites without WGS capabilities and can help define which strains require additional characterization using more discriminatory methods.

Dynamic Epidemiology and Virulence Characteristics of Carbapenem-Resistant Klebsiella pneumoniae in Wenzhou, China from 2003 to 2016.

PurposeTo investigate transitions in resistance mechanisms, virulence characteristics and molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) during 2003–2016 in a major Eastern Chinese medical center.Patients and MethodsFrom a total of 2299 K. pneumoniae clinical strains collected from 2003 to 2016, 214 were found to be CRKP isolates and were selected for further study. Characterization of these was conducted by molecular detection of antibiotic resistance markers and virulence determinants, modified carbapenem inactivation method and multilocus sequence typing (MLST).ResultsIn this study, the prevalence of CRKP was increasing over the 14-year period, mirroring a national trend. These CRKP strains were resistant to most of the tested, clinically relevant drugs. The majority of these CRKP strains were positive for carbapenemases, with the Klebsiella pneumoniae carbapenemase (KPC) found to be the dominant type (207/210, 98.6%). The carrier rates of virulence genes uge, entB, fimH, mrkD and ureA increased in 2016, while the ybtA, iucA and irp2 showed a relatively constant trend. From MLST data, ST11 (88.8%, 190/214) was the preponderant sequence type (ST), followed by ST15 (1.9%, 4/214) and ST656 (1.4%, 3/214). Several strains with less common STs (ST690, ST895, ST1823 and ST1384) were also detected, and these too showed high levels of antimicrobial resistance.ConclusionThe average national rise in CRKP across China is mirrored in this in-depth analysis of a single hospital, while the prevalence of hypervirulent CRKP (such as ST15) was relatively low as of 2016. Continuous monitoring is necessary to keep track of CRKP and should include the prospect of newly emerging strains with less common STs and the prospect of detecting carbapenem-resistant, carbapenemase-negative Klebsiella pneumoniae.

Comparing PFGE, MLST, and WGS in monitoring the spread of macrolide and rifampin resistant Rhodococcus equi in horse production

BackgroundRhodococcus equi (R. equi) infections are endemic in many horse facilities in the United States resulting significant economic loses annually. Currently, there is no commercial vaccine available and the emergence of isolates that are resistant to the current treatment and prophylaxis using antibiotics prompts closer surveillance of this pathogen. ObjectiveThis study compares three different genotyping techniques, Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST) and whole genome SNP-based phylogeny to determine the most accurate method to monitor the spread of macrolide-and-rifampin-resistant R. equi. Methods16 macrolide and rifampin-resistant and 6 susceptible R. equi and their Illumina Miseq whole genome sequences were used in this study. The isolates were sub-typed by PFGE with VspI and a dendrogram based on their similarities generated. Additionally, three phylogenetic trees were constructed using CSI phylogeny on (i) whole genome sequences (WGS), (ii) in silico MLST sequences and (iii) MLST sequences obtained after PCR-amplification and Sanger sequencing. ResultsPFGE identified 18 different genetic profiles and grouped the 22 isolates into 3 clusters independently of their susceptibilities. The phylogenetic trees built from WGS and MLST data showed similar topology, separating the isolates into 2 major clades in accordance with their susceptibility profiles (susceptible and resistant). However, only the trees generated with next generation sequencing data could detect the clonality of the resistant isolates.

Whole-genome sequence-based comparison and profiling of virulence-associated genes of Bacillus cereus group isolates from diverse sources in Japan

BackgroundThe complete genome sequences of 44 Bacillus cereus group isolates collected from diverse sources in Japan were analyzed to determine their genetic backgrounds and diversity levels in Japan. Multilocus sequence typing (MLST) and core-genome single-nucleotide polymorphism (SNP) typing data from whole-genome sequences were analyzed to determine genetic diversity levels. Virulence-associated gene profiles were also used to evaluate the genetic backgrounds and relationships among the isolates.ResultsThe 44 B. cereus group isolates, including soil- and animal-derived isolates and isolates recovered from hospitalized patients and food poisoning cases, were genotyped by MLST and core-genome SNP typing. Genetic variation among the isolates was identified by the MLST and core-genome SNP phylogeny comparison against reference strains from countries outside of Japan. Exploratory principal component analysis and nonmetric multidimensional scaling (NMDS) analyses were used to assess the genetic similarities among the isolates using gene presence and absence information and isolate origins as the metadata. A significant correlation was seen between the principal components and the presence of genes encoding hemolysin BL and emetic genetic determinants in B. cereus, and the capsule proteins in B. anthracis. NMDS showed that the cluster of soil isolates overlapped with the cluster comprising animal-derived and clinical isolates.ConclusionsMolecular and epidemiological analyses of B. cereus group isolates in Japan suggest that the soil- and animal-derived bacteria from our study are not a significant risk to human health. However, because several of the clinical isolates share close genetic relationships with the environmental isolates, both molecular and epidemiological surveillance studies could be used effectively to estimate virulence in these important pathogens.

Co-existence of virulence factors and antibiotic resistance in new Klebsiella pneumoniae clones emerging in south of Italy

BackgroundEndemic presence of Klebsiella pneumoniae resistant to carbapenem in Italy has been due principally to the clonal expansion of CC258 isolates; however, recent studies suggest an ongoing epidemiological change in this geographical area.Methods50 K. pneumoniae strains, 25 carbapenem-resistant (CR-Kp) and 25 susceptible (CS-Kp), collected from march 2014 to march 2016 at the Laboratory of Bacteriology of the Paolo Giaccone Polyclinic University hospital of Palermo, Italy, were characterized for antibiotic susceptibility and fully sequenced by next generation sequencing (NGS) for the in silico analysis of resistome, virulome, multi-locus sequence typing (MLST) and core single nucleotide polymorphism (SNP) genotypesResultsMLST in silico analysis of CR-Kp showed that 52% of isolates belonged to CC258, followed by ST395 (12%), ST307 (12%), ST392 (8%), ST348 (8%), ST405 (4%) and ST101 (4%). In the CS-Kp group, the most represented isolate was ST405 (20%), followed by ST392 and ST15 (12%), ST395, ST307 and ST1727 (8%). The in silico β-lactamase analysis of the CR-Kp group showed that the most detected gene was blaSHV (100%), followed by blaTEM (92%), blaKPC (88%), blaOXA (88%) and blaCTX-M (32%). The virulome analysis detected mrk operon in all studied isolates, and wzi-2 was found in three CR-Kp isolates (12%). Furthermore, the distribution of virulence genes encoding for the yersiniabactin system, its receptor fyuA and the aerobactin system did not show significant distribution differences between CR-Kp and CS-Kp, whereas the Klebsiella ferrous iron uptake system (kfuA, kfuB and kfuC genes), the two-component system kvgAS and the microcin E495 were significantly (p < 0.05) prevalent in the CS-Kp group compared to the CR-Kp group.Core SNP genotyping, correlating with the MLST data, allowed greater strain tracking and discrimination than MLST analysis.ConclusionsOur data support the idea that an epidemiological change is ongoing in the Palermo area (Sicily, Italy). In addition, our analysis revealed the co-existence of antibiotic resistance and virulence factors in CR-Kp isolates; this characteristic should be considered for future genomic surveillance studies.