Multilocus Sequence Typing Method Research Articles

Isolation and Genetic Characterization of Vancomycin-resistant and mecC+ Methicillin-resistant Staphylococcus aureus Strains in Clinical Samples of Bojnurd, Northeastern Iran

Background: The emergence of antibiotic-resistant Staphylococcus aureus strains is one of the major concerns about the various staphylococcal infections. Vancomycin is one the most important effective antibiotics on staphylococcal lethal infections. To date, vancomycin-resistant strains are increasingly isolated in different parts of the world, and it is alerting. Objectives: The current study was designed to evaluate the prevalence, and antibiotic susceptibility pattern of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) isolates in the main tertiary hospital of Bojnurd, Iran. Methods: Staphylococcus aureus isolates were collected from different clinical samples in Imam Reza Hospital of Bojnurd. After identification of isolates through using conventional methods, they were evaluated by agar screening, disk diffusion, and minimum inhibitory concentration (MIC) methods to determine resistance to vancomycin and methicillin. We also performed polymerase chain reaction (PCR) for the detection of mecA, mecC, vanA, and vanB genes. After confirmation of vancomycin resistance, genetic analysis was performed using SCCmec, agr, and spa typing, and multilocus sequence typing (MLST) methods on VRSA isolates. Results: We found four vancomycin-resistant isolates (1.29%). Also, 75% of isolates were resistant to cefoxitin. Using the PCR method, mecA was found in 73%, mecC in 0.64%, and vanA in 1.29% of isolates. Interestingly, we found two mecC positive isolates in MRSA isolates. The alpha-hemolysin (81.81%) and enterotoxin C (27%) had the highest and lowest toxins percentage, respectively. Among mecA positive isolates, SCCmec IV (37%), SCCmec III (31.27%), SCCmec I (14%), SCCmec II (11%), and SCCmec V (5.7%) were the most prevalent SCCmec types, respectively. It should be noted that the two mecC positive isolates belonged to SCCmec XI. Agr I (76.29%) was the highest agr type. We recognized t037 as the dominant spa type, and ST239, ST6, ST97, and ST8 were found in VRSA isolates. Conclusions: In our study, the frequency of mecA genes in MRSA isolates was very high. It seems that the resistant isolates belonged to endemic clones of Iran.

Establishment of a Publicly Available Core Genome Multilocus Sequence Typing Scheme for Clostridium perfringens.

ABSTRACTClostridium perfringens is a spore-forming anaerobic pathogen responsible for a variety of histotoxic and intestinal infections in humans and animals. High-resolution genotyping aiming to identify bacteria at strain level has become increasingly important in modern microbiology to understand pathogen transmission pathways and to tackle infection sources. This study aimed at establishing a publicly available genome-wide multilocus sequence-typing (MLST) scheme for C. perfringens. A total of 1,431 highly conserved core genes (1.34 megabases; 50% of the reference genome genes) were indexed for a core genome-based MLST (cgMLST) scheme for C. perfringens. The scheme was applied to 282 ecologically and geographically diverse genomes, showing that the genotyping results of cgMLST were highly congruent with the core genome-based single-nucleotide-polymorphism typing in terms of resolution and tree topology. In addition, the cgMLST provided a greater discrimination than classical MLST methods for C. perfringens. The usability of the scheme for outbreak analysis was confirmed by reinvestigating published outbreaks of C. perfringens-associated infections in the United States and the United Kingdom. In summary, a publicly available scheme and an allele nomenclature database for genomic typing of C. perfringens have been established and can be used for broad-based and standardized epidemiological studies.IMPORTANCE Global epidemiological surveillance of bacterial pathogens is enhanced by the availability of standard tools and sharing of typing data. The use of whole-genome sequencing has opened the possibility for high-resolution characterization of bacterial strains down to the clonal and subclonal levels. Core genome multilocus sequence typing is a robust system that uses highly conserved core genes for deep genotyping. The method has been successfully and widely used to describe the epidemiology of various bacterial species. Nevertheless, a cgMLST typing scheme for Clostridium perfringens is currently not publicly available. In this study, we (i) developed a cgMLST typing scheme for C. perfringens, (ii) evaluated the performance of the scheme on different sets of C. perfringens genomes from different hosts and geographic regions as well as from different outbreak situations, and, finally, (iii) made this scheme publicly available supported by an allele nomenclature database for global and standard genomic typing.

Whole-genome sequencing and ad hoc shared genome analysis of Staphylococcus aureus isolates from a New Zealand primary school

Epidemiological studies of communicable diseases increasingly use large whole-genome sequencing (WGS) datasets to explore the transmission of pathogens. It is important to obtain an initial overview of datasets and identify closely related isolates, but this can be challenging with large numbers of isolates and imperfect sequencing. We used an ad hoc whole-genome multi locus sequence typing method to summarise data from a longitudinal study of Staphylococcus aureus in a primary school in New Zealand. Each pair of isolates was compared and the number of genes where alleles differed between isolates was tallied to produce a matrix of “allelic differences”. We plotted histograms of the number of allelic differences between isolates for: all isolate pairs; pairs of isolates from different individuals; and pairs of isolates from the same individual. 340 sequenced isolates were included, and the ad hoc shared genome contained 445 genes. There were between 0 and 420 allelic differences between isolate pairs and the majority of pairs had more than 260 allelic differences. We found many genetically closely related S. aureus isolates from single individuals and a smaller number of closely-related isolates from separate individuals. Multiple S. aureus isolates from the same individual were usually very closely related or identical over the ad hoc shared genome. Siblings carried genetically similar, but not identical isolates. An ad hoc shared genome approach to WGS analysis can accommodate imperfect sequencing of the included isolates, and can provide insights into relationships between isolates in epidemiological studies with large WGS datasets containing diverse isolates.

Comparison of Conventional Molecular and Whole-Genome Sequencing Methods for Differentiating Salmonella enterica Serovar Schwarzengrund Isolates Obtained from Food and Animal Sources.

Over the last decade, Salmonella enterica serovar Schwarzengrund has become more prevalent in Asia, Europe, and the US with the simultaneous emergence of multidrug-resistant isolates. As these pathogens are responsible for many sporadic illnesses and chronic complications, as well as outbreaks over many countries, improved surveillance is urgently needed. For 20 years, pulsed-field gel electrophoresis (PFGE) has been the gold standard for determining bacterial relatedness by targeting genome-wide restriction enzyme polymorphisms. Despite its utility, recent studies have reported that PFGE results correlate poorly with that of closely related outbreak strains and clonally dominant endemic strains. Due to these concerns, alternative amplification-based molecular methods for bacterial strain typing have been developed, including clustered regular interspaced short palindromic repeats (CRISPR) and multilocus sequence typing (MLST). Furthermore, as the cost of sequencing continues to decrease, whole genome sequencing (WGS) is poised to replace other molecular strain typing methods. In this study, we assessed the discriminatory power of PFGE, CRISPR, MLST, and WGS methods to differentiate between 23 epidemiologically unrelated S. enterica serovar Schwarzengrund isolates collected over an 18-year period from distinct locations in Taiwan. The discriminatory index (DI) of each method for different isolates was calculated, resulting in values between 0 (not discriminatory) and 1 (highly discriminatory). Our results showed that WGS has the greatest resolution (DI = 0.982) compared to PFGE (DI = 0.938), CRISPR (DI = 0.906), and MLST (DI = 0.463) methods. In conclusion, the WGS typing approach was shown to be the most sensitive for S. enterica serovar Schwarzengrund fingerprinting.

The Emergence of Novel Sequence Type Strains Reveals an Evolutionary Process of Intraspecies Clone Shifting in ICU-Spreading Carbapenem-Resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an urgent public health problem worldwide, and its rapid evolution in the clinical environment has been a major concern. A total of 99 CRKP isolates spreading in the intensive care unit (ICU) setting were included and subjected to whole-genome sequencing, and their sequence types (STs), serotype loci, and virulence determinants were screened based on genome data. The phylogenetic structure was reconstructed based on the core genome multilocus sequence typing method. Regions of recombination were assessed. Biofilm formation, serum resistance assays, and a Galleria mellonella infection model were used to evaluate strain virulence. A novel ST, designated ST4496, emerged in the ICU and spread for 6 months before its disappearance. ST4496 was closely related to ST11, with only a single-allele variant, and ST11 is the most dominant clinical clone in China. Recombination events occurred at capsule biosynthesis loci and divided the strains of ST11 and its derivative ST4496 into three clusters, including ST11-KL47, ST11-KL64, and ST4496-KL47. The phylogenetic structure indicated that ST11-KL47 was probably the origin of ST11-related strain evolution and presented more diversity in terms of both sequence similarity and phenotypes. ST4496-KL47 cluster strains presented less virulence than ST11-KL64, which was probably one of the factors preventing the former from spreading widely. In conclusion, ST4496-KL47 was probably derived from ST11-KL47 via intraspecies shifting but was less competitive than ST11-KL64, which also evolved from ST11-KL47 and developed increased virulence via capsule biosynthesis locus recombination. ST11-KL64 has the potential to be the predominant CRKP clone in China.

Antimicrobial resistance profiling and molecular typing of ruminant-borne isolates of Clostridium perfringens from Xinjiang, China

ObjectivesClostridium perfringens (C. perfringens) can cause intestinal diseases in livestock and humans, which seriously threatens the healthy development of animal husbandry and human food safety. Here, the characteristics of antimicrobial resistance and molecular typing of ruminant-borne strains of C. perfringens in Xinjiang, China were explored and profiled. MethodsA total of 307 clinical feces collected from ruminants (cattle and sheep) with diarrheal symptoms were screened for C. perfringens. The recovered isolates were characterized in respect to their antimicrobial resistance pattern and molecular typing. ResultsA total of 109 isolates of C. perfringens were isolated from 307 clinical feces of ruminants, most of which displayed the multidrug resistance (MDR) phenotype. Demonstration of the quinolone-resistance gene was the highest among the isolates (70.6%). The multiplex PCR typing based on toxin genes showed that type A and type D strains made up 82.6% (90/109) and 17.4% (19/109), among which, the isolates carrying β2 gene occupied 43.3% (39/90) of type A strains and 31.6% (6/19) of type D strains. These isolates were divided into 6 genotypes (I-VI) by enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) method. A total of 33 ST types (ST1-ST33) were identified by multilocus sequence typing (MLST) method. ConclusionC. perfringens isolates with multidrug resistance (MDR) were frequent and circulating in ruminants. Among them, type A-Ⅰ-ST19 was the dominant genotype of C. perfringens, displaying obvious genetic diversity. This study provided important epidemiological data for the risk assessment of food safety associated with ruminant-borne C. perfringens in Xinjiang, China.

Prevalence and Characteristics of Metallo-beta-Lactamase-positive and High-risk Clone ST235 Pseudomonas aeruginosa at Ardabil Hospitals

Background: Carbapenems are the most commonly administered drugs for the treatment of multidrug-resistant Pseudomonas aeruginosa (MDR P. aeruginosa) infections. However, carbapenem-resistant P. aeruginosa is spreading rapidly and has led to a new threat to human health worldwide. Objectives: The current study aimed to determine the prevalence of imipenem-resistant P. aeruginosa, detect metallo-β-lactamase (MBL)-producer isolates, and evaluate their clonal relationships in strains isolated from patients referring to the hospitals of Ardabil city, Iran. Methods: The resistance rate to imipenem was evaluated using the disk diffusion method. Double-disk synergy test and PCR technique were used for phenotypic and genotypic screening of MBL-positive P. aeruginosa, respectively. Ultimately, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and multilocus sequence typing (MLST) methods were used for assessing clonal relatedness among the isolates. Results: The prevalence of imipenem-resistant P. aeruginosa strains was estimated at 57.1% (48 out of 84 isolates). In addition, 45 (93.7%) out of 48 imipenem-resistant P. aeruginosa isolates were phenotypically screened as MBL-positive, among which 16 (35.5%) and three (6.6%) isolates harbored blaIMP and blaVIM-1 genes, respectively. However, blaNDM, blaSIM-2, blaSPM, and blaGIM-1 genes were not detected in this study. MBL-producing P. aeruginosa strains were divided into 42 ERIC-PCR types. Based on the results of MLST, P. aeruginosa ST235 was the only identified sequence type. Conclusions: Our results revealed a high and alarming prevalence of imipenem-resistant and blaIMP-positive P. aeruginosa ST235 at Ardabil hospitals. Continuous monitoring is essential to control the further spread of this highly virulent and drug-resistant clone.

Association of sepM gene mutation with mutacin IV production by Streptococcus mutans

To investigate the types of sepM gene mutations and their distribution in clinical isolates of Streptococcus mutans (S. mutans) and explore the association of sepM gene mutation with the capacity of mutacin Ⅳ production by S. mutans. We assessed the capacity of mutacin Ⅳ production in 80 clinical isolates of S. mutans using an inhibition zone assay. The minimum spanning tree and phylogenetic tree of these isolates were constructed using core genome multilocus sequence typing and maximum likelihood method, respectively. GeneMarkS software was used to predict the coding genes of these isolates, and the predicted genes were blasted against the sepM gene sequence of the reference genome UA159 to determine sepM gene mutations and their distribution characteristics in the clinical isolates. The mutation types affecting mutacin Ⅳ production were identified by analyzing the differentially distributed mutations between mutacin Ⅳ-producing isolates and mutacin Ⅳ-free isolates and by comparing the inhibition zones between isolates with sepM gene mutations and those without mutations. Among the 80 clinical isolates of S. mutans, 25 isolates were capable of mutacin Ⅳ production and 55 did not produce mutacin Ⅳ. The minimum spanning tree showed that the allelic differences were less among the mutacin Ⅳproducing isolates than among the mutacin Ⅳ-free isolates, and the origins of the mutacin Ⅳ-producing isolates were similar. We identified a total of 34 single base mutations in the 80 clinical isolates, and among them, C31T (P=0.001), G533A (P < 0.001), C756T (P=0.025), and C1036T (P=0.003) showed significant differential distributions between the mutacin Ⅳ-producing and mutacin Ⅳ-free isolates. These differentially distributed mutations were positively correlated with the capacity of mutacin Ⅳ production of the bacteria. sepM gene mutations that affect the capacity of mutacin Ⅳ production are present in the clinical strains of S. mutans.

Multi-locus sequence typing analysis of Mycoplasma synoviae isolates reveals unique sequence types in China

Mycoplasma synoviae is a common pathogen affecting poultry and has important economic significance. Infectious synovitis is the most common clinical effect. Since 2010, the incidence of M. synoviae infection in China has rapidly risen, causing significant economic losses to the chicken industry; however, the cause of the disease outbreak remains unclear. Phylogenetic and evolutionary analyses of field strains will help unravel the mystery. The multi-locus sequence typing (MLST) method is typically utilized to conduct genotyping and traceability analysis of microorganisms. MLST of M. synoviae has previously been established and shown strong discriminatory power. In this study, 54 Chinese M. synoviae strains isolated from 2016 to 2020 were genotyped by MLST based on seven housekeeping genes. This study aimed to investigate the dominant genotypes of M. synoviae in China and reveal the genetic and evolutionary relationships of these isolates. All 54 isolates were found to have new allelic sequences, which may indicate new sequence types. The results of BURST analysis indicated that all 54 strains belonged to group 11, which is an independent phylogenetic branch, and were separated from any other reference strains (189 isolates) in the PubMLST database. In conclusion, the results of this study suggest that the M. synoviae strains circulating in China are relatively independent in terms of transmission and evolutionary relationships.

Genotypic diversity of Iranian Cryptococcus neoformans using multilocus sequence typing (MLST) and susceptibility to antifungals.

Cryptococcus species is an opportunistic yeast pathogen and classified into different molecular types according to typing techniques including multilocus sequence typing (MLST). The study aimed to investigate the genotypes of environmental Cryptococcus isolates using MLST and the relationship between the in vitro antifungal susceptibility and sequence types of isolates. Genotyping Cryptococcus isolates was performed by the MLST method at seven nuclear loci. Antifungal susceptibility was determined by using CLSI broth micro-dilution method for amphotericin B, fluconazole, itraconazole, voriconazole, flucytosine, and luliconazole. Seven sequence types (ST) were detected using MLST analysis, with the most frequent (50%) ST77, followed by ST4 (16.7%) among 30 C. neoformans isolates. All antifungals demonstrated excellent activity against isolates, except for itraconazole and amphotericin B that were non-wild type against 53.3% and 10% of isolates, respectively. Although seven sequence types belonging to C. neoformans isolates were detected, ST77 was the main sequence type in Ahvaz. Also, non-wild type isolates were only found against itraconazole and amphotericin B.

Multilocus Sequence-Typing of Anthrax Microbe Strains Isolated in Russia and Neighboring Countries

Objective – genotyping by multilocus sequence-typing (MLST) and phylogenetic analysis of 40 Bacillus anthracis strains isolated in Russia and neighboring countries.Materials and methods. In this study, the sequences of seven housekeeping genes of B. anthracis strains were assembled based on the data of a whole genome new generation sequencing, after which the identified mutations and their coordinates were described. The obtained sequences were used for genotyping of the investigated sample using the MLST method. The results are compared with the data presented in PubMLST database. A phylogenetic analysis was performed for the in silico fused sequences of the seven loci of the identified sequence types. The MEGA 7.0 software package was used to build the dendrograms.Results and discussion. Two sequence types (ST) have been found in the examined sample: 35 strains belong to ST-1, and five strains that differed by one common mutation at the glpF locus – to ST-3 (according to PubMLST coding), which emphasizes the genetic separation of this group of strains. One strain has a unique mutation in the gmk gene located outside the region used for MLST.

Analysis of blaCHDL Genes and Insertion Sequences Related to Carbapenem Resistance in Acinetobacter baumannii Clinical Strains Isolated in Warsaw, Poland

Acinetobacter baumannii is an important cause of nosocomial infections worldwide. The elucidation of the carbapenem resistance mechanisms of hospital strains is necessary for the effective treatment and prevention of resistance gene transmission. The main mechanism of carbapenem resistance in A. baumannii is carbapenemases, whose expressions are affected by the presence of insertion sequences (ISs) upstream of blaCHDL genes. In this study, 61 imipenem-nonsusceptible A. baumannii isolates were characterized using phenotypic (drug-susceptibility profile using CarbaAcineto NP) and molecular methods. Pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) methods were utilized for the genotyping. The majority of isolates (59/61) carried one of the following acquired blaCHDL genes: blaOXA-24-like (39/59), ISAba1-blaOXA-23-like (14/59) or ISAba3-blaOXA-58-like (6/59). Whole genome sequence analysis of 15 selected isolates identified the following intrinsic blaOXA-66 (OXA-51-like; n = 15) and acquired class D β-lactamases (CHDLs): ISAba1-blaOXA-23 (OXA-23-like; n = 7), ISAba3-blaOXA-58-ISAba3 (OXA-58-like; n = 2) and blaOXA-72 (OXA-24-like; n = 6). The isolates were classified into 21 pulsotypes using PFGE, and the representative 15 isolates were found to belong to sequence type ST2 of the Pasteur MLST scheme from the global IC2 clone. The Oxford MLST scheme revealed the diversity among these studied isolates, and identified five sequence types (ST195, ST208, ST208/ST1806, ST348 and ST425). CHDL-type carbapenemases and insertion elements upstream of the blaCHDL genes were found to be widespread among Polish A. baumannii clinical isolates, and this contributed to their carbapenem resistance.

Development and evaluation of a core genome multilocus sequence typing scheme for Paenibacillus larvae, the deadly American foulbrood pathogen of honeybees

Summary Paenibacillus larvae is the causative agent of the fatal American foulbrood disease in honeybees (Apis mellifera). Strain identification is vital for preventing the spread of the disease. To date, the most accessible and robust scheme to identify strains is the multilocus sequence typing (MLST) method. However, this approach has limited resolution, especially for epidemiological studies. As the cost of whole‐genome sequencing has decreased and as it becomes increasingly available to most laboratories, an extended MLST based on the core genome (cgMLST) presents a valuable tool for high‐resolution investigations. In this study, we present a standardized, robust cgMLST scheme for P. larvae typing using whole‐genome sequencing. A total of 333 genomes were used to identify, validate and evaluate 2419 core genes. The cgMLST allowed fine‐scale differentiation between samples that had the same profile using traditional MLST and allowed for the characterization of strains impossible by MLST. The scheme was successfully used to trace a localized Swedish outbreak, where a cluster of 38 isolates was linked to a country‐wide beekeeping operation. cgMLST greatly enhances the power of a traditional typing scheme, while preserving the same stability and standardization for sharing results and methods across different laboratories.

Characterization of Blood-isolated, Penicillin-Nonsusceptible Streptococcus pneumoniae From Children Between 2014 and 2018 in Bojnurd, Iran

Background: Streptococcus pneumoniae is one of the common bacterial pathogens in pediatrics. In this study, we performed antimicrobial susceptibility testing, serotyping, and molecular typing of blood-isolated strains of pneumococci in Bojnurd. Objectives: In the current study, blood-isolated, penicillin-nonsusceptible S. pneumoniae strains were subjected to antimicrobial susceptibility testing and typing of capsular polysaccharides using the quelling reaction and PCR method, as well as genotyping using the Multi Locus Sequence Typing (MLST) method. Methods: In this study, 51 S. pneumoniae strains were isolated from blood samples of children less than five-years-old in 2014 - 2018. Antibiogram was performed using the Kirby-Bauer method. All of the isolates were serotyped by the Quelling reaction and PCR. The MLST method was applied to determine the molecular types. Results: Our study revealed that the most common serotypes of blood-isolated pneumococci were 19A, 6A/B, 1, 23F, 19F, 14, 15B/C, and 15A, and the common serotypes in Penicillin-nonsusceptible pneumococci (PNSP) isolates were 19F, 19A, 23F, 14, and finally 15A, 6A/B, 1, and 15B/C. The MLST analysis of PNSP isolates revealed that three highly resistant isolates with MIC ≥ 16 belonged to Sweden15A-25-19A (ST63), Taiwan19F-14-1 (ST236), and Taiwan19F-14 (ST236) clones. Conclusions: Regarding the common serotypes in this study, it seems that PCV-13 is a suitable choice for vaccination in this area. We also observed a high prevalence of PNSP and multi-drug resistant (MDR) strains between 2014 and 2018. It seems that the Taiwan19F-14 clone and its related STs played an essential role in the diffusion of antibiotic-resistant S. pneumoniae isolates in Bojnurd.

Investigation of carbapenem resistant Acinetobacter baumannii ST2 in Iran.

This study investigated carbapenem resistance among Acinetobacter baumannii isolated from respiratory specimens. Epidemiological relationship of the isolates was also evaluated. In this study, 81 respiratory specimens of A. baumannii from AL Zahra Hospital were confirmed by phenotypic and genotypic methods. Antimicrobial susceptibility was performed by disc diffusion method. Carbapenem resistance genes were identified by PCR. The isolates were typed by RAPD-PCR and multilocus sequence typing (MLST) methods. All isolates were resistant to imipenem and 80 isolates to meropenem. Frequency of oxacillinase genes was as follows: blaOXA-23 gene was positive in 74 (91.3%), blaOXA-24 gene in 50 (61.7%) and blaOXA-58 was not found in any isolates. On the other hand 22 (27.2%) isolates contained blaIMP-1, 3 (3.7%) isolates contained blaIMP-2 gene, 5 (6.2%) isolates contained blaVIM-1, 4 (5%) isolates had blaVIM-2 and none of the isolates had blaSIM-1 gene. RAPD-PCR typing identified 16 different patterns, with one pattern being the most frequent one in 26 isolates. In MLST 6 different sequence types were identified, the most predominant being ST2 belonging to clonal complex 2. The results of this study showed high resistance to carbapenems as well as high abundance of oxacillinase genes.

Cytotoxicity profile of Cronobacter species isolated from food and clinical specimens in Brazil.

The objective of this study was to evaluate the cytotoxic activity of Cronobacter strains isolated from foods (n=50) and clinical samples (n=6) in Brazil and genotype selected strains (n=18) using multi-locus sequence typing (MLST) METHODS AND RESULTS: The cytotoxic activity of C. sakazakii (n=29), C. dublinensis (n=13), C. malonaticus (n=6), C. turicensis (n=6) and C. muytjensii (n=2) was screened using Vero, RK13, Hep2c, NCTC clone 929 and BHK-21 cell lines. Selected Cronobacter strains were assigned to C. sakazakii ST 21, C. turicensis ST 252, C. sakazakii ST 647, and three newly assigned STs: C. turicensis STs 738-740. The maximum death caused by non-heat-treated filtrates was 20·4, 86·2, 47·0 and 84·0%, in Vero, RK13, Hep2c and NCTC clone 929 cells, respectively. These were caused by C. sakazakii strains C291 and C292 (ST 494) which had been isolated during neonatal Cronobacter meningitis infection, and C110 (ST 395) isolated from flaxseed flour. Thermal treatment (100°C/20min) significantly reduced the cytotoxicity activity in NCTC clone 929 and Vero cells (P≤2×10-6 ), but not in RK13 (P=0·12) and Hep2c (P=0·85), indicating the cytotoxin(s) were probably proteinaceous. Electron microscopy revealed that cytotoxic compounds from C. sakazakii induced several cell death characteristics, including loss of cell-cell contact, microvilli reduction and cellular lysis. Autophagic vacuoles and mitochondrial damage were the most common ultrastructural features observed. It was concluded that Cronobacter strains, especially C. sakazakii, could produce heat-labile cytotoxic compounds in cell filtrates. This study providing insights into the pathogenesis of the Cronobacter genus. Cytotoxins were identified in excreted filtrates of C. sakazakii strains isolated from food and clinical specimens. The presence of Cronobacter strains that can produce cytotoxins in foods can be a potential threat to human health and highlight the need for high levels of hygiene.

Development of a New High-Throughput Multilocus Sequence Typing Method to Monitor Causative Agents of Nosocomial Infections

Background: Nosocomial infections cause 4%–56% mortality in newborns. Several epidemiological studies have shown that transmission of opportunistic pathogens from the sink to the patient, including Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Serratia marcescens are associated with nosocomial infections in neonatal intensive care units (NICUs). In this project, we aimed to develop fast, accurate, and high-throughput multilocus sequence typing assays (HiMLST-Illumina) to detect opportunistic pathogens to assess their distribution in the sink environment of NICUs and their transfer to patients. Methods: Genome sequences of P. aeruginosa (n = 45), S. maltophilia (n = 23) and S. marcescens (n = 34) strains were retrieved from public genome databases to build their pangenomes, using the open-source PGAdb-builder server. The core genome was identified for each opportunistic pathogen and was searched for genes displaying the highest polymorphism. The minimal number of loci to include in a HiMLST-Illumina assay was determined by comparing topology of phylogenetic trees of concatenated loci based on genome similarity, computed as the average nucleotide identity (ANI) score. The primers used for HiMLST-Illumina schemes were designed in silico on a conserved domain and were tested on reference strains of each species. Results: Bioinformatics analyses showed that 3–4 loci (&lt;300 base pairs per locus) distinguished strains with the same performances than ANI scores. The assays were tested using opportunistic pathogen isolates and environmental DNA originating from NICU sinks. The HiMLST-Illumina analysis of environmental DNA revealed the presence of at least 1 of the 3 studied opportunistic pathogens in 50% of sampled drains (n = 20). In a previous sampling, P. aeruginosa was isolated on selective culture media before and 48 hours after disinfection of a sink drain with chlorine. S. marcescens was also isolated from another sink 2 weeks after disinfection. Identification of the isolates was confirmed by HiMLST-Illumina analyses and will be typed to compare with clinical isolates. Conclusions: Initial in silico tests predict a high discriminating power of the HiMLST-Illumina method, suggesting that it would be possible to quickly identify strains of interest in a large number of samples. The power of this method is also in the possibility for molecular typing without a need for cultivation. Preliminary results suggest that sinks are readily colonized by opportunistic pathogens. This HiMLST-Illumina scheme will be applied in a 2-year intensive survey of NICUs in 3 hospitals in Montreal to evaluate the performance of new sink designs in limiting bioaerosol production and transmission of opportunistic pathogens to patients.Funding: NoneDisclosures: None

New tool for phylogenetic analysis of Helicobacter pylori

The aim of this study was to detect canonical insertion/deletion (INDEL) markers in the genome of Helicobacter pylori and offer INDEL-typing method for differentiation of H. pylori strains. For comparative analysis of the genomes of H. pylori presented in the GenBank database, a local database of nucleotide sequences of 69 H. pylori strains was created. For detecting all INDEL markers with a preset size of 6-20 bp a pairwise comparison of more than 1500 open reading frames (ORF) in the genomes of local database strains was performed. Ten loci containing INDEL markers were founded. The five most variable loci were tested in silico with 21 strains with known geographical origin from the most common populations of hpEurope, hspWAfrica, and hspEAsia. Fifteen individual genotypes with a high diversity index (DI=0.95) were identified. For cluster analysis, the minimal spanning tree (MST) method was used, which demonstrated a clear distribution of clusters according to the geographical origin of the strains tested. INDEL-typing of 21 regional strains from the Astrakhan region was performed in vitro. It was shown that an extensive majority of them belong to the population hpEurope. The findings in this study indicate that the proposed INDEL-typing method almost perfectly reflects the geographical distribution of H. pylori strains determined by the multilocus sequence typing (MLST) method, despite the fact that the primary object of research is completely different genes. Further research is needed to determine the geographical origin of H. pylori strains in Russia.

Emergence of vancomycin-resistant Enterococcus faecium ST1421 lacking the pstS gene in Korea.

Although multilocus sequence typing (MLST) has been used to study molecular epidemiology and to explore the population structure of Enterococcus faecium, vancomycin-resistant E. faecium (VREF) strains lacking the pstS gene that were non-typable using conventional MLST methods were reported recently. We found nationwide emergence of VREF isolates lacking pstS in Korea and hereby report the molecular characteristics of these isolates. Forty-six VREF isolates lacking the pstS gene were identified among 300 VREF rectal isolates collected from hospitalized patients between 2014 and 2015. MLST was performed and clonal relatedness was determined by pulsed-field gel electrophoresis (PFGE). Four VREF ST1421 isolates were whole-genome sequenced. Among the VREF rectal isolates lacking pstS, 98% were classified as ST1421, which has identical allelic profiles to ST17 for all housekeeping genes except pstS. PFGE pattern analyses revealed 32 pulsotypes. All isolates harbored Tn1546 components with various transposase and insertion sequences. The whole-genome sequencing of four VREF ST1421 isolates showed that the pstS gene region was deleted at various locations with considerable inversion. The pstS gene was also depleted in 12.1% of 33 VREF clinical isolates in 2006-2007 and in 11.8% of 59 clinical isolates in 2012-2013. VREF ST1421 strains lacking the pstS gene have emerged in Korea. The emergence and spread of pstS-deleted VREF strains pose a serious challenge for epidemiological investigation. Alternative molecular typing methods to MLST will be increasingly necessary.

Molecular epidemiology of clinically high-risk Pseudomonas aeruginosa strains: Practical overview.

In recent years, numerous outbreaks of multidrug-resistant Pseudomonas aeruginosa have been reported across the world. Once an outbreak occurs, besides routinely testing isolates for susceptibility to antimicrobials, it is required to check their virulence genotypes and clonality profiles. Replacing pulsed-field gel electrophoresis DNA fingerprinting are faster, easier-to-use, and less expensive polymerase chain reaction (PCR)-based methods for characterizing hospital isolates. P. aeruginosa possesses a mosaic genome structure and a highly conserved core genome displaying low sequence diversity and a highly variable accessory genome that communicates with other Pseudomonas species via horizontal gene transfer. Multiple-locus variable-number tandem-repeat analysis and multilocus sequence typing methods allow for phylogenetic analysis of isolates by PCR amplification of target genes with the support of Internet-based services. The target genes located in the core genome regions usually contain low-frequency mutations, allowing the resulting phylogenetic trees to infer evolutionary processes. The multiplex PCR-based open reading frame typing (POT) method, integron PCR, and exoenzyme genotyping can determine a genotype by PCR amplifying a specific insertion gene in the accessory genome region using a single or a multiple primer set. Thus, analyzing P. aeruginosa isolates for their clonality, virulence factors, and resistance characteristics is achievable by combining the clonality evaluation of the core genome based on multiple-locus targeting methods with other methods that can identify specific virulence and antimicrobial genes. Software packages such as eBURST, R, and Dendroscope, which are powerful tools for phylogenetic analyses, enable researchers and clinicians to visualize clonality associations in clinical isolates.