Multijurisdictional Outbreak of Enterohemorrhagic Escherichia coli O157 Caused by Consumption of Ready-to-Eat Grilled Skewered Meat in Niigata, Japan.

Enterohemorrhagic Escherichia coli O157 (EHEC) causes severe complications such as hemolytic uremic syndrome. Contaminated ready-to-eat (RTE) food is one of the vehicles of multijurisdictional outbreaks of foodborne disease worldwide. Multijurisdictional (covering cities, towns, and villages) outbreaks of EHEC are usually linked to an increase in cases, and here we describe such an outbreak involving 29 cases in October 2017 in the Niigata Prefecture. After prefecture-wide active case finding, we conducted a case-control study of 29 cases with eligible data who tested positive for EHEC. To determine the association of the outbreak with risk factors, we compared these cases with 38 controls selected from family and acquaintances who were both symptom free and tested negative for EHEC. The largest number of cases was in the 20-29-year age group (7/29; 24%) and most were women (20/29; 69%). All 29 cases had an identical or similar multilocus variable number tandem-repeat analysis (MLVA) profile. Of these, 76% (22/29) had consumed some type of grilled skewered meat. Also, 69% (20/29) had consumed grilled skewered meat produced by company X. EHEC infection was strongly associated with the consumption of grilled skewered meat produced by any food processing company (odds ratio [OR] = 11.8, confidence interval [95% CI]: 3.7-37.4) and by company X (OR = 9.8, 95% CI: 3.2-30.7). At company X, the skewered meat was grilled to 95°C and then removed from the grilling area to meat trays. The meat trays were not sufficiently washed and disinfected. Testing indicated that the facility was negative for EHEC but four asymptomatic employees tested positive for EHEC. Company X was temporarily closed and voluntarily recalled the foods. We recommend that all employees sufficiently wash and disinfect meat trays to prevent contamination of RTE food, avoid cross-contamination of grilled skewered meat through the environment by regularly cleaning the facility, and appropriately practice self-health care.

Genotypes of Bordetella pertussis isolated from infants in Xi'an and Shanghai.

OBJECTIVE: To compare the genotypes of Bordetella pertussis isolated from infants in Xi'an and Shanghai. METHODS: Samples were collected by nasopharyngeal swab from infants aged <1 year hospitalized with suspected pertussis in Xi'an and Shanghai during 2018 and 2019. Bordetella pertussis was isolated, and multilocus antigen sequence typing (MAST) and multilocus variable-number tandem repeat analysis (MLVA) were used to analyse the genotypes. RESULTS: A total of 1200 samples were collected from infants suspected of pertussis and 60 strains of Bordetella pertussis were isolated, including 34 strains in Xi'an and 26 strains in Shanghai. There were significant differences in the MAST types between Xi'an and Shanghai ( χ 2=18.642, P<0.01); the prn1/ ptxP1/ ptxA1/ fim3-1/ fim2-1 strains dominated in Xi'an (32/34, 94.12%), while the dominated MAST types in Shanghai were prn1/ ptxP1/ ptxA1/ fim3-1/ fim2-1 (13/26, 50.00%) and prn2/ ptxP3/ ptxA1/ fim3-1/ fim2-1 (11/26, 42.31%). The composition of MLVA type of pertussis strains was also significantly different between Xi'an and Shanghai ( χ 2=15.866, P<0.01); the MT195 (13/34, 38.24%), MT55 (10/34, 29.41%) and MT104 (9/34, 26.47%) strains dominated in Xi'an, while the MT27 (12/26, 46.15%) strain was most common in Shanghai. CONCLUSION: There are differences in molecular types of Bordetella pertussis isolated from infants with suspected persussis in Xi'an and Shanghai, indicating that further monitoring of Bordetella pertussis is necessary for better understanding the pathogen evolution in China.

Mycobacterium microti Infections in Free-Ranging Red Deer (Cervus elaphus).

Infections with Mycobacterium microti, a member of the M. tuberculosis complex, have been increasingly reported in humans and in domestic and free-ranging wild animals. At postmortem examination, infected animals may display histopathologic lesions indistinguishable from those caused by M. bovis or M. caprae, potentially leading to misidentification of bovine tuberculosis. We report 3 cases of M. microti infections in free-ranging red deer (Cervus elaphus) from western Austria and southern Germany. One diseased animal displayed severe pyogranulomatous pleuropneumonia and multifocal granulomas on the surface of the pericardium. Two other animals showed alterations of the lungs and associated lymph nodes compatible with parasitic infestation. Results of the phylogenetic analysis including multiple animal strains from the study area showed independent infection events, but no host-adapted genotype. Personnel involved in bovine tuberculosis-monitoring programs should be aware of the fastidious nature of M. microti, its pathogenicity in wildlife, and zoonotic potential.

Mycoplasma pneumoniae Genotypes and Clinical Outcome in Children.

Factors leading to the wide range of manifestations associated with Mycoplasma pneumoniae infection are unclear. We investigated whether M. pneumoniae genotypes are associated with specific clinical outcomes. We compared M. pneumoniae loads and genotypes of children with mucocutaneous disease to those of children with pneumonia, family members with upper respiratory tract infection (URTI), and carriers from a prospective cohort study (n = 47; 2016 to 2017) and to those of other children with mucocutaneous disease from a case series (n = 7; 2017 to 2020). Genotyping was performed using macrolide resistance determination, P1 subtyping, multilocus variable-number tandem-repeat analysis (MLVA), and multilocus sequence typing (MLST). Comparisons were performed with a pairwise Wilcoxon rank sum test and a Fisher exact test with corrections for multiple testing, as appropriate. M. pneumoniae loads did not statistically differ between patients with mucocutaneous disease and those with pneumonia or carriers. Macrolide resistance was detected in 1 (1.9%) patient with mucocutaneous disease. MLVA types from 2016 to 2017 included 3-5-6-2 (n = 21 [46.7%]), 3-6-6-2 (n = 2 [4.4%]), 4-5-7-2 (n = 14 [31.1%]), and 4-5-7-3 (n = 8 [17.8%]), and they correlated with P1 subtypes and MLST types. MLVA types were not associated with specific outcomes such as mucocutaneous disease, pneumonia, URTI, or carriage. They were almost identical within families but varied over geographic location. MLVA types in patients with mucocutaneous disease differed between 2016 to 2017 (3-5-6-2, n = 5 [62.5%]) and 2017 to 2020 (4-5-7-2, n = 5 [71.4%]) (P = 0.02). Our results suggest that M. pneumoniae genotypes may not determine specific clinical outcomes.

Multilocus Variable-Number Tandem-Repeat Analysis of Enterohemorrhagic Escherichia coli Serogroups O157, O26, and O111 Based on a De Novo Look-Up Table Constructed by Regression Analysis.

Multilocus variable-number tandem-repeat analysis (MLVA) is a widely accepted molecular typing tool for enterohemorrhagic Escherichia coli (EHEC). However, ensuring the accuracy of MLVA data among multiple laboratories remains difficult. We developed a method of constructing adjusted look-up tables, which are necessary for MLVA profiling, at each laboratory using a regression analysis based on electrophoresis data from 24 in-house reference strains. On performing MLVA against 51 EHEC O157 isolates, the repeat numbers of 46 isolates were determined accurately using the look-up table with a 99% prediction interval, an outcome superior to that when using a 95% prediction interval. For the remaining five isolates, although the electrophoresis size fell outside the look-up table, we were able to predict the repeat number accurately by extrapolation or the nearest values of the look-up table. Our approach provides more accurate results than a nonadjusted conventional look-up table for calibrating MLVA profiles.

Effective Surveillance Using Multilocus Variable-Number Tandem-Repeat Analysis and Whole-Genome Sequencing for Enterohemorrhagic Escherichia coli O157.

Due to the potential of enterohemorrhagic Escherichia coli (EHEC) serogroup O157 to cause large food borne outbreaks, national and international surveillance is necessary. For developing an effective method of molecular surveillance, a conventional method, multilocus variable-number tandem-repeat analysis (MLVA), and whole-genome sequencing (WGS) analysis were compared. WGS of 369 isolates of EHEC O157 belonging to 7 major MLVA types and their relatives were subjected to comprehensive in silico typing, core genome single nucleotide polymorphism (cgSNP), and core genome multilocus sequence typing (cgMLST) analyses. The typing resolution was the highest in cgSNP analysis. However, determination of the sequence of the mismatch repair protein gene mutS is necessary because spontaneous deletion of the gene could lead to a hypermutator phenotype. MLVA had sufficient typing resolution for a short-term outbreak investigation and had advantages in rapidity and high throughput. cgMLST showed less typing resolution than cgSNP, but it is less time-consuming and does not require as much computer power. Therefore, cgMLST is suitable for comparisons using large data sets (e.g., international comparison using public databases). In conclusion, screening using MLVA followed by cgMLST and cgSNP analyses would provide the highest typing resolution and improve the accuracy and cost-effectiveness of EHEC O157 surveillance.IMPORTANCE Intensive surveillance for enterohemorrhagic Escherichia coli (EHEC) serogroup O157 is important to detect outbreaks and to prevent the spread of the bacterium. Recent advances in sequencing technology made molecular surveillance using whole-genome sequence (WGS) realistic. To develop rapid, high-throughput, and cost-effective typing methods for real-time surveillance, typing resolution of WGS and a conventional typing method, multilocus variable-number tandem-repeat analysis (MLVA), was evaluated. Nation-level systematic comparison of MLVA, core genome single nucleotide polymorphism (cgSNP), and core genome multilocus sequence typing (cgMLST) indicated that a combination of WGS and MLVA is a realistic approach to improve EHEC O157 surveillance.

Different Resolution Power of Multilocus Variable-Number Tandem Repeat Analysis and Whole-Genome Sequencing in the Characterization of S. 1,4,[5],12:i:- Isolates.

Salmonella enterica serovar 1,4,[5],12:i:- has emerged over the last two decades as one of the most common serovars causing human salmonellosis in Europe. It is supposed to originate from Salmonella enterica serovar Typhimurium due to antigenic and genotypic similarities between the two serovars. Due to the high level of similarity, the multilocus variable-number tandem repeat analysis (MLVA) protocol designed for Salmonella Typhimurium routine typing is commonly used also for the characterization of S. 1,4,[5],12:i. Nevertheless, the Salmonella Typhimurium-based MLVA protocol often shows poor discriminatory power for S. 1,4,[5],12:i. Indeed, only a limited number of MLVA profiles have been described for S. 1,4,[5],12:i:-. Moreover, based on the MLVA clustering, S. 1,4,[5],12:i:- is supposed to display high clonality. The aim of the present work was to assess whether the five loci of Salmonella Typhimurium investigated by MLVA are sufficiently accurate to correctly assign S. 1,4,[5],12:i:- isolates. For this purpose, 38 epidemiologically unrelated S. 1,4,[5],12:i:- were subjected to whole-genome sequencing. Isolates were selected among a collection of monophasic strains isolated in Italy from different sources over the period 2014-2016 and belonging to the five most commonly detected MLVA profiles. Results confirmed the possible clonality for S. 1,4,[5],12:i:- serovar in the light of the scarce difference observed in terms of single-nucleotide polymorphisms (SNPs) among investigated isolates. Nevertheless, unrelated isolates on the basis of the difference of SNP number were characterized as indistinguishable by MLVA profile, thus suggesting an insufficient resolution of MLVA. Hence, we can conclude that MLVA-based approach does not seem a valuable proxy to deepen into the epidemiological relationship among S. 1,4,[5],12:i:- isolates. These evidences can be useful to avoid incorrect assignment especially when surveillance data are used for outbreak investigations.

Genetic relatedness of Vibrio cholerae isolates within and between households during outbreaks in Dhaka, Bangladesh.

BACKGROUND: Household contacts of cholera patients have a 100 times higher risk of developing a cholera infection than the general population. To compare the genetic relatedness of clinical and water source Vibrio cholerae isolates from cholera patients' households across three outbreaks, we analyzed these isolates using whole-genome-sequencing (WGS) and multilocus variable-number tandem-repeat analysis (MLVA). RESULTS: The WGS analyses revealed that 80% of households had source water isolates that were more closely related to clinical isolates from the same household than to any other isolates. While in another 20% of households an isolate from a person was more closely related to clinical isolates from another household than to source water isolates from their own household. The mean pairwise differences in single nucleotide-variant (SNV) counts for isolates from the same household were significantly lower than those for different households (2.4 vs. 7.7 p < 0.0001), and isolates from the same outbreak had significantly fewer mean pairwise differences compared to isolates from different outbreaks (mean: 6.2 vs. 8.0, p < 0.0001). Based on MLVA in outbreak 1, we observed that the majority of households had clinical isolates with MLVA genotypes related to other clinical isolates and unrelated to water source isolates from the same household. While in outbreak 3, there were different MLVA genotypes between households, however within the majority of households, the clinical and water source isolates had the same MLVA genotypes. The beginning of outbreak 2 resembled outbreak 1 and the latter part resembled outbreak 3. We validated our use of MLVA by comparing it to WGS. Isolates with the identical MLVA genotype had significantly fewer mean pairwise SNV differences than those isolates with different MLVA genotypes (mean: 4.8 vs. 7.7, p < 0.0001). Furthermore, consistent with WGS results, the number of pairwise differences in the five MLVA loci for isolates within the same household was significantly lower than isolates from different households (mean: 1.6 vs. 3.0, p < 0.0001). CONCLUSION: These results suggest that transmission patterns for cholera are a combination of person-to-person and water-to-person cholera transmission with the proportions of the two modes varying within and between outbreaks.

Significant Decrease in Pertactin-Deficient Bordetella pertussis Isolates, Japan.

Prevalence of pertactin-lacking Bordetella pertussis isolates has been observed worldwide. In Japan, however, we found that the frequency of pertactin-deficient isolates in 2014-2016 (8%) was significantly lower than the frequency in 2005-2007 (41%), 2008-2010 (35%), and 2011-2013 (25%). This reduction was closely associated with changes in genotypes.

Investigation of Outbreaks of Salmonella enterica Serovar Typhimurium and Its Monophasic Variants Using Whole-Genome Sequencing, Denmark.

Whole-genome sequencing is rapidly replacing current molecular typing methods for surveillance purposes. Our study evaluates core-genome single-nucleotide polymorphism analysis for outbreak detection and linking of sources of Salmonella enterica serovar Typhimurium and its monophasic variants during a 7-month surveillance period in Denmark. We reanalyzed and defined 8 previously characterized outbreaks from the phylogenetic relatedness of the isolates, epidemiologic data, and food traceback investigations. All outbreaks were identified, and we were able to exclude unrelated and include additional related human cases. We were furthermore able to link possible food and veterinary sources to the outbreaks. Isolates clustered according to sequence types (STs) 19, 34, and 36. Our study shows that core-genome single-nucleotide polymorphism analysis is suitable for surveillance and outbreak investigation for Salmonella Typhimurium (ST19 and ST36), but whole genome-wide analysis may be required for the tight genetic clone of monophasic variants (ST34).

Multilocus variable number tandem repeat analysis of Edwardsiella piscicida isolates pathogenic to fish.

This study describes a novel multilocus variable number tandem repeat analysis (MLVA) based on six variable number of tandem repeat (VNTR) loci for genotyping of 37 Edwardsiella piscicida (previously Edwardsiella tarda) isolates from multiple sources. The number of alleles identified for each of the six VNTR loci ranged from 3 to 5 with VNTR loci 1 (DI = 0.632) and 3 (DI = 0.644), displaying the highest degrees of polymorphism. MLVA typing of the 37 E. piscicida isolates resulted in the identification of five major clusters consistent with their geographical origins, and were designated as MLVA types I, II, III, IV and V. Types III and V were resolved further into subtypes largely consistent with outbreak source. An MLVA profile comprising a string of integers representing the number of tandem repeats for each allele provided a unique identification for each MLVA type and/or strain. The MLVA protocol described in the current study is robust, relatively simple, has a higher power of resolution than multilocus sequence analysis (MLSA) and is capable of discriminating closely related isolates.

Molecular typing methods to characterize Brucella spp. from animals: A review.

Brucellosis is an infectious disease of animals that can infect humans. The disease causes significant economic losses and threatens human health. A timely and accurate disease diagnosis plays a vital role in the identification of brucellosis. In addition to traditional diagnostic methods, molecular methods allow diagnosis and typing of the causative agent of brucellosis. This review will discuss various methods, such as Bruce-ladder, Suiladder, high-resolution melt analysis, restriction fragment length polymorphism, multilocus sequence typing, multilocus variable-number tandem repeat analysis, and whole-genome sequencing single-nucleotide polymorphism, for the molecular typing of Brucella and discuss their advantages and disadvantages.

A simple approach to obtain comparable Shigella sonnei MLVA results across laboratories.

Multilocus variable-number tandem repeat analysis (MLVA) is a promising subtyping tool to complement pulsed-field gel electrophoresis for discriminating closely related strains of some monomorphic organisms, including Shigella sonnei, which is one of the major foodborne pathogens. However, MLVA results are usually difficult to compare directly between laboratories, impeding the application of MLVA as a subtyping tool for disease surveillance and investigation of common outbreaks across regions or countries. It has long been a big challenge in seeking an approach that can be implemented to obtain comparable MLVA results across laboratories. By implementing a panel of calibration strains in each participating laboratory for data normalization, the MLVA results of 20 test strains were comparable even though some analytical conditions were different among the laboratories. This approach is simple, protocol independent, and easy to implement in every laboratory, and a small calibration set is sufficient to generate mathematical equations for accurate copy number conversion.

Identification of the molecular characteristics of Bacillus anthracis (1982-2020) isolates in East Indonesia using multilocus variable-number tandem repeat analysis.

Background and Aim: Anthrax is one of the endemic strategic diseases in East Indonesia, particularly in the provinces of South Sulawesi, West Sulawesi, Gorontalo, East Nusa Tenggara, and West Nusa Tenggara. Anthrax is an important disease due to its zoonotic and economic impact on the livestock industry. This study aimed to identify the molecular characteristics of Bacillus anthracis in East Indonesia using multilocus variable-number tandem repeat (VNTR) analysis (MLVA). Materials and Methods: Isolates were obtained from an investigation of anthrax outbreaks in five provinces of East Indonesia from 1982 to 2020. Conventional polymerase chain reaction for B. anthracis was used to identify MLVA-8. Deoxyribonucleic acid sequencing analysis was based on MLVA-8 primers for VNTR identification of the phylogenetic relationship among 24 isolates of B. anthracis obtained from 17 distinct districts/cities in East Indonesia. Tandem Repeats Finder was used for VNTR identification, and Molecular Evolutionary Genetics Analysis X was used to construct phylogenetic analysis. Results: In this study, 24 isolates were classified as genotype or lineage A. There were four subgroups of B. anthracis circulating in East Indonesia based on eight molecular marker loci sequence results. Conclusion: The findings of this study show that MLVA-8 typing might be useful as a subtyping tool for the epidemiological investigation of identical genotypes and low genetic diversity of B. anthracis. No other lineage of B. anthracis was circulating in East Indonesia. Other molecular methods are needed, such as extended MLVA, whole-genome sequencing, and canonical single-nucleotide polymorphism, for a more precise study of B. anthracis genetic diversity.

Staphylococcal Cassette Chromosome mec Typing and Multilocus Variable Number Tandem Repeat Analysis of Methicillin Resistant Staphylococcus aureus Clinical Isolates with Vancomycin Creep Phenomenon.

Background: The association of treatment failure and mortality with vancomycin minimum inhibitory concentration creep (MIC) is a matter of serious concern in patients with severe methicillin resistant Staphylococcus aureus (MRSA) infections. The purpose of the study was to identify and characterize staphylococcal cassette chromosome mec (SCCmec) and clonal types of MRSA strains, exhibiting the vancomycin MIC creep phenomenon. Methods: A total of 3305 S. aureus strains were isolated from various clinical samples of Lahore General Hospital, Lahore, Pakistan. MRSA strains were identified by cefoxitin resistant (≤21mm) followed by mecA and mecC gene genotyping. Vancomycin MIC creep was determined by E-test. Isolates having MIC values >1.5 µg/mL were further subjected for SCCmec typing (I-V and XI) and multiple-locus variable number tandem repeat analysis (MLVA) by amplification of spa, sspA, clfA, clfB, and sdrCDE genes. A dendrogram was created based on the similarity index using bioneumerics software. Results: About 13.3% (440/3305) isolates were MRSA with 99.3% (437/440) and 0.7% (3/440) carried mecA and mecC genes, respectively. In 120 MRSA isolates, the MIC of vancomycin was >1.5µg/mL. In MRSA isolates with high vancomycin MIC (>1.5µg/mL), the most common SCCmec type was SCCmec III (38.3%), followed by SCCmec IVa (15.8%), SCCmec IIIa (13.3%,), SCCmec IVc (7.5%), SCCmec IVe (5.8%), SCCmec IVd (5.8%), SCCmec IVb (4.2%), SCCmec II (2.5%), SCCmec V (1.7%), SCCmec I (1.7%) and SCCmec XI (1.7%). MLVA revealed 60 genotypic groups of MRSA isolates having a 92% similarity index. Conclusion: SCCmec III was the most common type in genetically related MRSA isolates showing vancomycin MIC creep. The presence of SCCmec XI may further add burden to infection control measures.

Macrolide susceptibility and molecular characteristics of Bordetella pertussis.

OBJECTIVE: To analyse macrolide resistance and molecular characteristics of Bordetella pertussis clinical isolates from western China, and to explore the relationship between macrolide-resistance and genotypes. METHODS: Susceptibilities of B. pertussis clinical isolates to erythromycin, azithromycin and clarithromycin were determined by epsilometer test (E-test). Isolated strains were sequenced to ascertain the presence of the 23S rRNA gene A2047G mutation. Strains were typed using multilocus antigen sequence typing, multilocus variable-number tandem-repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). RESULTS: Of 58 B. pertussis strains isolated in this study, 46 were macrolide-resistant and 12 were macrolide sensitive. All macrolide-resistant strains carried the A2047G mutation and were the prn1/ptxP1/ptxA1/fim3-1/fim2-1 genotype; the MLVA types were MT195 (19/58), MT55 (13/58) and MT104 (14/58), and the PFGE profiles were classified into BpSR23 (17/58) and BpFINR9 (29/58) types. None of the macrolide-sensitive strains carried the A2047G mutation; genotypes were (prn9 or prn2)/ptxP3/ptxA1/fim3-1/fim2-1, and all were MT27. PFGE profiles differed from the macrolide-resistant strains. CONCLUSIONS: B. pertussis clinical isolates from western China were severely resistant to macrolides. Genotypes differed between macrolide-resistant and macrolide-sensitive strains, and there may be a correlation between acquisition of macrolide resistance and changes in specific molecular types.

Genotyping and macrolide-resistant mutation of Bordetella pertussis in East and South-East Asia.

OBJECTIVES: Macrolide-resistant Bordetella pertussis (MRBP) has been emerging and prevailing in mainland China since 2011. In this study, we aimed to investigate the genotype and macrolide resistance of circulating B. pertussis in East and Southeast Asia using genetic analyses. METHODS: A total of 302 DNA extracts from clinical specimens and isolates from 2010 to 2020 were analyzed: 145 from Vietnam, 76 from Cambodia, 48 from Taiwan, and 33 from Japan. Genotypes were determined by multilocus variable-number tandem-repeat analysis (MLVA). Macrolide-resistant A2047G mutation in B. pertussis 23S rRNA was investigated using the duplex Cycleave real-time polymerase chain reaction (PCR) assay. Whole-genome sequencing was performed on two MRBP isolates that were identified for the first time in Taiwan. RESULTS: Overall, 286 DNA extracts (95%) generated a complete MLVA genotype and 283 DNA extracts (94%) yielded a complete result for the A2047G mutation analysis. The A2047G mutation was detected in 18 DNA extracts: fourteen from Vietnam, one from Cambodia, two from Taiwan, and one from Japan. Most of them (78%) showed the genotypes MT104 and MT195, which have previously been reported in Chinese MRBP isolates. Further, the Taiwanese MRBP isolates were classified into the MT104 clade of Chinese MRBP isolates. CONCLUSION: After MRBP emerged and spread in mainland China, it may have spread to East and Southeast Asia in the 2010s. Continued surveillance targeting the A2047G mutation of MRBP is needed to prevent further spread of this emerging pathogen.

Molecular characterization of Vibrio cholerae O1 isolates obtained from outbreaks in the Philippines, 2015-2016.

Introduction. The Philippines, comprising three island groups, namely, Luzon, Visayas and Mindanao, experienced an increase in cholera outbreaks in 2016. Previous studies have shown that Vibrio cholerae isolates obtained from the Philippines are novel hybrid El Tor strains that have evolved in the country and are clearly distinct from those found in Mozambique and Cameroon.Gap statement. The characterization of the strains isolated from outbreaks has been limited to phenotypic characteristics, such as biochemical and serological characteristics, in most previous studies.Aim. We performed multilocus variable-number tandem repeat (VNTR) analysis (MLVA) for V. cholerae isolates obtained from 2015 to 2016 to further characterize and understand the emergence and dissemination of the strains in the Philippines.Methodology. A total of 139 V. cholerae O1 Ogawa biotype El Tor isolates were obtained from the Philippines during diarrhoeal outbreaks in 18 provinces between 2015 and 2016. VNTR data were analysed to classify the MLVA profiles where the large-chromosome types (LCTs) were applied for grouping.Results. We identified 50 MLVA types among 139 isolates originating from 18 provinces, and 14 LCTs. The distribution of the LCTs was variable, and a few were located in specific areas or even in specific provinces. Based on eBURST analysis, 99 isolates with 7 LCTs and 32 MLVA types belonged to 1 group, suggesting that they were related to each other. LCT A was predominant (n=67) and was isolated from Luzon and Visayas. LCT A had 14 MLVA types; however, it mostly emerged during a single quarter of a year. Eight clusters were identified, each of which involved specific MLVA type(s). The largest cluster involved 23 isolates showing 3 MLVA types, 21 of which were MLVA type A-14 isolated from Negros Occidental during quarter 4 of 2016. Comparative analysis showed that almost all isolates from the Philippines were distinct from those in other countries.Conclusions. The genotypic relationship of the V. cholerae isolates obtained during outbreaks in the Philippines was studied, and their emergence and dissemination were elucidated. MLVA revealed the short-term dynamics of V. cholerae genotypes in the Philippines.

Multilocus Variable-Number Tandem-Repeat Analysis for Geno-typing of Escherichia coli Strains Isolated from Hospital Wastewater, Tehran, Iran.

BACKGROUND: Escherichia coli is one of the most frequent causes of many common bacterial infections. As a potential reservoir, hospital wastewater is considered for the dissemination of bacterial pathogens such as E. coli. Therefore, research on hospital waste's bacteria by low-cost, rapid and easy molecular typing methods such as multilocus variable-number tandem-repeat analysis (MLVA) can be helpful for the study of epidemics. METHODS: E. coli strains were isolated from hospital wastewater sources in Tehran, Iran, over a 24-month sampling period (Jun 2014- Jun 2016) and identified by standard bacteriological methods. The diversity of repeated sequences of seven variable-number tandem-repeat (VNTR) loci was studied by MLVA method base on polymerase chain reaction (PCR). RESULTS: Overall, 80 E. coli isolates were discriminated into 51 different genotypes. Analysis of the MLVA profiles using a minimum spanning tree (MST) algorithm showed two clonal complexes with 71 isolates and only nine isolates were stayed out of clonal complexes in the form of a singleton. High genotypic diversity was seen among E. coli strains isolated from hospital wastewaters; however, a large number of isolates showed a close genetic relationship. CONCLUSION: MLVA showed to be a rapid, inexpensive and useful tool for the analysis of the phylogenetic relationships between E. coli strains under the study.

A novel multilocus variable-number tandem repeat analysis for Bordetella parapertussis.

Purpose. Human-adapted Bordetella parapertussis is one of the causative agents of whooping cough; however, there are currently no genotyping systems with high discriminatory power for this bacterial pathogen. We therefore aimed to develop a multilocus variable-number tandem repeat analysis (MLVA) for human-adapted B. parapertussis.Methodology. Four highly polymorphic variable number tandem repeat (VNTR) loci in the B. parapertussis genome were selected and amplified by multiplex PCR. MLVA was performed based on the number of tandem repeats at VNTR loci. The discriminatory power of MLVA was evaluated with three laboratory reference strains and 50 human isolates of B. parapertussis.Results. Multiplex PCR-based MLVA characterized 53 B. parapertussis reference strains and isolates into 25 MLVA types and the Simpson diversity index was 0.91 (95 % confidence interval, 0.86-0.97). The three reference strains exhibited different MLVA types. Thirty-one Japanese isolates, ten French isolates and three Taiwanese isolates belonged to fourteen, nine and three MLVA types, respectively. In contrast, all five Australian isolates belonged to the same type. Two Japanese isolates collected from patients with known epidemiological links had the same type.Conclusion. Our novel MLVA method has high discriminatory power for genotyping human B. parapertussis. Regarding this organism, this genotyping system is a promising tool for epidemiological surveillance and investigating outbreaks.