Multiple-locus Variable-number Tandem Repeat Research Articles

Investigation of the Relationships Between Clinical and Environmental Isolates of Aspergillus fumigatus by Multiple-locus Variable Number Tandem Repeat Analysis During Major Demolition Work in a French Hospital.

Genotyping is needed to explore the link between clinical cases from colonization of invasive aspergillosis (IA) and major building construction. Attempts to correlate Aspergillus fumigatus strains from clinical infection or colonization with those found in the environment remain controversial due to the lack of a large prospective study. Our aim in this study was to compare the genetic diversity of clinical and environmental A. fumigatus isolates during a demolition period. Fungal contamination was monitored daily for 11 months in 2015. Environmental surveillance was undertaken indoors and outdoors at 8 locations with automatic agar samplers. IA infection cases were investigated according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group criteria. Isolates were identified by amplification and sequencing of the β- tubulin gene. They were genotyped by multiple-locus variable number tandem repeat analysis (MLVA). The phylogenetic relationships between isolates were assessed by generating a minimum spanning tree. Based on 3885 samples, 394 A. fumigatus isolates (383 environmental and 11 clinical) were identified and genotyped using MLVA. Clinical isolates were collected from patients diagnosed as having probable IA (n = 2), possible IA (n = 1), or bronchial colonization (n = 6). MLVA generated 234 genotypes. Seven clinical isolates shared genotypes identical to environmental isolates. Among the diversity of genotypes described, similar genotypes were found in clinical and environmental isolates, indicating that A. fumigatus infection and colonization may originate from hospital environments.

Investigation and source-tracing of an anthrax outbreak in Gansu Province, China.

Anthrax is an endemic disease in China. Cases are reported every year, especially in the northwestern areas. In August 2016, an outbreak of 21 cutaneous anthrax cases was reported in Min County, Gansu Province, China. In this study, the general characteristics of the anthrax outbreak are described. Two molecular typing methods, canonical single-nucleotide polymorphism (canSNP) and multiple-locus variable-number tandem repeat analysis with 15 markers (MLVA15), were used to investigate the possible source of transmission and to identify the genetic relationship among the strains/samples isolated in this outbreak as well as previous isolates. In this outbreak, all patients were infected through contact with diseased livestock or contaminated animal products. Livestock had been introduced into the local area shortly before the outbreak from Gannan Prefecture (in Gansu Province), Sichuan and Qinghai Provinces. In the molecular typing analysis, there were two canSNP subgroups found in Gansu, A.Br.001/002 and A.Br.Ames, and five MLVA15 genotypes were observed. The strains collected from the anthrax outbreak in Min County in 2016 belonged to the A.Br.001/002 canSNP subgroup and the MLVA15-28 and MLVA15-30 genotype. Strains previously isolated from Sichuan, Inner Mongolia and Maqu County (in Gannan Prefecture, Gansu Province) were clustered with these outbreak-related strains/samples according to the MLVA15-30 genotype. The MLVA15-28 genotype was found in strains isolated from Gansu and Xinjiang in previous studies. Combining the epidemiological investigation and molecular typing results, we conclude that the patients in this outbreak were infected by a local pathogen present in the adjoining area of Gansu, Sichuan and Qinghai Provinces.

Source tracing of the Yersinia pestis strains isolated from Heqing county, Yunnan province in 2017

Objective: To understand the genotype of the Yersinia (Y.) pestis strains isolated from Heqing county, Yunnan province in 2017 and provide evidence for the prevention and control of plague in this area. Methods: Ten Y. pestis strains isolated from Heqing were typed by the detections of different region (DFR) and clustered regularly interspaced short palindromic repeats (CRISPRs) as well as multiple-locus variable-number tandem repeat analysis (MLVA). And the results were compared with those of the 93 Y. pestis strains from the adjacent plague foci of Heqing obtained from the established database for clustering analysis. Results: The results showed that Heqing strains had the same type of DFR (Genomovar 05) and CRISPRs (Cluster Ca7, Type 22) with isolates from the plague focus in Lijiang. Heqing strains and Lijiang strains were in the same cluster in MST and only VNTR loci N2117 and M23 of Heqing strains were different from that of Lijiang strains. Conclusion: The Y. pestis strains isolated from Heqing in 2017 were highly homogenous with the strains isolated from wild rodents in plague focus in Lijiang, and Heqing plague might be the result of further southward spread of Lijiang plague.

Genotyping Mycoplasma hyopneumoniae isolates based on multi-locus sequence typing, multiple-locus variable-number tandem repeat analysis and analysing gene p146

Mycoplasma hyopneumoniae is a swine pathogen bacterium, causing significant economic losses worldwide. Epidemiological investigations based on molecular typing methods support the prevention and eradication strategies for the control of M. hyopneumoniae, through tracing the spreading of the pathogen. The present study describes the genotyping of 44 M. hyopneumoniae strains isolated from Hungarian, Czech and Slovakian porcine lung samples by multi-locus sequence typing (MLST), multiple-locus variable-number tandem repeat analysis (MLVA) and analysing gene p146, and the evaluation of the used methods. The resolution of the three-gene (adk, rpoB, tpiA) and the seven-gene (efp, metG, pgiB, recA, adk, rpoB, tpiA) based MLST systems was identical with 27 sequence types. MLVA utilising loci P97-RR1 and Locus1 extended with the serine repeat numbers of gene p146 showed the highest resolution power among the studied methods differentiating 40 genotypes. The independent analysis of gene p146 revealed 31 different types among the isolates.High variability of M. hyopneumoniae strains was detected by the used typing methods. The results confirmed that utilization of the minimal MLST is suitable for phylogenetic analyses of M. hyopneumoniae strains. The MLVA method extended with the evaluation of serine repeat numbers of gene p146 is adequate for the resolution of genetic relationships within MLST groups. Examination of the p146 gene is suitable to complement both MLST and MLVA methods in order to refine closer genetic relationships.

Phenotypic Characterization and Genotypic Subtyping of Salmonella enterica Serovars Enteritidis and Gallinarum Isolated from Human and Poultry-Related Samples

ABSTRACTThis study aimed to assess the serotypes, antibiotic susceptibility, and molecular subtyping of Salmonella enterica isolated from food products and human patients with gastroenteritis. A total of 59 isolates were investigated, and the results revealed a predominance of S. Enteritidis with 57.63% (34/59) S. Gallinarum held second with 15.62% (5/32) of total food borne. While, isolates from humans showed 18.51% (5/27) of S. Typhimurium. High level of resistance to nalidixic acid was noted among food strains and 35.29% of human isolates were found to be multidrug resistant. Eight representative isolates were subtyping using three molecular approaches, ribotyping, Multilocus sequence typing (MLST) and Multiple-locus variable number tandem repeat analysis (MLVA). MLST showed three sequence types corresponding to two clonal complexes, (ST-78, CC-4) for S. Gallinarum, (ST-11, CC-4) for S. Enteritidis and (S-367, CC-37) for S. Cerro. While, MLVA generated six different profiles targeting nine loci for S. Enteritidis and S. Gallinarum.

The predominance and clustering of Clostridioides (Clostridium) difficile PCR ribotype 001 isolates in three hospitals in Eastern Slovakia, 2017.

This study aimed to implement a toxigenic culture as an optional third diagnostic step for glutamate dehydrogenase (GDH)-positive and toxin A/B-negative diarrheal stool samples into a diagnostic algorithm for Clostridioides (Clostridium) difficile infection (CDI), and to characterise C. difficile isolates for epidemiological purposes. During the 5-month study, 481 diarrhoeal stool samples from three Slovak hospitals were investigated and 66 non-duplicated GDH-positive stool samples were found. Of them, 36 were also toxin A/B-positive. Twenty-three GDH-positive and toxin A/B-negative stool samples were shown subsequently to be positive following toxigenic culture (TC). Molecular characterisation of C. difficile isolates showed the predominance of PCR ribotype (RT) 001 (n = 37, 56.1%) and the occurrence of RT 176 (n = 3, 4.5%). C. difficile RT 001 isolates clustered to eight clonal complexes (CCs) using multiple-locus variable-number tandem repeats analysis (MLVA). Interestingly, one third of RT 001 isolates clustering in these CCs were cultured from toxin A/B-negative stool samples. Our observations highlight the need of use multiple step diagnostic algorithm in CDI diagnosis in order to detect all CDI cases and to avoid the spread of C. difficile in healthcare settings.

Genetic Analysis Method for Staphylococcus chromogenes Associated with Goat Mastitis.

Mastitis in goats is mainly caused by coagulase-negative Staphylococcus (CNS). The identification methods for this group are based on evaluating the expression of phenotypic characteristics such as the ability to metabolize various substrates; however, this is disadvantageous as these methods are dependent on gene expression. In recent years, genotyping methods such as the Multiple Locus Variable-Number Tandem Repeat Analysis (MLVA) and gene identification have been useful for epidemiological study of several bacterial species. To develop a genotyping method, the genome sequence of Staphylococcus chromogenes MU970 was analysed. The analysis showed nine virulence genes described in Staphylococcus aureus. The MLVA was developed using four loci identified in the genome of S. chromogenes MU970. This genotyping method was examined in 23 strains of CNS isolated from goat mastitis. The rate of discrimination for MLVA was 0.8893, and the highest rates of discrimination per the index of Simpson and Hunter-Gaston were 0.926 and 0.968 for the locus 346_06, respectively. The virulence genes were present in all strains of S. chromogenes but not in other CNS. The genotyping method presented in this paper is a viable and easy method for typifying CNS isolates from mastitis cases in different regions and is an ideal mean of tracking this disease.

Rapid and safe one-step extraction method for the identification of Brucella strains at genus and species level by MALDI-TOF mass spectrometry.

Brucellosis is essentially a disease of domesticated livestock; however, humans can also be infected via the consumption of contaminated meat or dairy products, underlying the need for rapid and accurate identification methods. Procedures for microbiological identification and typing of Brucella spp. are expensive, time-consuming, and must be conducted in biohazard containment facilities to minimize operator risk. The development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based assay has reduced the processing time while maintaining performance standards. In this study, to improve the identification accuracy and suitability of the MALDI-TOF-based assay for routine diagnosis, we developed a new protein extraction protocol and generated a custom reference database containing Brucella strains representative of the most widespread species. The reference library was then challenged with blind-coded field samples isolated from infected animals. The results indicated that the database could be used to correctly identify 99.5% and 97% of Brucella strains at the genus and species level, respectively, indicating that the performance of the assay was not affected by the different culture conditions used for microbial isolation. Moreover, the inactivated samples were stored and shipped to reference laboratories with no ill effect on protein stability, thus confirming the reliability of our method for routine diagnosis. Finally, we evaluated the epidemiological value of the protocol by comparing the clustering analysis results of Brucella melitensis strains obtained via multiple locus variable-number tandem repeat analysis or MALDI-TOF MS. The results showed that the MALDI-TOF assay could not decipher the true phylogenetic tree, suggesting that the protein profile did not correspond with the genetic evolution of Brucella.

Multiple-locus variable-number tandem repeat analysis using multiplex polymerase chain reaction and next-generation sequencing - A novel high-throughput method for subtyping Listeria strains

Listeria monocytogenes is significantly associated with listeriosis infection. Several species of Listeria other than L. monocytogenes directly affect the food industry. In the present study we developed a novel subtyping method for tracking the source of L. monocytogenes and L. innocua in large-scale investigations using multiple-locus variable-number tandem repeat analysis (MLVA) coupled with next-generation sequencing (NGS). Forty-eight L. monocytogenes and L. innocua strains isolated from a food processing plant and the environment were used for the amplification of 15 specific variable number tandem repeat (VNTR) loci. The results exhibited 100% concordance for amplicon detection using both the subtyping methods. Coupling of MLVA with capillary electrophoresis (CE) or NGS detected 38 and 89 different alleles, respectively. MLVA coupled with NGS showed significantly higher discriminating power than MLVA coupled with CE. In MLVA coupled with NGS method, locus JLR1 of L. monocytogenes had 54% discrimination while locus TR3 had 100% discrimination solely in L. innocua. In addition, MLVA coupled with NGS method had significantly higher discrimination power (DI = 1.0 with 48 MLVA patterns) than that of MLVA coupled with CE (DI = 0.79 with 15 MLVA patterns). These results indicate the advantage of MLVA coupled with NGS method in detecting not only the length but also sequence polymorphism in a single read run. MLVA coupled with NGS led to significant reduction in labor and cost than CE and the traditional Sanger sequencing. Thus, MLVA coupled with NGS is an effective and feasible technique for the molecular typing of Listeria species in food industries.

Antimicrobial susceptibility and MLVA analysis of S. Typhimurium strains isolated from human and poultry samples in Tunisia.

Salmonella enterica infections are a significant public health concern worldwide, being Salmonella Typhimurium one of the most prevalent serovars. Human salmonellosis is typically associated with the consumption of contaminated foods, such as poultry, eggs and processed meat. The extensive use of antimicrobials in humans and animals has led to an increase in multidrug resistance among Salmonella strains, becoming multidrug-resistant (MDR) strains a major public health concern. This study was designed to investigate the antimicrobial susceptibility and the genotypic diversity of Salmonella Typhimurium strains isolated in Tunisia from human and poultry sources from 2009 to 2015. Fortyfive strains were analyzed by disk-diffusion test to determine the antimicrobial susceptibility. The presence of antimicrobial resistance genes was tested by PCR, and genotyping was performed using multiple-locus variable-number tandem repeats analysis (MLVA). About 50% of the strains were resistant to at least 3 antibiotics (multidrug-resistant strains, MDR). The most frequent resistance profile in clinical strains was AMP-TIC-TET-MIN-SXT (n = 7) and TET-MIN in poultry origin strains (n = 7). The MLVA typing grouped the strains in 2 main clusters. Cluster I was mostly formed by human isolates, whereas in cluster II both human and poultry isolates were grouped. Simpson's diversity index was 0.870 and 0.989 for antimicrobial resistance profiles and MLVA, respectively. Multiresistance is common in Salmonella Typhimurium isolated from human and poultry sources in Tunisia. The genotyping results suggest that some strains isolated from both sources may descend from a common subtype.

Genotyping of pathogenic leptospira by Multiple Locus Variable-number Tandem Repeat Analysis (MLVA)

ObjectiveTo introduce the method of molecular genotyping (MLVA) to determine the genotype of field isolates of leptospira.IntroductionLeptospirosis (ictherohemoglobinuria, Leptospirosis biliousness) is a natural focal and zoonotic infectious disease dangerous for humans and farm animals. It is important to identify specific leptospira strains isolated from rodents or sick and suspicious animals by the serotype or genotype. In comparison with serotyping using micro agglutination test (MAT), molecular genotyping makes it possible to accurately identify a specific pathogen strain. The genetic classification now becomes more significant than the phenotypic classification.MethodsSpecific oligonucleotide primers, which flank fragments of the genome locus of pathogenic leptospira varies in terms of the number of tandem repeats VNTR-4, -7, -10 specific for L.interrogans, L.kirschneri, and L.borgpetersenii were used. The amplification products were detected using agar gel electrophoresis with the following identification of the fragment length with a molecular weight marker and comparison with the collection of VNTR profiles of the strains described in the literature.ResultsIt was established that the method of leptospira molecular genotyping by determining the number of variable tandem repeats of a locus (VNTR-variable number tandem repeats analysis) is suitable for molecular epizootology studies in Ukraine. The advantages of the method are the simplicity of performance and availability for diagnostic and research laboratories in Ukraine compared to other pathogen genome sequencing based genotyping methods, in particular Multilocus sequence typing (MLST) or Multispacer Sequence Typing (MST), which require complex equipment and operating conditions. The reference strain of Leptospira M20 serotype Copengageni serogroup Icterohaemorrhagiae from the NAAS IVM collection of was studied and its VNTR profile was identified with the genotype of the strain Fiocruz L1-130 that is described in the literature as a serotype of Copengageni serogroup Icterohaemorrhagiae. The genotype of the leptospira field isolate obtained from a rat in Lviv Oblast of Ukraine was specified and its identity was established in the aforementioned genotype. The obtained data support the prospects of using MLVA genotyping method to study the distribution of different genotypes of leptospira. The research will continue to study the specificities of molecular epizootology of leptospirosis in Ukraine.ConclusionsThe method of leptospira molecular genotyping by multilocus analysis of the number of variable tandem repeats has been tested in the Leptospirosis Research Laboratory in collaboration with the Museum of Microorganisms at the National Academy of Sciences, the Ukraine Institute of Veterinary Medicine, the ELISA and PCR Research Laboratory, and the Bila Tserkva National Agrarian University. The genotype of the reference strain has been correlated with its serological profile; identification of the genotype of the field isolate pathogenic leptospira has been completed. The tested method is planned to be implemented in surveillance and control over leptospirosis spreading in Ukraine, and aimed to help in development and improvement of leptospirosis vaccine formulations. Additionally, method of Multiple-Locus Variable number tandem repeat Analysis will be used for molecular epidemiology research in Ukraine.ReferencesSalaün L, Mérien F, Gurianova S, Baranton G, Picardeau M. Application of multilocus variable-number tandem-repeat analysis for molecular typing of the agent of leptospirosis. J Clin Microbiol. 2006;44(11):3954-3962. doi:10.1128/JCM.00336-06.Caimi K, Repetto SA, Varni V, Ruybal P. Infection , Genetics and Evolution Leptospira species molecular epidemiology in the genomic era. Infect Genet Evol. 2017;54(July):478-485. doi:10.1016/j.meegid.2017.08.013.Ayral F, Zilber AL, Bicout DJ, Kodjo A, Artois M, Djelouadji Z. Distribution of leptospira interrogans by multispacer sequence typing in urban Norway rats (Rattus norvegicus): A survey in France in 2011-2013. PLoS One. 2015;10(10):1-14. doi:10.1371/journal.pone.0139604.

Resurgence of Pertussis and Emergence of the Ptxp3 Toxin Promoter Allele in South Italy.

Despite universal immunization programs, pertussis remains a major public health concern. This study aimed to describe the pertussis epidemiology in the Puglia region in 2006-2015 and to identify recent polymorphisms in Bordetella pertussis virulence-associated genes. The pertussis cases in 2006-2015 were identified from the National Hospital Discharge Database and the Information System of Infectious Diseases. Samples of pertussis cases in 2014-2016 that were confirmed by the Regional Reference Laboratory were subjected to ptxA, ptxP and prn gene sequencing and, in 10 cases, multiple-locus variable-number tandem repeat analysis. In Puglia in 2006-2015, the pertussis incidence rose from an average of 1.39/100,000 inhabitants in 2006-2013 to 2.56-2.54/100,000 in 2014-2015. In infants <1 year of age, the incidence rose from an average of 60.4/100,000 infants in 2006-2013 to 149.9/100,000 in 2015. Of the 661 cases recorded in 2006-2015, 80.3% required hospitalization; of these, 45.4% were <1 year of age. Of the 80 sequenced samples, the allelic profile ptxA1-ptxP3-prn2 was detected in 74. This variant was detected in both vaccinated and unvaccinated people. Six Bordetella pertussis samples were prn deficient. The multiple-locus variable-number tandem repeat analysis cases exhibited multiple-locus variable-number tandem repeat analysis-type 27. The pertussis incidence in Puglia has risen. The hypervirulent strain was also found in vaccinated people. This suggests bacterial adaptation to the vaccine and raises questions about acellular vaccine effectiveness. Prevention of infant pertussis cases is best achieved by immunizing the pregnant mother. Enhanced surveillance and systematic laboratory confirmation of pertussis should be improved in Italy.

Characterisation of Streptococcus pneumoniae isolates from invasive disease in adults following the introduction of PCV10 in Brazil.

Purpose. Invasive pneumococcal disease (IPD) in the elderly is an important public health issue due to the increased proportion of this population in many countries including Brazil. We aimed to characterise pneumococci isolates in adults >50 years with IPD, following the introduction of the 10-valent pneumococcal conjugate vaccine (PCV10) as part of the National Childhood Immunisation Program for children ≤2 years in March 2010.Methodology. Between 2013 and 2015, pneumococcal isolates were collected and serotypes were determined using multiplex PCR and/or Quellung reaction. Antimicrobial susceptibility was defined by E-test (bioMérieux); genetic diversity was determined using Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) and, in selected isolates, Multi Locus Sequence Typing (MLST) was performed.Results/Key findings. Among 102 pneumococcal isolates, the most frequent serotypes were 19A, 13 of 102 (12.7 %) and 22F, 10 of 102 (9.8 %). Ninety-eight isolates were tested for antimicrobial susceptibility. Intermediate resistance to penicillin was present in 2/98 (2.0 %), ceftriaxone in 7/98 (7.1 %) and meropenem in 7/95 (7.4 %) of the isolates (non-meningitis breakpoint: 4 µg ml-1/2 µg ml-1/0.5 µg ml-1, respectively). Resistance to penicillin (meningitis breakpoint ≥0.12 µg ml-1) was observed in 31/98 (31.6 %) of the isolates. Genetic analysis presented two relevant clonal groups, belonging to non-PCV10 serotypes: 19A (ST320, linked to non-susceptibility) and 22F (ST6403).Conclusion. Our data suggest a predominance of non-PCV10 serotypes among IPD in the elderly population in circulating strains ca. 3 to 5 years after the introduction of PCV10 in Brazil.

Enzootic situation and molecular epidemiology of Brucella in livestock from 2011 to 2015 in Qingyang, China

A large-scale survey was conducted in domestic animal populations from 2011 to 2015 in Qingyang, China. A total of 448,398 animals from different districts of Qingyang were tested for the presence of Brucella-specific antibodies using the Rose Bengal Plate Test (RBPT) and the Standard Agglutination Test (SAT). From 2011 to 2015, the yearly average positive rates were between 0.04 and 4.75% in the eight counties tested. In addition, the prevalence rates were between 0 and 9.96% in these eight counties. Sheep was the dominant host of Brucella in Qingyang, and the prevalence rate in sheep (2.74%) was higher than those in the other animals tested. Identification of 10 Brucella isolates from sheep confirmed that the epidemic strains were B. melitensis biovar 3 (n = 9) and B. melitensis biovar 1 (n = 1). MLVA-11 (multiple-locus variable-number tandem repeat analysis) analysis of the 10 isolates showed three genotypes: genotype 116 (n = 8), genotype 115 (n = 1) and genotype 136 (n = 1). Furthermore, analysis of the whole-genome sequences of the representative B. melitensis strain QY1 indicated that this isolate was closely related to isolates from China and India. The results of serum epidemiology confirmed that the region of northern Qingyang was a critical Brucella epidemic area and that the disease showed a rising trend, especially from 2013 to 2015. An analysis of the isolate genotypes suggested that sheep brucellosis mainly resulted from conventional B. melitensis (East Mediterranean group), although the external strain (American group) also occurred in Qingyang.

Genotyping and Phylogenetic Analysis of Group B Streptococcus by Multiple Locus Variable Number Tandem Repeat Analysis in Iran

Background: Group B streptococcus (GBS), also known as Streptococcus agalactiae, is well known as a causative agent for neonatal invasive diseases; it is also a major pathogen in adults. Analytic epidemiology is required to monitor the clinical isolates of GBS. However, there is insufficient information on the genetic background of GBS in Iran, and this information is needed to guide and develop a GBS vaccine. Materials and Methods: In total, 90 well-characterized GBS isolates were collected from April 2014 to August 2015. In this study, molecular typing was used to disclose a relationship between the multiple-locus variable number tandem repeat analysis (MLVA) types, serotyping, and pilus islands. The isolates were characterized by the types of capsular polysaccharides and pilus islands and were examined by MLVA to study the epidemiological relationship of isolates. Results: The results indicate that there is a significant relationship between the distribution of serotypes and pilus island genes; GBS isolates were differentiated into 12 types by capsular polysaccharides and pilus islands analysis. The discriminatory power of an MLVA analysis was high based on the five most variable numbers of tandem repeat loci and 44 MLVA types that were identified. Conclusions: This study has provided useful insights into the genetic heterogeneity of GBS isolates in Tehran and Alborz, Iran. The extensive distribution of pilus islands in various serotypes and MLVA types throughout the GBS population refers to the advancement of the pilus-based GBS vaccines. [GMJ.2018;7:e1121]

Genotyping and Phylogenetic Analysis of Group B Streptococcus by Multiple Locus Variable Number Tandem Repeat Analysis in Iran

Background: Group B streptococcus (GBS), also known as Streptococcus agalactiae, is well known as a causative agent for neonatal invasive diseases; it is also a major pathogen in adults. Analytic epidemiology is required to monitor the clinical isolates of GBS. However, there is insufficient information on the genetic background of GBS in Iran, and this information is needed to guide and develop a GBS vaccine. Materials and Methods: In total, 90 well-characterized GBS isolates were collected from April 2014 to August 2015. In this study, molecular typing was used to disclose a relationship between the multiple-locus variable number tandem repeat analysis (MLVA) types, serotyping, and pilus islands. The isolates were characterized by the types of capsular polysaccharides and pilus islands and were examined by MLVA to study the epidemiological relationship of isolates. Results: The results indicate that there is a significant relationship between the distribution of serotypes and pilus island genes; GBS isolates were differentiated into 12 types by capsular polysaccharides and pilus islands analysis. The discriminatory power of an MLVA analysis was high based on the five most variable numbers of tandem repeat loci and 44 MLVA types that were identified. Conclusions: This study has provided useful insights into the genetic heterogeneity of GBS isolates in Tehran and Alborz, Iran. The extensive distribution of pilus islands in various serotypes and MLVA types throughout the GBS population refers to the advancement of the pilus-based GBS vaccines. [GMJ.2018;7:e1121]

Genotyping and Phylogenetic Analysis of Group B Streptococcus by Multiple Locus Variable Number Tandem Repeat Analysis in Iran.

Background: Group B streptococcus (GBS), also known as Streptococcus agalactiae, is well known as a causative agent for neonatal invasive diseases; it is also a major pathogen in adults. Analytic epidemiology is required to monitor the clinical isolates of GBS. However, there is insufficient information on the genetic background of GBS in Iran, and this information is needed to guide and develop a GBS vaccine. Materials and Methods: In total, 90 well-char - acterized GBS isolates were collected from April 2014 to August 2015. In this study, molecular typing was used to disclose a relationship between the multiple-locus variable number tandem repeat analysis (MLVA) types, serotyping, and pilus islands. The isolates were characterized by the types of capsular polysaccharides and pilus islands and were examined by MLVA to study the epidemiological relationship of isolates. Results: The results indicate that there is a significant relationship between the distribution of serotypes and pilus island genes; GBS isolates were differentiated into 12 types by capsular polysaccharides and pilus islands analysis. The discriminatory power of an MLVA analysis was high based on the five most variable numbers of tandem repeat loci and 44 MLVA types that were identified. Conclusion: This study has provided useful insights into the genetic heterogeneity of GBS isolates in Tehran and Alborz, Iran. The extensive distribution of pilus islands in various serotypes and MLVA types throughout the GBS population refers to the advancement of the pilus-based GBS vaccines.

Molecular epidemiology of Shiga toxin-producing O113:H21 isolates from cattle and meat.

The serotype O113:H21 is considered one of the relevant non-O157 STEC serotypes associated with severe human infections. Due to the increased detection of O113 strains and their relationship with clinical cases, which emphasizes the importance of this serogroup as an emerging pathogen, our aim was to determine the characteristics of STEC O113:H21 strains circulating in bovine cattle and retail meat from Argentina. For this purpose, we determined the presence and combinations of various virulence genes (and their variants) related to adhesion and toxicity in a collection of 34 isolates. Their genetic relatedness using multiple-locus variable-number tandem repeat analysis (MLVA) was also studied. Subtyping of stx genes indicated that O113:H21 strains circulating in Argentina mainly present stx2a alone or together with stx2c or, less frequent, with stx2d , all of which are subtypes associated with human disease. We found plasmid markers, such as saa, ehxA and subA, in a higher proportion than previous studies, and five variants of saa, two of which were novel ones. In relation to MLVA subtyping, we detected a limited diversity among the isolates considering that several loci were not discriminative and, that in some farms, the same clone seemed to remain circulating throughout the year. The O113:H21 strains studied harbour several toxin and adhesion genes (saa, espP, fimCD, ehaA, iha, hcpA, elfA, lpfO113, ecpA, subA, cdt-V) and Stx subtypes associated with human disease. Results also highlighted that subtyping of stx and saa is useful to discriminate O113:H21 strains that share virulence genes. In conclusion, this study shows that a number of O113:H21 strains that occur in foods and bovines could be pathogenic for humans. This situation calls for further attention in the prevention and control of foodborne disease caused by these strains.

Molecular characterization, toxin detection and resistance testing of human clinical Clostridium difficile isolates from Lebanon

Clostridium (Clostridioides) difficile is the main cause for nosocomial diarrhoea in industrialised nations. Epidemiologic data on the pathogen’s occurrence in other world regions are still scarce. In this context we characterized with phenotypic and molecular genetic methods C. difficile isolates stemming from hospitalised patients with diarrhoea in Lebanon.From 129 stool samples of symptomatic patients at a tertiary care University hospital in Lebanon, a total of 107 C. difficile strains were cultivated and underwent ribotyping, toxin gene detection and antibiotic resistance testing.Ribotype 014 (RT014, 16.8%) predominated, followed by RT002 (9.3%), RT106 (8.4%) and RT070 (6.5%). Binary toxin gene-positive isolates (RT023, RT078 and RT126) were rarely detected and RT027 was absent. Interestingly, within one isolate only the toxin A gene (tcdA) was detected. Multiple-locus variable-number tandem repeat analysis (MLVA) revealed strong strain diversity in most RTs. The isolates were sensitive to metronidazole and vancomycin, and only a small proportion of strains displayed resistance against moxifloxacin, rifampicin, and clarithromycin (5.6%, 1.9%, and 2.8%), respectively.The data indicate that the genetic strain composition of Lebanese strains differs markedly from the situation seen in Europe and North America. Especially the epidemic RTs seen in the latter regions were almost absent in Lebanon. Interestingly, most strains showed almost no resistance to commonly used antibiotics that are suspected to play a major role in the development of C. difficile infection, despite frequent use of these antibiotics in Lebanon. Thus, the role of antimicrobial resistance as a major driving force for infection development remains uncertain in this area.

Molecular epidemiology of Neisseria gonorrhoeae strains circulating in Indonesia using multi-locus variable number tandem repeat analysis (MLVA) and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) techniques

BackgroundControl of gonorrhea in resource-limited countries, such as Indonesia, is mostly unsuccessful. Examining Neisseria gonorrhoeae (Ng) transmission networks using strain typing might help prioritizing public health interventions.MethodsIn 2014, urogenital Ng strains were isolated from clients of sexually transmitted infection clinics in three Indonesian cities. Strains were typed using Multiple-Locus Variable Number Tandem Repeat (VNTR) Analysis (MLVA) and Ng Multi-Antigen Sequence Typing (NG-MAST) at the Public Health Service, Amsterdam, the Netherlands, and compared to Dutch strains collected from 2012 to 2015. Minimum spanning trees (MSTs) were constructed using MLVA profiles incorporating demographics and NG-MAST genogroups. A cluster was defined as ≥5 strains differing in ≤1 VNTR locus. The concordance between MLVA and NG-MAST was examined with the adjusted Wallace coefficients (AW).ResultsWe collected a total of 78 Indonesian strains from Yogyakarta (n = 44), Jakarta (n = 25), and Denpasar (n = 9). Seven MLVA clusters and 16 non-clustered strains were identified. No cluster was specific for any geographic area, risk group or age group.Combined with 119 contemporary Dutch strains, 8 MLVA clusters were identified, being four clusters of Indonesian strains, two of Dutch strains, and two of both Indonesian and Dutch strains. Indonesian strains (79.5%) were more often clustered compared to Dutch strains (24.3%).The most prevalent NG-MAST genogroups among Indonesian strains was G1407 (51.3%) and among Dutch strains was G2992 (19.3%). In Indonesian strains, the AW [95% confidence interval] for MLVA to NG-MAST was 0.07 [0.00–0.27] and for NG-MAST to MLVA was 0.03 [0.00–0.12].ConclusionIndonesian Ng strains are more often clustered than Dutch strains, but show no relation with geographical area, risk group, or age group, suggesting a more clonal Ng epidemic in Indonesia. Some similar Ng strains circulate in both Indonesia and the Netherlands.