Multilocus Variable-number Tandem-repeat Analysis Profiles Research Articles

Whole genome sequencing of Ethiopian Brucella abortus isolates expands the known diversity of an early branching sub-Saharan African lineage.

Brucellosis remains one of the most significant zoonotic diseases globally, responsible for both considerable human morbidity and economic losses due to its impacts on livestock productivity. Despite this, there remain significant evidence gaps in many low- and middle-income countries, including those of sub-Saharan Africa. Here we report the first molecular characterisation of Brucella sp. from Ethiopia. Fifteen Brucella sp. isolates from an outbreak in cattle from a herd in central Ethiopia were identified as Brucella abortus, using bacterial culture and molecular methods. Sequencing of the Ethiopian B. abortus isolates allowed their phylogenetic comparison with 411 B. abortus strains of diverse geographical origins, using whole genome single nucleotide polymorphisms (wgSNP). The Ethiopian isolates belonged to an early-branching lineage (Lineage A) previously only represented by data from two strains, both of sub-Saharan African origin (Kenya and Mozambique). A second B. abortus lineage (Lineage B), also comprised solely of strains originating from sub-Saharan Africa, was identified. The majority of strains belonged to one of two lineages of strains originating from a much broader geographical range. Further analyses based on multi-locus sequence typing (MLST) and multi-locus variable-number tandem repeat analysis (MLVA) expanded the number of B. abortus strains available for comparison with the Ethiopian isolates and were consistent with the findings from wgSNP analysis. MLST profiles of the Ethiopian isolates expanded the sequence type (ST) diversity of the early branching lineage of B. abortus, equivalent to wgSNP Lineage A. A more diverse cluster of STs, equivalent to wgSNP Lineage B, was comprised solely of strains originating from sub-Saharan Africa. Similarly, analysis of B. abortus MLVA profiles (n = 1891) confirmed that the Ethiopian isolates formed a unique cluster, similar to only two existing strains, and distinct from the majority of other strains of sub-Saharan African origin. These findings expand the known diversity of an under-represented lineage of B. abortus and suggest a potential evolutionary origin for the species in East Africa. In addition to providing information concerning Brucella species extant within Ethiopia this work serves as the basis for further studies on the global population structure and evolutionary history of a major zoonotic pathogen.

Application of a new multi-locus variable number tandem repeat analysis (MLVA) scheme for the seasonal investigation of Cryptosporidium parvum cases in Wales and the northwest of England, spring 2022

The protozoan Cryptosporidium parvum is an important cause of gastroenteritis in humans and livestock, and cryptosporidiosis outbreaks are common. However, a multi-locus genotyping scheme is not widely adopted. We describe the further development and application of a seven-locus multi-locus variable number of tandem repeats analysis (MLVA) scheme. From 28th March to 31st July 2022, confirmed C. parvum stools (n = 213) from cryptosporidiosis patients (cases) in Wales (n = 95) and the north west of England (n = 118) were tested by MLVA. Typability (defined as alleles identified at all seven loci in a sample) was 81.2% and discriminatory power estimated by Hunter Gaston Discriminatory Index was 0.99. A MLVA profile was constructed from the alleles, expressed in chromosomal order. Profiles were defined as simple (single allele at each locus) or mixed (more than one allele at any locus). A total of 161 MLVA profiles were identified; 13 were mixed, an additional 38 simple profiles contained null records, and 110 were complete simple profiles. A minimum spanning tree was constructed of simple MLVA profiles and those identical at all seven loci defined genetic clusters of cases (here, null records were considered as an allele); 77 cases formed 25 clusters, ranging from two to nine (mode = two) cases. The largest cluster, following epidemiological investigation, signalled a newly-identified outbreak. Two other cases with mixed profiles that contained the outbreak alleles were included in the outbreak investigation. In another epidemiologically-identified outbreak of six initial cases, MLVA detected two additional cases. In a third, small outbreak of three cases, identical MLVA profiles strengthened the microbiological evidence. Review of the performance characteristics of the individual loci and of the seven-locus scheme suggested that two loci might be candidates for review, but a larger dataset over a wider geographical area and longer timeframe will help inform decision-making about the scheme by user laboratories and stakeholders (such as public health agencies). This MLVA scheme is straightforward in use, fast and cheap compared to sequence-based methods, identifies mixed infections, provides an important tool for C. parvum surveillance, and can enhance outbreak investigations and public health action.

Characterization of Salmonella Typhimurium and monophasic Salmonella Typhimurium isolated in Abruzzo and Molise regions, Italy, from 2012 to 2017.

Salmonellosis is currently the second most common zoonosis in European Union but in the 6-years periods, between 2012 and 2017, there has been a significant decrease trend in the yearly number of infections caused by Salmonella. In Italy, S. Typhimurium and monophasic S. Typhimurium represent the most prevalent serotypes. In this paper, we investigated these two serovars isolated from 2012 to 2017 in Abruzzo and Molise regions. A set of 345 strains isolated from human sporadic cases, surface water, food and animals were collected and analyzed. Monophasic S. Typhimurium strains were found to be resistant to streptomycin, sulfisoxazole, ampicillin, tetracycline and nalidixic acid, while S. Typhimurium isolates showed high levels of resistance to tetracycline, sulfisoxazole and ampicillin. The 5-loci Multilocus Variable-Number Tandem Repeat Analysis (MLVA) identified 88 genotypes (GT), six of which were common for the two serovars. Several MLVA profiles were shared by human and non-human isolates. MLVA had sufficient typing resolution for epidemiological studies of S. Typhimurium but demonstrated poor discriminatory in trace-back study of monophasic S. Typhimurium.

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.

Multilocus Variable-Number Tandem-Repeat Analysis for Genotyping 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.

Erratum for Cadel-Six et al., "Draft Genome Sequences of Salmonella enterica subsp. enterica Serovar Dublin Strains from Raw Milk Cheeses Characterized by Multilocus Variable-Number Tandem-Repeat Analysis Profiles Associated with Two Outbreaks in France".

Volume 8, no. 1, e01361-18, 2019, [https://doi.org/10.1128/MRA.01361-18][1]. Page 1: The article title should read as given above. Page 1, abstract, line 2: “St. Nectaire and Morbier cheeses” should read “two raw milk cheeses.” Page 1, abstract, lines 3 and 4: “fatal outbreaks” should

Linked seasonal outbreaks of Salmonella Typhimurium among passerine birds, domestic cats and humans, Sweden, 2009 to 2016.

In 2016, an outbreak of Salmonella Typhimurium (STm) with multilocus variable-number tandem repeat analysis (MLVA) profiles historically associated with passerine birds (2-[11-15]-[3-4]-NA-212) occurred among passerines, cats and humans in Sweden. Our retrospective observational study investigated the outbreak and revisited historical data from 2009–16 to identify seasonality, phylogeography and other characteristics of this STm variant. Outbreak isolates were analysed by whole-genome single nucleotide polymorphism (SNP) typing. The number of notified cases of passerine-associated STm among passerines, cats and humans per month and county, and their MLVA profiles, were compared to birdwatchers’ counts of passerines. Seasonal trend decomposition and correlation analysis was performed. Outbreak isolates did not cluster by host on SNP level. Passerine-associated STm was seasonal for birds, cats and humans, with a peak in March. Cases and counts of passerines at bird feeders varied between years. The incidence of passerine-associated STm infections in humans was higher in the boreal north compared with the southern and capital regions, consistent with passerine population densities. Seasonal mass migration of passerines appears to cause STm outbreaks among cats certain years in Sweden, most likely via predation on weakened birds. Outbreaks among humans can follow, presumably caused by contact with cats or environmental contamination.

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis.

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, etc.) of this bacterium have been lacking. A Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay was therefore developed as an easily accessible and unambiguous tool for high-resolution genotyping of recovered isolates. For the MLVA assay presented here, DNA is extracted from cultured Y. ruckeri samples by boiling bacterial cells in water, followed by use of supernatant as template for PCR. Primer-pairs targeting ten Variable-number tandem-repeat (VNTR) loci, interspersed throughout the Y. ruckeri genome, are distributed equally amongst two five-plex PCR reactions running under identical cycling conditions. Forward primers are labelled with either of three fluorescent dyes. Following amplicon confirmation by gel electrophoresis, PCR products are diluted and subjected to capillary electrophoresis. From the resulting electropherogram profiles, peaks representing each of the VNTR loci are size-called and employed for calculating VNTR repeat counts in silico. Resulting ten-digit MLVA profiles are thenused to generate Minimum spanning trees enabling epizootiological evaluation by cluster analysis. The highly portable output data, in the form of numerical MLVA profiles, can rapidly be compared across labs and placed in a spatiotemporal context. The entire procedure from cultured colony to epizootiological evaluation may be completed for up to 48 Y. ruckeri isolates within a single working day.

Multilocus variable number tandem repeat analysis and antimicrobial susceptibility pattern of Staphylococcus epidermidis isolates in Tehran, Iran

Objective: Detection of sources of outbreaks caused by coagulase-negative Staphylococcus relies on molecular epidemiology methods. Little is known about the genetic diversity of the Staphylococcus epidermidis isolates isolated from various sources in Iran. We assessed the molecular epidemiology of S. epidermidis isolates collected from clinical and nonclinical sources from Tehran counties during 2014 to 2016 using MLVA (multilocus variable number tandem repeat analysis). Methods: One hundred and three clinical and nonclinical S. epidermidis isolates were collected from two hospitals in Tehran. Antibiotic susceptibility testing of isolates was evaluated for cefoxitin, tetracycline, erythromycin, clindamycin, mupirocin, vancomycin and linezolid according to Clinical and Laboratory Standards Institute, as well as prevalence of mecA gene was evaluated by PCR method. In addition, genetic relatedness of isolates was assessed by MLVA method. Results: Resistant rate to cefoxitin, tetracycline, erythromycin, clindamycin and mupirocin were 64, 36, 72, 44 and 23% in all isolates. All clinical and nonclinical isolates were susceptible to linezolid and vancomycin. In all, 49.5% of S. epidermidis isolates were multidrug resistant. Prevalence of mecA was 64%. The MLVA profile consists of a series of allele numbers, corresponding to the number of repeats at each variable number tandem repeat locus. The results of MLVA showed 64 types among all 103 isolates. There were 16 MLVA types that were common in two hospitals and 15 MLVA types were existed in various sources of S. epidermidis isolates. The diversity index obtained with MLVA was 0.989 (95% confidence interval [0.984–0.993]) for the 103 S. epidermidis isolates. A range of one to six alleles was identified at variable number tandem repeats loci with Simpson's diversity values between 0.526 and 0.781. Conclusion: Our study demonstrated presence of high molecular diversity among S. epidermidis isolates. In addition, the main conclusion was that some MLVA types can be disseminated over the wards and between the hospitals. In other hand, resistance to various antibiotics in S. epidermidis isolates and prevalence of methicillin-resistant S. epidermidis and multidrug resistant S. epidermidis isolates to be increasing.

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.

Draft Genome Sequences of Salmonella enterica subsp. enterica Serovar Dublin Strains from St. Nectaire and Morbier Cheeses Characterized by Multilocus Variable-Number Tandem-Repeat Analysis Profiles Associated with Two Fatal Outbreaks in France.

We report here the draft genome sequences of 2 Salmonella enterica subsp. enterica serovar Dublin strains from St. Nectaire and Morbier cheeses having multilocus variable-number tandem-repeat analysis (MLVA) profiles identified during the fatal outbreaks that occurred in France in 2012 and 2015 to 2016, respectively. These draft genome sequences will help uncover the virulence determinants in invasive S. Dublin strains.

Genetic diversity of Leptospira interrogans circulating isolates and vaccine strains in China from 1954–2014

ABSTRACTLeptospirosis is one of the most important but neglected, infectious tropical diseases worldwide. Leptospira interrogans is now recognized as a leading cause of the disease. Little is known of the genetic diversity and phylogenetic characteristics of L. interrogans within China. To better understand the transmission and genetic diversity of L. interrogans populations, we characterized 271 isolates and seven vaccine strains from China during 1954–2014 using multilocus variable-number tandem repeat analysis (MLVA). 110 different L. interrogans MLVA profiles (MTs) were identified, of which five were predominant, reflecting a high level of genetic diversity in L. interrogans population in China. Different from that of circulating isolates, seven vaccine strains have different MT, of which some are phylogenetically away from the circulating isolates. The results showed that Icterohaemorrhagiae, Hebdomadis, and Canicola ranked as the top three serogroups among L. interrogans strains tested. The cluster analysis demonstrate the clonal links between rodent and human isolates, suggesting the rodent species played a key role in the transmission of leptospirosis to humans, and contributed to the circulation of the pathogen in humans. Taken together, these findings should provide insight into a better knowledge of the epidemiology and molecular evolution of L. interrogans in China. Furthermore, the results should facilitate the selection of candidate vaccine strains in the future.

Molecular epidemiology of Bordetella pertussis in Greece, 2010–2015

PurposeTo determine the predominant strains of Bordetella pertussis in Greece during 2010–2015.MethodologyInfants and children (n=1150) (15 days to 14 years) of Greek, Roma and immigrant origin with different vaccination statuses were hospitalized in Athens, Greece with suspected pertussis infection. IS481/IS1001 real-time PCR confirmed Bordetella spp./B. pertussis infection in 300 samples. A subset of samples (n=153) were analysed by multi-locus variable number tandem repeat analysis (MLVA) and (n=25) by sequence-based typing of the toxin promotor region (ptxP) on DNA extracted from clinical specimens.Results/Key findingsA complete MLVA profile was determined in 66 out of 153 samples; the B. pertussis MLVA type 27 (n=55) was the dominant genotype and all tested samples (n=25) expressed the ptxP3 genotype. The vaccine coverage in the Greek population was 90 %; however, the study population expressed complete coverage in 2 out of 264 infants (0–11 months) and in 20 out of 36 children (1–14 years). Roma and immigrant minorities represent 7 % of the Greek population, but make up 50 % of the study population, indicating a low vaccine coverage among these groups.ConclusionsThe B. pertussis MT27 and ptxP3 genotype is dominant in Greek, Roma and immigrant infants and children hospitalized in Greece. Thus, the predominant MLVA genotype in Greece is similar to other countries using acellular vaccines.

MLVA subtyping of Listeria monocytogenes isolates from meat products and meat processing plants

Listeria monocytogenes is widely distributed in meat products and the meat-processing industry thus posing a risk to consumers. The aim of this study was to evaluate the suitability of the multilocus variable-number tandem-repeat analysis (MLVA) for use as a L. monocytogenes subtyping technique for surveillance and routine control in meat products and meat processing plants. A collection of 113 isolates (including control strains and isolates from meat products and meat processing plants) were subject to MLVA analysis using two different platforms for fragment sizing: 1.) ABI 3730xl DNA analyzer (Life Technologies) as the reference method and 2.) The QIAxcel Advanced System (Qiagen). Although discrepancies in fragment sizing were observed it was possible to standardize results in order to assign the same allele for a given fragment independently of the platform used for fragment sizing. A total of 27 different MLVA profiles were obtained considering all the isolates (N=113), 24 of them corresponding to the meat industry isolates (N=106). MLVA and multilocus sequence typing (MLST) results were compared and yielded Simpson's diversity indices of 0.907 and 0.872, respectively. The congruence between both typing methods was measured with the adjusted Wallace coefficient (AW). Using MLVA as the primary method, AW=0.946 suggested that MLVA can predict the sequence type with high accuracy. Given its discriminatory power and high throughput, MLVA could be considered a rapid, reliable, and high-throughput alternative to existing subtyping methods for surveillance and control of L. monocytogenes in the meat-processing industry.

Molecular epidemiology of Bordetella pertussis in Cambodia determined by direct genotyping of clinical specimens

ObjectivesThis study sought to determine the genotypes of circulating Bordetella pertussis, the causative agent of pertussis, in Cambodia by direct molecular typing of clinical specimens. MethodsDNA extracts from nasopharyngeal swabs obtained from 82 pertussis patients in 2008–2016 were analyzed by multilocus variable-number tandem repeat analysis (MLVA). B. pertussis virulence-associated allelic genes (ptxA, prn, and fim3) and the pertussis toxin promoter ptxP were also investigated by DNA sequence-based typing. ResultsForty-four DNA extracts (54%) yielded a complete MLVA profile, and these were sorted into 8 MLVA types (MT18, MT26, MT27, MT29, MT43, MT72, MT95, and MT200). MT27 and MT29, which are common in developed countries, were the predominant strain types (total 73%). The predominant profile of virulence-associated allelic genes was the combination of ptxP3/ptxA1/prn2/fim3A (48%). MT27 strains were detected during the entire study period, whereas MT29 strains were only found in 2014–2016. ConclusionsThe B. pertussis population in Cambodia, where a whole-cell pertussis vaccine (WCV) has been continuously used, resembled those observed previously in developed countries where acellular pertussis vaccines are used. Circulating B. pertussis strains in Cambodia were distinct from those in other countries using WCVs.

Molecular Characterization of Mycoplasma pneumoniae Infections in Two Rural Populations of Thailand from 2009 to 2012.

Studies on Mycoplasma pneumoniae in Thailand have focused on urban centers and have not included molecular characterization. In an attempt to provide a more comprehensive understanding of this organism, we conducted a systematic random sampling to identify 3,000 nasopharyngeal swab specimens collected from January 2009 through July 2012 during population-based surveillance for influenza-like illness in two rural provinces. M. pneumoniae was detected by real-time PCR in 175 (5.8%) specimens. Genotyping was performed using the major adhesion protein (P1) and multilocus variable-number tandem-repeat analysis (MLVA). Of the 157 specimens typed, 97 were P1 type 1 and 60 were P1 type 2. Six different MLVA profiles were identified in 149 specimens, with 4/5/7/2 (40%) and 3/5/6/2 (26%) predominating. There was no discrete seasonality to M. pneumoniae infections. Examination of the 23S rRNA sequence for known polymorphisms conferring macrolide resistance revealed that all 141 tested to possess the genotype associated with macrolide susceptibility.

Within-Flock Population Dynamics of Dichelobacter nodosus.

Footrot causes 70–90% of lameness in sheep in Great Britain. With approximately 5% of 18 million adult sheep lame at any one time, it costs the UK sheep industry £24–84 million per year. The Gram-negative anaerobe Dichelobacter nodosus is the causative agent, with disease severity influenced by bacterial load, virulence, and climate. The aim of the current study was to characterize strains of D. nodosus isolated by culture of swabs from healthy and diseased feet of 99 ewes kept as a closed flock over a 10-month period and investigate persistence and transmission of strains within feet, sheep, and the flock. Overall 268 isolates were characterized into strains by serogroup, proline–glycine repeat (pgr) status, and multi-locus variable number tandem repeat analysis (MLVA). The culture collection contained 87 unique MLVA profiles and two major MLVA complexes that persisted over time. A subset of 189 isolates tested for the virulence marker aprV2 were all positive. The two MLVA complexes (76 and 114) comprised 62 and 22 MLVA types and 237 and 28 isolates, respectively. Serogroups B, and I, and pgrB were associated with MLVA complex 76, whereas serogroups D and H were associated with MLVA complex 114. We conclude that within-flock D. nodosus evolution appeared to be driven by clonal diversification. There was no association (P > 0.05) between serogroup, pgr, or MLVA type and disease state of feet. Strains of D. nodosus clustered within sheep and were transmitted between ewes over time. D. nodosus was isolated at more than one time point from 21 feet, including 5 feet where the same strain was isolated on two occasions at an interval of 1–33 weeks. Collectively, our results indicate that D. nodosus strains persisted in the flock, spread between sheep, and possibly persisted on feet over time.

Multi-laboratory validation study of multilocus variable-number tandem repeat analysis (MLVA) for Salmonella enterica serovar Enteritidis, 2015.

Multilocus variable-number tandem repeat analysis (MLVA) is a rapid and reproducible typing method that is an important tool for investigation, as well as detection, of national and multinational outbreaks of a range of food-borne pathogens. Salmonella enterica serovar Enteritidis is the most common Salmonella serovar associated with human salmonellosis in the European Union/European Economic Area and North America. Fourteen laboratories from 13 countries in Europe and North America participated in a validation study for MLVA of S. Enteritidis targeting five loci. Following normalisation of fragment sizes using a set of reference strains, a blinded set of 24 strains with known allele sizes was analysed by each participant. The S. Enteritidis 5-loci MLVA protocol was shown to produce internationally comparable results as more than 90% of the participants reported less than 5% discrepant MLVA profiles. All 14 participating laboratories performed well, even those where experience with this typing method was limited. The raw fragment length data were consistent throughout, and the inter-laboratory validation helped to standardise the conversion of raw data to repeat numbers with at least two countries updating their internal procedures. However, differences in assigned MLVA profiles remain between well-established protocols and should be taken into account when exchanging data.

Severe pertussis infection in infants less than 6 months of age: Clinical manifestations and molecular characterization

ABSTRACTWe conducted a study to determine the main traits of pertussis among unimmunized infants less than 6 months of age. From August 2012 to March 2015, 141 nasopharyngeal aspirates (NPAs) were collected from infants with respiratory symptoms attending 2 major hospitals in Rome. Clinical data were recorded and analyzed. Lab-confirmation was performed by culture and realtime PCR. B. pertussis virulence-associated genes (ptxP, ptxA and prn), together with multilocus variable-number tandem repeat analysis (MLVA), were also investigated by the sequence-based analysis on the DNAs extracted from positive samples. Antibiotic susceptibility with Etest was defined on 18 viable B. pertussis isolates. Samples from 73 infants resulted positives for B. pertussis. The median age of the patients was 45 d (range 7–165); 21 infants were treated with macrolides before hospital admission. Cough was reported for a median of 10 d before admission and 18 d after hospital discharge among infected infants, 84% of whom showed paroxysmal cough. No resistance to macrolides was detected. Molecular analysis identified MT27 as the predominant MLVA profile, combined with ptxP3-ptxA1-prn2 associated virulence genes.Although our data may not be generalized to the whole country, they provide evidence of disease severity among infants not vaccinated against pertussis. Moreover, genetically related B. pertussis strains, comprising allelic variants of virulence associated genes, were identified.

A genetic diversity study of antifungal Lactobacillus plantarum isolates

Lactobacillus plantarum is a lactic acid bacterium commonly found on fruits and vegetables and also used in a variety of food fermentations. Strains from this species are also regularly reported as having antifungal or probiotic activity. Genotyping methods can be used to differentiate strains of the same species thus determining if strains are related or not. However for L. plantarum, the currently used methods have limitations including DNA band profile interpretation difficulty and cost. In this study, a new genotyping method based on multi-locus variable number tandem repeat analysis (MLVA) was developed and compared to a previously reported randomly amplified polymorphic DNA-PCR (RAPD-PCR) method for L. plantarum. With a selection of 13 antifungal strains of L. plantarum isolated from heterogeneous sources (cheese, silage, sauerkraut, vegetables and a probiotic product), RAPD-PCR revealed 9 different profiles resulting in a Hunter-Gaston discrimination index (D-value) of 0.94. The new MLVA method which compares the lengths of 4 repetitive regions within LPXTG motif-containing surface protein genes differentiated the 13 L. plantarum strains into 10 different subtypes leading to a D-value of 0.95. Interestingly 11 additional L. plantarum isolates obtained in a previous study during a screen for antifungal activity against the common cheese spoilage mould Penicillium commune all possessed the same RAPD-PCR and MLVA profile as each other and the commercial probiotic strain L. plantarum 299v. This study demonstrates that the new MLVA method can be used to simply and inexpensively differentiate L. plantarum strains and provide information regarding strain relatedness and thus potential insight into strain properties.