Multiple-locus Variable-number Tandem-repeat (VNTR) Analysis Research Articles

Prevalence of Multidrug-Resistant Pseudomonas aeruginosa and Risk Factors for their Infections at Intensive Care Units of a Tertiary Hospital in Southern China

Pseudomonas aeruginosa (PA) is highly significant opportunistic pathogens causing healthcare associated infections (HAIs) in hospital settings, notably at intensive care units (ICUs). The aim of this study was to retrospectively analyze the infection status, prevalence and antimicrobial resistance (AMR) of PA at ICUs of a tertiary care hospital in southern China during a one-year period (2016) and examine the clinical risk factors for HAIs by PA. Multiple-locus variablenumber tandem-repeat (VNTR) analysis (MLVA) method was employed to analyze clonality of the strains. Our results suggested that the resistance of PA in ICUs were higher than in other wards. In terms of resistance to carbapenems, the resistance gene island (blaOXA-1+blaIMP+ant(2’’)-Ia+aac(6’)-Ib) carried in IntI was a salient feature among AMR genes. While PA infections at local ICUs seemed frequent, there were no obvious trends suggestive of outbreaks. Some epidemic strains have apparently thrived locally for substantial periods, as carriers of major AMR genes and virulence factors. For risk factors for HAIs, inappropriate treatment was found to impact empiric antibiotic therapy of PA infections, especially in the case of carbapenems, where patients often did not get proper treatment during hospitalization of more than 30 days. Multifactor analysis shows that ventilator-associated pneumonia (VAP) was an independent risk factor for increasing the 30-day mortality rate in patients. In addition, the use of antimicrobials, duration of hospitalization and use of mechanical ventilation before isolation were independent risk factors for HAIs.

Molecular analysis of Pseudomonas aeruginosa strains isolated from cystic fibrosis patients

Pseudomonas aeruginosa is a severe bacterial pathogen. Due to the genetic flexibility among strains, chronic airways infection can lead to mortality among cystic fibrosis (CF) patients. It is essential to develop patient-specific therapy which will rely on phenotypic and genomic diversity. The primary objective of this study was to assess the genomic variability of P. aeruginosa strains, using two different molecular techniques for tracking the epidemiological transmissions. This study applied a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) for an efficient genotyping of clinical P. aeruginosa strains isolated from CF patients and compared results with a TRS-PCR typing. The percentage similarity analysis was performed using the categorical multi-state coefficient and UPGMA method. Based on the MLVA and TRS-PCR group assessment, 43 P. aeruginosa strains/variants were detected among the 63 clinical isolates from eight CF patients. The study of P. aeruginosa isolates has revealed that during chronic bacterial infections, CF patients harbor different P. aeruginosa strains or variants within the same host over the years. P. aeruginosa genotypes diversity may result from infection with several strains and result from a microevolution process of an initially acquired strain. The TRS-PCR method proposed in this work can complement the MLVA scheme. It can also be used as a preliminary method for genetic typing of P. aeruginosa isolates in CF patients.

Polyclonal spread of multiple genotypes of Mycoplasma pneumoniae in semi-closed settings in Yamagata, Japan.

To clarify the spread of Mycoplasma pneumoniae infections in semi-closed settings such as schools and family homes using molecular typing methods. We retrospectively searched for school- and family-based clusters of M. pneumoniae infections based on information regarding patients from whom M. pneumoniae strains had been isolated between 2011 and 2013 in Yamagata, Japan. The molecular typing profile, including the P1 type and the four-locus (Mpn13, 14, 15 and 16) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) type, was obtained from our previous study. We identified 11 school-based clusters involving 71 patients and 16 family-based clusters involving 38 patients, including 14 duplications between these types of clusters. A total of 95M. pneumoniae strains isolated from those patients were divided into 4 genotypes: 33 strains of type 4-5-7-2, 1; 31 of type 4-5-7-3, 1; 24 of type 3-5-6-2, 2c; and 7 of type 3-5-6-2, 2a. Of the 11 school-based clusters, 6 clusters (54.5%) consisted of multiple genotypes, and the remaining 5 clusters consisted of a single genotype. Moreover, the presence of multiple genotypes was identified in three classrooms of a school. On the other hand, in 14 (87.5%) of the 16 family-based clusters, the genotypes of the M. pneumoniae strains isolated from each family member were identical. The spread of M. pneumoniae infection in schools is likely polyclonal, since M. pneumoniae strains are brought into schools from various sites, such as family homes, which are important sites of disease transmission.

Improvement of Multiple-Locus VNTR Analysis Typing Scheme for Helicobacter pylori

Aims: To improve a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) assay for Helicobacter pylori typing.
 Materials and Methods: Polymorphic VNTRs were searched by Gene Expert. The distribution and polymorphism of each VNTR locus were analyzed in 18 H. pylori genomes from the NCBI genome database by BLAST and were compared with a collection of 15 clinical H. pylori strains. The MLVA assay was compared with MLST-typing for discriminating H. pylori isolates.
 Results: Twelve VNTR loci were identified by bioinformatic screening of H. pylori genomes, and five of them were highly polymorphic. Therefore, an MLVA assay composed of five VNTR loci was developed with greatest discriminatory power.
 Conclusion: MLVA typing is a faster and more standardized method for studying the genetic relatedness of H. pylori isolates. At preliminary stage it is sufficient to use only 3 VNTR loci for the differentiation of H. pylori strains.

Molecular Subtyping of Salmonella enterica Serovar Typhi by Pulsed-Field Gel Electrophoresis and Multiple-Locus Variable-Number Tandem-Repeat Analysis in India: Their Association with Antimicrobial Resistance Profiles.

Molecular subtyping and DNA sequencing-based methods, which are commonly used for discriminating Salmonella enterica serovar Typhi (S. Typhi) isolates, lead to improved molecular epidemiological investigations for prevention and control of typhoid fever. We obtained S. Typhi blood isolates (n = 66) from India during 2007-14 for molecular subtyping by pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) in association with antibiotic resistance profiles. Genotypic diversity was observed more by MLVA (Simpson's index of diversity, D value = 0.997) than PFGE (D value = 0.864). Two prevalent pulsotypes containing nalidixic acid-resistant (NALR) and NALR-ciprofloxacin-resistant (CIPR) S. Typhi isolates circulated in India. Multidrug-resistant (MDR), NALR-CIPR, and most NALR isolates were found to be clonal by PFGE. MLVA could differentiate the clonal isolates. Most of the MDR and NALR-CIPR isolates showed variation in single or double VNTR loci, whereas NALR isolates varied in more than 2 loci, reflecting higher genetic diversity among the NALR isolates. Of the 6 VNTR loci, TR4,699 (D value = 0.838) and Sal02 (D value = 0.890) loci played important roles as MLVA cluster-supporting alleles. The rapid turnaround time and high-level discriminatory power of MLVA may be useful for tracking and controlling the transmission of S. Typhi isolates during epidemiological investigations.

Feasibility of Using Multiple-Locus Variable-Number Tandem-Repeat Analysis for Epidemiology Study of Vibrio parahaemolyticus Infections

Vibrio parahaemolyticus causes foodborne gastroenteritis, which is often associated with the consumption of raw or undercooked shellfish. Molecular typing can provide critical information for detecting outbreaks and for source attribution. In this study, we describe the development and evaluation of an optimized multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) for the characterization of V. parahaemolyticus isolates. The discriminatory power of MLVA was compared to that of pulsed-field gel electrophoresis (PFGE) by typing 73 sporadic isolates. Epidemiologic concordance was evaluated by typing 23 isolates from five epidemiologically well-characterized outbreaks. The optimized MLVA was applied in early warning, epidemiological surveillance, and source tracking for V. parahaemolyticus infections. There was no significant difference in the discriminatory power of PFGE and MLVA with six or eight VNTR loci for the sporadic isolates. All isolates within an outbreak were indistinguishable by MLVA with six loci, except for one outbreak. Typically, the epidemiological survey could be initiated according to PFGE clusters. We applied MLVA with six loci on 22 isolates in two PFGE clusters. Isolates in one PFGE cluster were distinguished by MLVA. Although a follow-up investigation showed that both clusters had no epidemiological concordance, MLVA decreased the frequency of initiation of epidemiological surveys, thereby reducing labor costs. The ability of MLVA to trace the source of infection was evaluated by isolates from two outbreaks and shrimp samples. The isolates from one of outbreaks and a shrimp had the same MLVA type, suggesting that an epidemiological survey was initiated. Data from the epidemiological investigation subsequently indicated that contaminated shrimp from a nearby city (Dongguan) might be the source of the outbreak. In conclusion, these results indicate that the optimized MLVA may be a promising tool for early warning and epidemiological surveillance of V. parahaemolyticus infections.

Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Acinetobacter pittii and Development of an Optimized Multiple-Locus VNTR Analysis Typing Scheme

ObjectiveTo develop a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) assay for Acinetobacter pittii typing. MethodsPolymorphic VNTRs were searched by Tandem Repeats Finder. The distribution and polymorphism of each VNTR locus were analyzed in all the A. pittii genomes deposited in the NCBI genome database by BLAST and were evaluated with a collection of 20 well-characterized clinical A. pittii strains and one reference strain. The MLVA assay was compared with pulsed-field gel electrophoresis (PFGE) for discriminating A. pittii isolates. ResultsTen VNTR loci were identified upon bioinformatic screening of A. pittii genomes, but only five of them showed full amplifiability and good polymorphism. Therefore, an MLVA assay composed of five VNTR loci was developed. The typeability, reproducibility, stability, discriminatory power, and epidemiological concordance were excellent. Compared with PFGE, the new optimized MLVA typing scheme provided the same and even greater discrimination. ConclusionCompared with PFGE, MLVA typing is a faster and more standardized alternative for studying the genetic relatedness of A. pittii isolates in disease surveillance and outbreak investigation.

Comparative analysis of subtyping methods against a whole-genome-sequencing standard for Salmonella enterica serotype Enteritidis.

A retrospective investigation was performed to evaluate whole-genome sequencing as a benchmark for comparing molecular subtyping methods for Salmonella enterica serotype Enteritidis and survey the population structure of commonly encountered S. enterica serotype Enteritidis outbreak isolates in the United States. A total of 52 S. enterica serotype Enteritidis isolates representing 16 major outbreaks and three sporadic cases collected between 2001 and 2012 were sequenced and subjected to subtyping by four different methods: (i) whole-genome single-nucleotide-polymorphism typing (WGST), (ii) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA), (iii) clustered regularly interspaced short palindromic repeats combined with multi-virulence-locus sequence typing (CRISPR-MVLST), and (iv) pulsed-field gel electrophoresis (PFGE). WGST resolved all outbreak clusters and provided useful robust phylogenetic inference results with high epidemiological correlation. While both MLVA and CRISPR-MVLST yielded higher discriminatory power than PFGE, MLVA outperformed the other methods in delineating outbreak clusters whereas CRISPR-MVLST showed the potential to trace major lineages and ecological origins of S. enterica serotype Enteritidis. Our results suggested that whole-genome sequencing makes a viable platform for the evaluation and benchmarking of molecular subtyping methods.

A single-tube multiple-locus variable-number tandem-repeat analysis of Mycoplasma pneumoniae clinical specimens by use of multiplex PCR-capillary electrophoresis.

In this study, we developed a single-tube multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) assay to type Mycoplasma pneumoniae directly from respiratory samples collected from children with respiratory infections. The multiplex PCR included four fluorescently primed VNTRs (Mpn13, Mpn14, Mpn15, and Mpn16) and was carried out in a single tube. A total of 137 M. pneumoniae-positive specimens, collected in 2013 from Beijing, China, were divided among four types (M4-5-7-2, M4-5-6-2, M3-5-6-2, and M5-5-7-2) using the amended MLVA system. The most prevalent genotype was M4-5-7-2. No correlation was found between macrolide resistance in the M. pneumoniae samples and the MLVA types. To our knowledge, this is the first study to type and analyze M. pneumoniae clinical specimens using multiplex PCR-capillary electrophoresis in a single tube. This novel low-cost method can be used to rapidly type M. pneumoniae clinical specimens directly and shows great potential for monitoring outbreaks of M. pneumoniae.

High-resolution in situ genotyping of Legionella pneumophila populations in drinking water by multiple-locus variable-number tandem-repeat analysis using environmental DNA.

Central to the understanding of infections by the waterborne pathogen Legionella pneumophila is its detection at the clonal level. Currently, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) of L. pneumophila isolates can be used as a tool for high-resolution genotyping. Since L. pneumophila is difficult to isolate, the isolation of outbreak strains often fails due to a viable but nonculturable (VBNC) state of the respective environmental population. Therefore, we developed a cultivation-independent approach to detect single clones in drinking water. This approach is based on the extraction of DNA from drinking water followed by PCR using a set of eight VNTR primer pairs necessary for MLVA genotyping of L. pneumophila. The PCR amplicons were analyzed by single-strand conformation polymorphism (SSCP) and capillary electrophoresis to obtain the respective MLVA profiles. Parallel to the high-resolution analysis, we used the same environmental DNA to quantify the number of L. pneumophila cells in drinking water using real-time PCR with 16S rRNA gene-targeted primers. We used a set of drinking water samples from a small-scale drinking water network to test our approach. With these samples we demonstrated that the developed approach was directly applicable to DNA obtained from drinking water. We were able to detect more L. pneumophila MLVA genotypes in drinking water than we could detect by isolation. Our approach could be a valuable tool to identify outbreak strains even after the outbreak has occurred and has the potential to be applied directly to clinical material.

Characterization of Novel Brucella Strains Originating from Wild Native Rodent Species in North Queensland, Australia

We report on the characterization of a group of seven novel Brucella strains isolated in 1964 from three native rodent species in North Queensland, Australia, during a survey of wild animals. The strains were initially reported to be Brucella suis biovar 3 on the basis of microbiological test results. Our results indicated that the rodent strains had microbiological traits distinct from those of B. suis biovar 3 and all other Brucella spp. To reinvestigate these rodent strains, we sequenced the 16S rRNA, recA, and rpoB genes and nine housekeeping genes and also performed multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA). The rodent strains have a unique 16S rRNA gene sequence compared to the sequences of the classical Brucella spp. Sequence analysis of the recA, rpoB, and nine housekeeping genes reveals that the rodent strains are genetically identical to each other at these loci and divergent from any of the currently described Brucella sequence types. However, all seven of the rodent strains do exhibit distinctive allelic MLVA profiles, although none demonstrated an amplicon for VNTR 07, whereas the other Brucella spp. did. Phylogenetic analysis of the MLVA data reveals that the rodent strains form a distinct clade separate from the classical Brucella spp. Furthermore, whole-genome sequence comparison using the maximal unique exact matches index (MUMi) demonstrated a high degree of relatedness of one of the seven rodent Brucella strains (strain NF 2653) to another Australian rodent Brucella strain (strain 83-13). Our findings strongly suggest that this group of Brucella strains isolated from wild Australian rodents defines a new species in the Brucella genus.

Population-based molecular epidemiology of leprosy in Cebu, Philippines.

To address the persisting problem of leprosy in Cebu, Philippines, we compiled a database of more than 200 patients who attend an established referral skin clinic. We described the patient characteristics in conventional demographic parameters and also applied multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) and single nucleotide polymorphism (SNP) typing for Mycobacterium leprae in biopsied skin lesion samples. These combined approaches revealed that transmission is ongoing, with the affected including the young Cebuano population under 40 years of age in both crowded cities and rural areas of the island. The emergence of multicase families (MCF) is indicative of infection unconstrained by standard care measures. For the SNPs, we designed a low-cost PCR-restriction fragment length polymorphism typing method. MLVA in M. leprae was highly discriminatory in this population yet could retain broad groups, as defined by the more stable SNPs, implying temporal marker stability suitable for interpreting population structures and evolution. The majority of isolates belong to an Asian lineage (SNP type 1), and the rest belong to a putative postcolonial lineage (SNP type 3). Specific alleles at two VNTR loci, (GGT)5 and 21-3, were highly associated with SNP type 3 in this population. MLVA identified M. leprae genotype associations for patients with known epidemiological links such as in MCFs and in some villages. These methods provide a molecular database and a rational framework for targeted approaches to search and confirm leprosy transmission in various scenarios.

Rapid Variable-Number Tandem-Repeat Genotyping for Mycobacterium leprae Clinical Specimens

Mycobacterium leprae is the noncultivable pathogen of leprosy. Since the genome sequence of an isolate of M. leprae has become available, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been explored as a tool for strain typing and identification of chains of transmission of leprosy. In order to discover VNTRs and develop methods transferable to clinical samples, MLVA was applied to a global collection of M. leprae isolates derived from leprosy patients and propagated in armadillo hosts. PCR amplification, agarose gel electrophoresis, and sequencing methods were applied to DNA extracts from these infected armadillo tissues (n = 21). We identified polymorphisms in 15 out of 25 short-tandem-repeat (STR) loci previously selected by in silico analyses of the M. leprae genome. We then developed multiplex PCR for amplification of these 15 loci in four separate PCRs suitable for fluorescent fragment length analysis and demonstrated STR profiles highly concordant with those from the sequencing methods. Subsequently, we extended this method to DNA extracts from human clinical specimens, such as skin biopsy specimens (n = 30). With these techniques, mapping of multiple loci and differentiation of genotypes have been possible using total DNA extracts from limited amounts of clinical samples at a reduced cost and with less time. These practical methods are therefore available and applicable to answer focused epidemiological questions and to allow monitoring of the transmission of M. leprae in different countries where leprosy is endemic.

Multiple-Locus Variable-Number Tandem-Repeat Analysis for Clonal Identification of Vibrio parahaemolyticus Isolates by Using Capillary Electrophoresis

Epidemics of Vibrio parahaemolyticus in Chile have occurred since 1998. Direct genome restriction enzyme analysis (DGREA) using conventional gel electrophoresis permitted discrimination of different V. parahaemolyticus isolates obtained from these outbreaks and showed that this species consists of a highly diverse population. A multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) approach was developed and applied to 22 clinical and 91 environmental V. parahaemolyticus isolates from Chile to understand their clonal structures. To this end, an advanced molecular technique was developed by applying multiplex PCR, fluorescent primers, and capillary electrophoresis, resulting in a high-resolution and high-throughput (HRHT) genotyping method. The genomic basis of this HRHT method was eight VNTR loci described previously by Kimura et al. (J. Microbiol. Methods 72:313-320, 2008) and two new loci which were identified by a detailed molecular study of 24 potential VNTR loci on both chromosomes. The isolates of V. parahaemolyticus belonging to the same DGREA pattern were distinguishable by the size variations in the indicative 10 VNTRs. This assay showed that these 10 VNTR loci were useful for distinguishing isolates of V. parahaemolyticus that had different DGREA patterns and also isolates that belong to the same group. Isolates that differed in their DGREA patterns showed polymorphism in their VNTR profiles. A total of 81 isolates was associated with 59 MLVA groups, providing fine-scale differentiation, even among very closely related isolates. The developed approach enables rapid and high-resolution analysis of V. parahaemolyticus with pandemic potential and provides a new surveillance tool for food-borne pathogens.

Multiple-Locus Variable-Number Tandem-Repeat Analysis for Longitudinal Survey of Sources of Pseudomonas aeruginosa Infection in Cystic Fibrosis Patients

In order to identify the source of infection by Pseudomonas aeruginosa in patients with cystic fibrosis (CF), systematic genotyping of isolates is necessary. Multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) was used to survey the sources of P. aeruginosa infections in a French (Paris, France) pediatric CF center. Between January 2004 and December 2006, 108 patients ages 2 to 21 years who were regularly monitored at the center provided sputum for culture. P. aeruginosa was detected in 46 children, 17 of whom had primary colonization. A total of 163 isolates were recovered. MLVA was improved from a previously published method by the addition of new, informative, and easily typeable markers. Upon genotyping with 15 VNTRs, a total of 39 lineages composed of indistinguishable or closely related isolates, were observed. One of them corresponds to "clone C," which is widely distributed in Europe, and another corresponds to reference strain PA14. Six patients were colonized with two different strains, and the remaining 40 patients were colonized with a single strain. Strains from seven lineages were shared by at least two and up to four patients among a total of 20 patients. The study demonstrates that MLVA is an efficient, easy, and rapid molecular method for epidemiological surveillance for P. aeruginosa infection. The resulting data and strain genetic profiles can be queried on http://bacterial-genotyping.igmors.u-psud.fr.

Identification and distribution of Mycobacterium leprae genotypes in a region of high leprosy prevalence in China: a 3-year molecular epidemiological study.

Multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been proposed as a means of strain typing for tracking the transmission of leprosy. However, empirical data for a defined population are lacking. To this end, a study was initiated to assess the diversity and distribution of prevalent Mycobacterium leprae strains in Qiubei County, Yunnan Province, People's Republic of China, where the annual detection rate of leprosy is 10-fold higher than the national average rate. Sixty-eight newly diagnosed leprosy patients were included in the study. MLVA at eight M. leprae loci was applied using DNA extracts from skin biopsies. The number of alleles per locus ranged from 4 to 24, providing adequate strain discrimination. MLVA strain typing identified several clusters of patients whose M. leprae specimens shared similar VNTR profiles. Two of these clusters were comprised of patients who resided predominantly in the north and northwest parts of Qiubei County. Furthermore, it was found that multicase families are common in this county: 23 of the 68 patients were from 11 families. Intrafamilial VNTR profiles closely matched within six families, although they were different between the families. Moreover, VNTR patterns related to those found in some multicase families were also detected in patients in the same or adjacent townships, indicating the utility of VNTR strain typing to identify and detect short-range transmission events. Social contact through village markets is proposed as a means of transmission.