A multiple typing approach is recommended for the laboratory-based surveillance of salmonellosis in Romania

In Romania, Salmonella enterica serovar Typhimurium isolates are currently typed by antimicrobial resistance profiles and phage typing, as part of the national laboratory-based surveillance system of human enteric infections. The aim of the present study was to assess the added value of complementing this approach with molecular fingerprinting, namely pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeats analysis (MLVA). Thirty-six S. Typhimurium isolates received by the Reference Center for Human Salmonella Infections for confirmation and typing from the Microbiology Departments of three Public Health Authorities, were selected for this study. Phage typing revealed that 14 isolates (39%) were nontypeable (NT). Twenty-two isolates were assigned to 5 phage types: DT193 (11 isolates), U302 (7 isolates), DT116 (2 isolates), DT41 (1 isolate) and DT86 (1 isolate). Antimicrobial susceptibility testing showed that all the NT and DT116 isolates were multidrug resistant and extended-spectrum betalactamase producers. All the examined isolates were typeable when using the molecular approach. Both methods gave conclusive and comparable results, documenting the genetic relatedness and discriminating the outbreak isolates from sporadic cases. We conclude that in order to improve outbreak investigation and surveillance of salmonellosis in Romania, the current routine typing of Salmonella isolates should be complemented with at least one of these DNA fingerprinting methods.     

MLVA as a Tool for Public Health Surveillance of Human Salmonella Typhimurium: Prospective Study in Belgium and Evaluation of MLVA Loci Stability

Surveillance of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is generally considered to benefit from molecular techniques like multiple-locus variable-number of tandem repeats analysis (MLVA), which allow earlier detection and confinement of outbreaks. Here, a surveillance study, including phage typing, antimicrobial susceptibility testing and the in Europe most commonly used 5-loci MLVA on 1,420 S. Typhimurium isolates collected between 2010 and 2012 in Belgium, was used to evaluate the added value of MLVA for public health surveillance. Phage types DT193, DT195, DT120, DT104, DT12 and U302 dominate the Belgian S. Typhimurium population. A combined resistance to ampicillin, streptomycin, sulphonamides and tetracycline (ASSuT) with or without additional resistances was observed for 42.5% of the isolates. 414 different MLVA profiles were detected, of which 14 frequent profiles included 44.4% of the S. Typhimurium population. During a serial passage experiment on selected isolates to investigate the in vitro stability of the 5 MLVA loci, variations over time were observed for loci STTR6, STTR10, STTR5 and STTR9. This study demonstrates that MLVA improves public health surveillance of S. Typhimurium. However, the 5-loci MLVA should be complemented with other subtyping methods for investigation of possible outbreaks with frequent MLVA profiles. Also, variability in these MLVA loci should be taken into account when investigating extended outbreaks and studying dynamics over longer periods.     

Heterogeneity in expression of lipopolysaccharide by strains of Salmonella enterica serotype Typhimurium definitive phage type 104 and related phage types

AIMS: To investigate lipopolysaccharide (LPS) expression in Salmonella enterica serotype Typhimurium definitive phage type 104 (Salmonella Typhimurium DT104) and related phage types. METHODS AND RESULTS: Isolates were examined for the expression of LPS by SDS-PAGE and silver staining and subtyped by Pulsed Field Gel Electrophoresis (PFGE). The 100 isolates expressed one of two LPS profiles designated A (72%) and B (28%). LPS profiling was able to discriminate between isolates of identical PFGE type. Among 10 groups of outbreak isolates examined, each group was of a single LPS profile: A, 8/10 and B, 2/10. All 10 outbreaks were identical by PFGE analysis. CONCLUSIONS: Isolates of Salmonella Typhimurium DT104 and related phage types expressed one of two distinct LPS profiles. The two LPS profiles appear similar but shifted and in phase with one another, suggesting that the heterogeneity is due to changes in the LPS core region rather than among the repeating oligosaccharide units of the long-chain LPS. SIGNIFICANCE AND IMPACT OF THE SUTDY: LPS profiling provides a useful adjunct to PFGE and other molecular methods for the subtyping of this group of bacteria in epidemiological investigations.     

Application of pulsed-field gel electrophoresis to laboratory surveillance of small community outbreaks of Salmonella enterica serovar Typhimurium

Infectious diarrhea syndrome is an important cause of human morbidity around the world, and Salmonella genus remains one of the most prevalent etiology. Salmonella enterica serovar Typhimurium outbreak-associated isolates received by the Laboratory for Enteric Pathogens from N.I.R.D.M.I. "Cantacuzino" for confirmation and typing were analyzed by genomic pulsed-field gel electrophoresis (PFGE) and phage susceptibility testing to establish their relatedness. Both typing methods proved to have similar discriminatory power. The isolates originating from the same outbreak belonged to the same phage type and showed indistinguishable PFGE profiles. The molecular characterization of autochthonal Salmonella enterica Typhimurium outbreak human isolates provided laboratory evidence that epidemiologically related isolates collected from community outbreaks of disease were also genetically related. In order to improve the national and international surveillance of major foodborne pathogens the reference laboratory centers are required to establish and maintain the capacity to perform a wide range of both phenotypic and genotypic methods to support outbreak investigations.     

Use of multiple‐locus variable number tandem repeat analysis and phage typing for subtyping ofSalmonella Enteritidis from sporadic human cases in the United States

Aims: To investigate the genetic diversity among S. Enteritidis isolates from different geographic regions to evaluate the relationship between phage types (PTs) and variable number tandem repeat analysis (VNTR) loci. Methods and Results: We performed multiple‐locus variable number tandem repeat analysis (MLVA) and phage typing on 245 S. Enteritidis isolates collected from sporadic human clinical cases in Michigan, Minnesota, New York, and Washington states between 2000 and 2007. Ninety‐four MLVA types and 22 different PTs were identified. Specific PTs were associated with a predominant allele for certain VNTR loci. Cluster analysis using a minimum‐spanning tree demonstrated two major clusters (I, II) and one minor cluster of isolates. PTs 8, 13a, 13 and 34 were significantly associated with MLVA cluster I. Phage types 1, 4, 6a, and 18 were significantly associated with MLVA cluster II. Conclusions: We found significant association between MLVA‐based clusters and PTs. Certain VNTR loci were associated with specific PTs and could serve as useful molecular markers for S. Enteritidis in epidemiological investigations. Significance and Impact of the Study: MLVA genotyping in combination with phage typing can be used for effective characterization of S. Enteritidis isolates. It can also be useful for tracing possible sources during investigations of sporadic and outbreak cases of S. Enteritidis.     

Use of multiple‐locus variable‐number tandem‐repeats analysis (MLVA) typing to characterize Salmonella Typhimurium DT41 broiler breeder infections

Aims: To characterize isolates of Salmonella Typhimurium DT41 obtained from infected flocks of broiler breeders by multiple‐locus variable‐number tandem‐repeats analysis (MLVA) and compare results with a diverse strain collection from Germany and United Kingdom and isolates from Danish patients. Methods and Results: A total of 102 isolates of Salm. Typhimurium phage type DT41 were MLVA typed. MLVA typing showed 4, 12, 25, 9 and 8 different alleles at the five MLVA loci 9, 5, 6, 10 and 3, respectively. A dendrogram based on MLVA types was constructed, and one large group, nine minor groups and 29 more unrelated MLVA types were obtained. The major group included 20 of the 30 human isolates. Isolates obtained from broiler breeders demonstrated major diversity, indicating the existence of several independent introductions of DT41 at farm level. When comparison was made to isolates included from Germany and England, DT41 seems to be ubiquitous in the wild fauna which might represent a risk factor for poultry. Conclusions: Transmission from Danish broilers to humans was not demonstrated, neither was the transmission from rearing farms to broiler breeder farms. Sources of infection at broiler breeder farm level remained unidentified. Significance and Impact of the Study: Major diversity was demonstrated for DT41 MLVA types. A persisting problem with infection of broiler breeder flocks with DT41 was not reflected in broiler flocks originating from these flocks.     

Characterization of Salmonella enterica serovar Enteritidis isolates recovered from blood and stool specimens in Thailand

ABSTRACT: BACKGROUND: Bacteremia due to Salmonella spp. is a life-threatening condition and is commonly associated with immune compromise. A 2009 observational study estimated risk factors for the ten most common non-typhoidal Salmonella (NTS) serovars isolated from Thai patients between 2002-2007. In this study, 60.8% of Salmonella enterica serovar Enteritidis isolates (n = 1517) were recovered from blood specimens and infection with Salmonella serovar Enteritidis was a statistically significant risk factor for bacteremia when compared to other NTS serovars. Based on this information, we characterized a subset of isolates collected in 2008 to determine if specific clones were recovered from blood or stool specimens at a higher rate. Twenty blood isolates and 20 stool isolates were selected for antimicrobial resistance testing (MIC), phage typing, PFGE, and MLVA. Result Eight antibiogrammes, seven MLVA types, 14 XbaI/BlnI PFGE pattern combinations, and 11 phage types were observed indicating considerable diversity among the 40 isolates characterized. Composite analysis based on PFGE and MLVA data revealed 22 genotypes. Seven of the genotypes containing two or more isolates were from both stool and blood specimens originating from various months and zones. Additionally, those genotypes were all further discriminated by phage type and/or antibiogramme. Ninety percent of the isolates were ciprofloxacin resistant. CONCLUSIONS: The increased percentage of bloodstream infections as described in the 2009 observational study could not be attributed to a single clone. Future efforts should focus on assessing the immune status of bacteriaemic patients and identifying prevention and control measures, including attribution studies characterizing non-clinical (animal, food, and environmental) isolates.     

Genotypic and PFGE/MLVA analyses of Vibrio cholerae O1: geographical spread and temporal changes during the 2007-2010 cholera outbreaks in Thailand

Background

Vibrio cholerae O1 El Tor dominated the seventh cholera pandemic which occurred in the 1960s. For two decades, variants of V. cholerae O1 El Tor that produce classical cholera toxin have emerged and spread globally, replacing the prototypic El Tor biotype. This study aims to characterize V. cholerae O1 isolates from outbreaks in Thailand with special reference to genotypic variations over time.

Methods/Findings

A total of 343 isolates of V. cholerae O1 from cholera outbreaks from 2007 to 2010 were investigated, and 99.4% were found to carry the classical cholera toxin B subunit (ctxB) and El Tor rstR genes. Pulsed-field gel electrophoresis (PFGE) differentiated the isolates into 10 distinct pulsotypes, clustered into two major groups, A and B, with an overall similarity of 88%. Ribotyping, multiple-locus variable-number tandem-repeat analysis (MLVA), and PCR to detect Vibrio seventh pandemic island II (VSP-II) related genes of randomly selected isolates from each pulsotype corresponded to the results obtained by PFGE. Epidemiological investigations revealed that MLVA type 2 was strongly associated with a cholera outbreak in northeastern Thailand in 2007, while MLVA type 7 dominated the outbreaks of the southern Gulf areas in 2009 and MLVA type 4 dominated the outbreaks of the central Gulf areas during 2009–2010. Only MLVA type 16 isolates were found in a Thai-Myanmar border area in 2010, whereas those of MLVA types 26, 39, and 41 predominated this border area in 2008. Type 39 then disappeared 1–2 years later as MLVA type 41 became prevalent. Type 41 was also found to infect an outbreak area.

Conclusions

MLVA provided a high-throughput genetic typing tool for understanding the in-depth epidemiology of cholera outbreaks. Our epidemiological surveys suggest that some clones of V. cholerae O1 with similar but distinctive genetic traits circulate in outbreak sites, while others disappear over time.     

Multi Locus Variable-Number Tandem Repeat (MLVA) Typing Tools Improved the Surveillance of Salmonella Enteritidis: A 6 Years Retrospective Study

Surveillance of Salmonella enterica subsp. enterica serovar Enteritidis is generally considered to benefit from molecular techniques like multiple-locus variable-number of tandem repeats analysis (MLVA), which allow early detection and confinement of outbreaks. Here, a surveillance study, including phage typing, antimicrobial susceptibility testing and MLVA on 1,535 S. Enteritidis isolates collected between 2007 and 2012, was used to evaluate the added value of MLVA for public health surveillance in Belgium. Phage types PT4, PT8, PT21, PT1, PT6, PT14b, PT28 and PT13 dominate the Belgian S. Enteritidis population. The isolates of S. Enteritidis were most frequently susceptible to all antibiotics tested. 172 different MLVA profiles were detected, of which 9 frequent profiles included 67.2% of the S. Enteritidis population. During a serial passage experiment on selected isolates to investigate the in vitro stability of the 5 MLVA loci, no variations over time were observed indicating that the MLVA profiles were stable. The MLVA profile of isolates originating from different outbreaks in the Democratic Republic of the Congo (DRC) between 2010 and 2011 were distinct from any of the MLVA profiles found in Belgian isolates throughout the six year observational period and demonstrates that MLVA improves public health surveillance of S. Enteritidis. However, MLVA should be complemented with other subtyping methods when investigating outbreaks is caused by the most common MLVA profile.     

Combined Use of Bacteriophage Typing and Pulsed-Field Gel Electrophoresis in the Epidemiological Analysis of Japanese Isolates of Enterohemorrhagic Escherichia coli O157:H7

A total of 236 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates in Japan were investigated by bacteriophage typing, and the results were compared with those of pulsed-field gel electrophoresis (PFGE). Seven phage types (PTs) were observed in 71 isolates which were derived from 22 outbreaks. All of the isolates from ten outbreaks in the Kinki region (midwestern part of Japan) in July-August 1996 were grouped into the same PFGE type (IIa) and PT 32, while among total isolates, there were such varieties as PFGE type IIa containing five phage types and PT32 containing two PFGE types. These results suggest that the ten outbreaks should be considered to be a single outbreak, and show that the combined use of bacteriophage typing and PFGE enhances reliability in epidemiological surveys.     

Genetic and phenotypic variability among Salmonella enterica serovar Typhimurium isolates from California dairy cattle and humans

Fifty-six human and 24 adult dairy cattle isolates of Salmonella enterica serovar Typhimurium from a single county in California were compared using ribotyping, insertion sequence typing (IS200), pulsed-field gel electrophoresis, plasmid typing, phage typing, and antimicrobial resistance testing. The majority of the isolates fell into one of two groups which were phage types DT104 and DT193. Combining the information from all typing methods, a total of 45 different "clusters" were defined, with 35 of those including only a single isolate. The library of isolates had a high degree of variability, but antibiotic resistance and plasmid typing each defined single clusters in which human or bovine isolates predominated (chi2, P < 0.05).     

Cost-effective application of pulsed-field gel electrophoresis to typing of Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.     

Use of WARD et al's scheme for bacteriophage typing of Salmonella enteritidis strains isolated in Poland

The typing phages set of Ward et al. was used to type a total of 517 strains of Salmonella Enteritidis isolated in Poland in 1986-1995. According to the Ward et al. scheme, 56.5% of the strains tested were assigned to 14 different phage types. Phage types 8, 4, 1 and 4a were placed first, second, third and fourth, respectively. They were dominated both in the outbreak isolates and in the isolates from the other sources. Ten phage types were represented by single strains. Other strains reacted with phages without showing any of the designated phage type (37.1%) or were untypable (6.4%). The Ward et al. scheme seems to demonstrate not enough high degree of strain discrimination for Salmonella Enteritidis isolates in Poland. It seems that the Ward et al. scheme is not enough useful for the epidemiological investigations of Salmonella Enteritidis in Poland.     

AFLP analysis of Salmonella enterica serovar Typhimurium isolates of phage types DT 9 and DT 135: diversity within phage types and its epidemiological significance

Amplified fragment length polymorphism (AFLP) was applied to 35 and 34 isolates, respectively, of Salmonella enterica serovar Typhimurium phage types DT 9 and DT 135, using eight primer pair combinations. Eight and 17 AFLP types were observed in DT 9 and DT 135, respectively. DT 9 is rare in the UK and common in Australia, but one AFLP form dominated with 28 isolates, comprising 22 of 25 UK isolates, four of five Australian isolates, one Jamaican and one Spanish isolate. Of the others, two UK isolates are closely related to the major form, two from elsewhere are in the major cluster and three isolates from different countries are in a separate cluster. For DT 135, two closely related AFLP types of seven and 11 isolates form the major cluster, which also includes 11 isolates, mostly in single-isolate AFLP types, while five isolates from different countries form a well-separated minor cluster. For both DTs all isolates are grouped together if only the phage type specific bands identified earlier are used, confirming their value for molecular-based 'phage typing'. Polymorphic markers identified in this study could also be used for subtyping within both phage types. The value of AFLP is in locating DNA fragments useful for typing, but implementation of a replacement typing scheme would probably involve multiplex PCR or microarray technologies.     

Cost-Effective Application of Pulsed-Field Gel Electrophoresis to Typing of Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.     

Can MLVA Differentiate among Endemic-Like MRSA Isolates with Identical Spa-Type in a Low-Prevalence Region?

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Norway is low, but an endemic-like MRSA clone with Staphylococcal protein A (spa)-type t304 has been established especially in nursing homes in the Oslo region causing several large outbreaks. The challenge was that spa-typing and the gold standard Pulsed-Field Gel Electrophoresis (PFGE) were inadequate in discriminating isolates in outbreak investigations. Additional higher resolution genotyping methods were needed. The aims of this study were a) to evaluate whether Multiple-Locus Variable number of tandem repeat Analysis (MLVA) could differentiate within the PFGE clusters between epidemiologically related and unrelated endemic-like ST8-MRSA-IV-t304-PVL-neg (MRSA-t304) isolates and b) investigate the evolution of the endemic-like MRSA-t304 clone over a 15-year time period. All MRSA-t304 isolates detected in the region from 1998 through April 2013 were included. In total, 194 of 197 isolates were available for PFGE and MLVA analyses. PFGE results on isolates from 1998–2010 have been published previously. Two PFGE clusters subdivided into eight MLVA types were detected. One major outbreak clone (PFGE cluster C2/ MLVA type MT5045) appeared from 2004 to 2011 causing long-lasting and large outbreaks in seven nursing homes and one hospital. Five new MLVA types (N = 9 isolates) differing in only one VNTR compared to the outbreak clone C2/MT5045 were detected, but only one (C2/MT5044) was seen after 2011. We suggest that MLVA can replace PFGE analysis, but MLVA may not be the optimal method in this setting as it did not discriminate between all epidemiologically unrelated isolates. The results may indicate that all eight outbreaks in different locations within the PFGE C2 cluster may be branches of one large regional outbreak. The major outbreak strain C2/MT5045 may now, however, be under control, extinguished or has moved geographically.     

Multiple-locus variable-number tandem repeat analysis of Salmonella Enteritidis isolates from human and non-human sources using a single multiplex PCR

Simplified multiple-locus variable-number tandem repeat analysis (MLVA) was developed using one-shot multiplex PCR for seven variable-number tandem repeats (VNTR) markers with high diversity capacity. MLVA, phage typing, and PFGE methods were applied on 34 diverse Salmonella Enteritidis isolates from human and non-human sources. MLVA detected allelic variations that helped to classify the S. Enteritidis isolates into more evenly distributed subtypes than other methods. MLVA-based S. Enteritidis clonal groups were largely associated with sources of the isolates. Nei's diversity indices for polymorphism ranged from 0.25 to 0.70 for seven VNTR loci markers. Based on Simpson's and Shannon's diversity indices, MLVA had a higher discriminatory power than pulsed field gel electrophoresis (PFGE), phage typing, or multilocus enzyme electrophoresis. Therefore, MLVA may be used along with PFGE to enhance the effectiveness of the molecular epidemiologic investigation of S. Enteritidis infections.     

Streptococcus suis outbreak investigation using multiple‐locus variable tandem repeat number analysis

Two outbreaks of Streptococcus suis ST7 occurred in humans in 1998 and 2005 in China. PFGE of chromosome restriction fragments found all ST7 isolates to be indistinguishable. Due to the genetic homogeneity of ST7 isolates, development of a rapid sub‐typing method with high discriminatory power for ST7 isolates is required. In this study, a novel method, MLVA, was developed to type S. suis serotype 2 strains. Further, this method was used to analyze outbreak‐associated ST7 strains in China. A total of 144 ST7 S. suis isolates were sub‐typed into 34 MLVA types. Among these, eight isolates from the 1998 outbreak were sub‐typed into five MLVA types, of which four MLVA types were also detected in Sichuan in 2005. These data indicate that the pathogens responsible for the two outbreaks had the same origin. In addition, some observations also provided molecular evidence for the transmission route, possibly indicating that the MLVA method has usefulness in epidemiology. The developed MLVA scheme for S. suis has greater discriminative power than PFGE. The method described here may be useful for identifying the source of S. suis infection and monitoring its spread.     

Phage type 193 of Salmonella typhimurium contains different chromosomal genotypes and multiple IS 200 profiles

Abstract The common phage type 193 of Salmonella typhimurium was analyzed with respect to molecular markers of chromosomal genotype. Three profiles of the 16S rRNA genes and seven profiles of the DNA insertion element of IS2 00 were found among tne representative strains of DT193. The IS 200 profiles found within this single phage type were highly diverse, confirming that DT193 is a composite phage type containing several distinct clones and hybrid lines. IS2 00 profiling is thus appropriate both for primary strain discrimination, and for subdivision within certain phage types of S. typhimurium , such as DT193. This rapid molecular definition of clonality will be useful for the epidemiological investigation of food poisoning outbreaks.     

Genotypes of multidrug-resistant Salmonella enterica serotype typhimurium from two regions of Kenya

A combination of phage typing and pulsed-field gel electrophoresis of Xbal-digested chromosomal DNA has been used to study the epidemiological relationships of multidrug-resistant Salmonella enterica serotype typhimurium from Nairobi (64 isolates) and Kilifi (40 isolates) collected over the period 1994-1997. Isolates from Nairobi belonged to 11 definitive phage types (DTs) encompassing eight different PFGE patterns. In contrast, isolates from Kilifi were mainly DT 56 (60%) and all fell into a single PFGE pattern. The remaining isolates did not conform to a recognisable phage type. We conclude that multidrug-resistant S. typhimurium infections from Nairobi were caused by multiple strains while those from Kilifi were likely to be from a microepidemic caused by a single clone.