Serotype and phage type distribution of salmonella strains isolated from humans,cattle, pigs,and chickens in the Netherlands from 1984 to 2001

We studied serotypes and phage types of Salmonella strains isolated from humans and animals in The Netherlands over the period 1984 to 2001. All human strains (n = 59,168) were clinical isolates. The animal strains (n = 65,567) were from clinical and nonclinical infections. All isolates were serotyped, and Salmonella enterica serovar Typhimurium and serovar Enteritidis strains were further phage typed. The most prevalent serotypes were as follows: in humans, serovars Typhimurium and Enteritidis; in cattle, serovars Typhimurium and Dublin; in pigs, serovar Typhimurium; and in chickens, serovars Enteritidis, Infantis, and Typhimurium. Serovar Enteritidis phage type 4 (pt 4) was the most common phage type in humans and chickens. Serovar Typhimurium pt 510 was the most prevalent serovar Typhimurium phage type in humans and pigs, pt 200 was the most prevalent serovar Typhimurium phage type in cattle, and pt 150 was the most prevalent serovar Typhimurium phage type in chickens. Analysis of the distribution of sero- and phage types during the study period indicated that types shifted over time in humans and animals. Serovar Typhimurium DT 104 emerged in 1991 in humans, cattle, pigs, and chickens and became the most common serovar Typhimurium phage type in 2001. In general, similar sero- and phage types were found in humans and animals, although distinct types were more common in animals. Between the animal species, the sero- and phage type distributions varied considerably.     

Molecular typing of Salmonella enterica serovar typhi isolates from various countries in Asia by a multiplex PCR assay on variable-number tandem repeats

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.     

Use of biochemical kinetic data to determine strain relatedness among Salmonella enterica subsp. enterica isolates

Classical biotyping characterizes strains by creating biotype profiles that consider only positive and negative results for a predefined set of biochemical tests. This method allows Salmonella subspecies to be distinguished but does not allow serotypes and phage types to be distinguished. The objective of this study was to determine the relatedness of isolates belonging to distinct Salmonella enterica subsp. enterica serotypes by using a refined biotyping process that considers the kinetics at which biochemical reactions take place. Using a Vitek GNI+ card for the identification of gram-negative organisms, we determined the biochemical kinetic reactions (28 biochemical tests) of 135 Salmonella enterica subsp. enterica strains of pig origin collected in Spain from 1997 to 2002 (59 Salmonella serotype Typhimurium strains, 25 Salmonella serotype Typhimurium monophasic variant strains, 25 Salmonella serotype Anatum strains, 12 Salmonella serotype Tilburg strains, 7 Salmonella serotype Virchow strains, 6 Salmonella serotype Choleraesuis strains, and 1 Salmonella enterica serotype 4,5,12:-:- strain). The results were expressed as the colorimetric and turbidimetric changes (in percent) and were used to enhance the classical biotype profile by adding kinetic categories. A hierarchical cluster analysis was performed by using the enhanced profiles and resulted in 14 clusters. Six major clusters grouped 94% of all isolates with a similarity of > or =95% within any given cluster, and eight clusters contained a single isolate. The six major clusters grouped not only serotypes of the same type but also phenotypic serotype variations into individual clusters. This suggests that metabolic kinetic reaction data from the biochemical tests commonly used for classic Salmonella enterica subsp. enterica biotyping can possibly be used to determine the relatedness between isolates in an easy and timely manner.     

Detection of multidrug-resistant Salmonella enterica serovar typhimurium phage types DT102, DT104, and U302 by multiplex PCR

Salmonella enterica serovar Typhimurium is a common cause of nontyphoidal salmonellosis in humans and animals. Multidrug-resistant serovar Typhimurium phage type DT104, which emerged in the 1990s, has become widely distributed in many countries. A total of 104 clinical isolates of Salmonella serogroup B were collected from three major hospitals in Taiwan during 1997 to 2003 and were examined by a multiplex PCR targeting the resistance genes and the spv gene of the virulence plasmid. A total of 51 isolates (49%) were resistant to all drugs (ACSSuT [resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline]), and all contained a 1.25-kb PCR fragment of integron that is part of the 43-kb Salmonella genomic island 1 (SGI1). The second group was resistant to SSu (28%), and the third was susceptible to all five drugs (13%). Fifty-nine isolates were serotyped to be serovar Typhimurium by the tube agglutination method using H antisera. The virulence plasmid was found in 54 (91.5%) of the 59 serovar Typhimurium isolates. A majority (94.1%) of the Salmonella serogroup B isolates with the ACSSuT resistance pattern harbored a virulence plasmid. Phage typing identified three major phage types: DT104, DT120, and U302. Analysis of the isolates by pulsed-field gel electrophoresis showed six genotypes. We found two genotypes in DT104 strains, two in DT120, and the other two in U302. The presence of a monophasic serovar (4,5,12:i:-) has added difficulty in the determination of the serovars of multidrug-resistant Salmonella serogroup B isolates. Nevertheless, the multiplex PCR devised in the present study appears to be efficient and useful in the rapid identification of ACSSuT-type serovar Typhimurium with SGI1, irrespective of their phage types.     

Characterization of Salmonella enterica serotype Typhimurium isolates from human, food, and animal sources in the Republic of Ireland

A potential epidemic clone of Salmonella enterica serotype Typhimurium DT104, and the possible emergence of S. enterica serotype Typhimurium DT104b, has been identified from the characterization of 67 S. enterica serotype Typhimurium strains from three sources, human gastroenteritis isolates, isolates from food samples, and veterinary isolates, by antimicrobial resistance profiling, phage typing, and pulsed-field gel electrophoresis. Resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline was found in 77.6% of these strains.     

Multiplex PCR-based method for identification of common clinical serotypes of Salmonella enterica subsp. enterica

A multiplex PCR method has been developed to differentiate between the most common clinical serotypes of Salmonella enterica subsp. enterica encountered in Washington State and the United States in general. Six genetic loci from S. enterica serovar Typhimurium and four from S. enterica serovar Typhi were used to create an assay consisting of two five-plex PCRs. The assays gave reproducible results with 30 different serotypes that represent the most common clinical isolates of S. enterica subsp. enterica. Of these, 22 serotypes gave unique amplification patterns compared with each other and the other 8 serotypes were grouped into four pairs. These were further resolved by two additional PCRs. We compared the data from PCR serotyping with conventional serotyping and found that PCR serotyping was nearly as discriminatory as conventional serotyping was. The results from a blind test screening 111 clinical isolates revealed that 97% were correctly identified using the multiplex PCR assay. The assay can be easily performed on multiple samples with final results in less than 5 h and, in conjunction with pulsed-field gel electrophoresis, forms a very robust test method for the molecular subtyping of Salmonella enterica subsp. enterica.     

DNA fingerprinting of Salmonella enterica subsp. enterica serovar typhimurium with emphasis on phage type DT104 based on variable number of tandem repeat loci

Seventy-eight human and environmental strains of Salmonella enterica subsp. enterica serovar Typhimurium, as well as 18 isolates of other Salmonella serovars and 6 isolates of Escherichia coli, were subjected to a novel variable number of tandem repeats (VNTR)-based fingerprinting method that showed high discrimination and reproducibility for typing serovar Typhimurium isolates. The method is based on capillary separation of PCR products from fluorescence-labeled VNTR in the serovar Typhimurium genome. The serovar Typhimurium isolates displayed 54 VNTR patterns, and the VNTR assay correctly identified strains from a well-characterized outbreak. Among 37 serovar Typhimurium phage type DT104 isolates, 28 distinct VNTR patterns were found. This VNTR-based method is fast and suitable for complete automation. Our VNTR-based method was capable of high discrimination within the homogeneous serovar Typhimurium DT104 phage type and can be used to trace outbreaks and to monitor DT104 as well as other phage types. The VNTR assay was compared to XbaI pulsed-field gel electrophoresis, amplified fragment length polymorphism analysis, integron-cassette profiles and gene PCR of intI1, qacEDelta1, sulI1, and floR. The VNTR assay showed greatly improved resolution compared to all other tested methods in this study.     

Prophage sequences defining hot spots of genome variation in Salmonella enterica serovar Typhimurium can be used to discriminate between field isolates

Sixty-one Salmonella enterica serovar Typhimurium isolates of animal and human origin, matched by phage type, antimicrobial resistance pattern, and place of isolation, were analyzed by microbiological and molecular techniques, including pulsed-field gel electrophoresis (PFGE) and plasmid profiling. PFGE identified 10 profiles that clustered by phage type and antibiotic resistance pattern with human and animal isolates distributed among different PFGE profiles. Genomic DNA was purified from 23 representative strains and hybridized to the composite Salmonella DNA microarray, and specific genomic regions that exhibited significant variation between isolates were identified. Bioinformatic analysis showed that variable regions of DNA were associated with prophage-like elements. Subsequently, simple multiplex PCR assays were designed on the basis of these variable regions that could be used to discriminate between S. enterica serovar Typhimurium isolates from the same geographical region. These multiplex PCR assays, based on prophage-like elements and Salmonella genomic island 1, provide a simple method for identifying new variants of S. enterica serovar Typhimurium in the field.     

Diversity of strains of Salmonella enterica serotype enteritidis from English poultry farms assessed by multiple genetic fingerprinting

Reliable and sufficiently discriminative methods are needed for differentiating individual strains of Salmonella enterica serotype Enteritidis beyond the phenotypic level; however, a consensus has not been reached as to which molecular method is best suited for this purpose. In addition, data are lacking on the molecular fingerprinting of serotype Enteritidis from poultry environments in the United Kingdom. This study evaluated the combined use of classical methods (phage typing) with three well-established molecular methods (ribotyping, macrorestriction analysis of genomic DNA, and plasmid profiling) in the assessment of diversity within 104 isolates of serotype Enteritidis from eight unaffiliated poultry farms in England. The most sensitive technique for identifying polymorphism was PstI-SphI ribotyping, distinguishing a total of 22 patterns, 10 of which were found among phage type 4 isolates. Pulsed-field gel electrophoresis of XbaI-digested genomic DNA segregated the isolates into only six types with minor differences between them. In addition, 14 plasmid profiles were found among this population. When all of the typing methods were combined, 54 types of strains were differentiated, and most of the poultry farms presented a variety of strains, which suggests that serotype Enteritidis organisms representing different genomic groups are circulating in England. In conclusion, geographical and animal origins of Salmonella serotype Enteritidis isolates may have a considerable influence on selecting the best typing strategy for individual programs, and a single method cannot be relied on for discriminating between strains.     

Evaluation of a modified single-enzyme amplified fragment length polymorphism (SE-AFLP) technique for subtyping Salmonella enterica serotype Enteritidis

Salmonella enterica subsp. enterica serotype Enteritidis is not readily subtyped beyond the level of phage type (PT). Pulsed field gel electrophoresis (PFGE) is generally acknowledged to be the most discriminating typing method for Salmonella, but only a restricted variety of PFGE types has been described for S. enterica serotype Enteritidis. In the present study, a modification of the SE-AFLP typing method was used to investigate both outbreak and apparently sporadic isolates of S. enterica serotype Enteritidis belonging to different PTs and/or PFGE types. The method proved to be as discriminatory as PFGE when combined with phage typing, and provided subtyping data consistent with epidemiological information. Although the modified SE-AFLP typing method did not prove to achieve a superior discriminatory ability in resolving clusters, it has a high enough throughput for use in outbreak investigations. This method can be used in combination with other typing methods to obtain epidemiologically relevant subtyping data on S. enterica serotype Enteritidis.     

DNA sequence-based subtyping and evolutionary analysis of selected Salmonella enterica serotypes

While serotyping and phage typing have been used widely to characterize Salmonella isolates, sensitive subtyping methods that allow for evolutionary analyses are essential for examining Salmonella transmission, ecology, and evolution. A set of 25 Salmonella enterica isolates, representing five clinically relevant serotypes (serotypes Agona, Heidelberg, Schwarzengrund, Typhimurium, and Typhimurium var. Copenhagen) was initially used to develop a multilocus sequence typing (MLST) scheme for Salmonella targeting seven housekeeping and virulence genes (panB, fimA, aceK, mdh, icdA, manB, and spaN). A total of eight MLST types were found among the 25 isolates sequenced. A good correlation between MLST types and Salmonella serotypes was observed; only one serotype Typhimurium var. Copenhagen isolate displayed an MLST type otherwise typical for serotype Typhimurium isolates. Since manB, fimA, and mdh allowed for the highest subtype discrimination among the initial 25 isolates, we chose these three genes to perform DNA sequencing of an additional 41 Salmonella isolates representing a larger diversity of serotypes. This "three-gene sequence typing scheme" allowed discrimination of 25 sequence types (STs) among a total of 66 isolates; STs correlated well with serotypes and allowed within-serotype differentiation for 9 of the 12 serotypes characterized. Phylogenetic analyses showed that serotypes Kentucky and Newport could each be separated into two distinct, statistically well supported evolutionary lineages. Our results show that a three-gene sequence typing scheme allows for accurate serotype prediction and for limited subtype discrimination among clinically relevant serotypes of Salmonella. Three-gene sequence typing also supports the notion that Salmonella serotypes represent both monophyletic and polyphyletic lineages.     

Blind comparison of traditional serotyping with three multiplex PCRs for the identification of Salmonella serotypes

Salmonella serotypes are defined on the basis of somatic (O) antigens which define the serogroup and flagellar (H) factor antigens, both of which are present in the cell wall of Salmonella. Most Salmonella organisms alternatively express phase-1 or phase-2 flagellar antigens encoded by fliC and fljB genes, respectively. Our group previously published two multiplex PCRs for distinguishing the most common first- and second-phase antigens. In this paper we describe a third multiplex PCR to identify the most common serogroups (O:B; O:C1; O:C2; O:D and O:E). The combination of these three PCRs enabled us to completely serotype organisms belonging to the Salmonella species. This multiplex PCR includes 10 primers. A total of 67 Salmonella strains belonging to 32 different serotypes were tested. Each strain generated one serogroup-specific fragment ranging between 162 and 615bp. Twenty-eight strains belonging to 21 serotypes, with a serogroup different from those tested in this work, did not generate any fragments. To compare molecular serotyping with traditional serotyping, 500 strains, received according to the order of arrival in the laboratory, were serotyped using both methods. The three multiplex PCRs were able to serotype 84.6% of the tested strains. This method was found to be very helpful in our laboratory as an alternative method for typing strains causing outbreaks, and it can be used to supplement conventional serotyping, since it is also applicable to motionless and rough strains.     

Identification of DT104 and U302 phage types among Salmonella enterica serotype typhimurium isolates by PCR

A DNA sequence was identified in isolates of Salmonella enterica serotype Typhimurium definitive type 104 (DT104). The PCR amplification of an internal segment of this sequence identified DT104 and the closely related U302 phage type among 146 isolates of S. enterica serotype Typhimurium tested, thus providing a tool for rapid identification of DT104 and related isolates.     

Epidemiologic typing of Salmonella enterica serotype enteritidis in a Canada-wide outbreak of gastroenteritis due to contaminated cheese

A major Canada-wide outbreak of gastroenteritis due to Salmonella enterica serotype Enteritidis phage type (PT) 8 occurred in 1998, and this was traced to contaminated cheese in a commercial lunch pack product. Phage typing and pulsed-field gel electrophoresis linked the clinical and cheese isolates of serotype Enteritidis but failed to differentiate outbreak from nonoutbreak PT 8 strains. Further differentiation was made by biotyping based on melibiose fermentation.     

An attempt to identify the evolutionary origin of a novel serotype of Salmonella enterica isolated from harbour porpoises

The isolation since 1991 of a new serotype of Salmonella enterica (antigenic formula 4,12:a:-) from harbour porpoises (Phocoena phocoena) at post-mortem examination raised the question of its evolutionary origin. Representative strains of S. enterica serotype 4,12:a:- and strains of eight other serotypes of serogroup 04 with phase-1 flagellar antigen H 'a' were examined by EcoRI ribotyping, IS200 fingerprinting and PCR-based profiling. Statistical analysis of results of multiple typing showed that strains of Salmonella serotype 4,12:a:- were genetically distant from those of antigenically similar salmonella serotypes, none of which seemed likely to be the progenitor of the 'porpoise' serotype.     

A comparison of antimicrobial susceptibilities in nontyphoidal salmonellas from humans and food animals in England and Wales in 2000

A joint study by the Public Health Laboratory Service and the Veterinary Laboratories Agency of resistance to antimicrobials in isolates of Salmonella enterica serotypes Enteritidis, Typhimurium, Hadar, and Virchow from humans and food-producing animals in England and Wales in 2000 has demonstrated that resistance was most common in Typhimurium, particularly in strains of definitive phage type (DT) 104. However resistance was also common in other phage types, particularly DTs 193 and 208 and phage type U302. Multiresistant strains of DT208 appeared to be predominantly associated with pigs; for the other phage types, the human/food-producing animal relationships of drug-resistant isolates were more complex. For Enteritidis, Virchow, and Hadar, there were substantial differences in the resistance spectra of isolates from humans and food-producing animals, suggesting that food-producing animals bred in England and Wales may not be the primary sources of drug-resistant strains of these serotypes causing infections in humans. Further phenotypic and molecular comparison of drug-resistant isolates of these serotypes may be required to ascertain the sources of strains responsible for infections in humans.     

Pilot study to evaluate microarray hybridization as a tool for Salmonella enterica serovar Typhimurium strain differentiation

In developed countries, Salmonella enterica subspecies 1 serovars Enteritidis and Typhimurium range among the most common causes of bacterial food-borne infections. The surveillance and typing of epidemic Salmonella strains are important tools in epidemiology. Usually, Salmonella enterica subspecies 1 serovars are differentiated by serotyping for diagnostic purposes. Further differentiation is done by phage typing as well as molecular typing techniques. Here we have designed and evaluated a prototype DNA microarray as a tool for serovar Typhimurium strain differentiation. It harbors 83 serovar Typhimurium probes obtained by differential subtractive hybridization and from the public database. The microarray yielded reproducible hybridization patterns in repeated hybridizations with chromosomal DNA of the same strain and could differentiate five serovar Typhimurium reference strains (DT204, DT104, DT208, DT36, and LT2). Furthermore, the microarray identified two distinct groups among 13 serovar Typhimurium DT104 strains. This correlated with observations from pulsed-field gel electrophoresis analysis. Twenty-three further serovar Typhimurium strains were analyzed to explore future directions for optimization of the simple 83-probe DNA microarray. The data presented here demonstrate that DNA microarrays harboring small numbers of selected probes are promising tools for serovar Typhimurium strain typing.     

Involvement of a Salmonella genomic island 1 gene in the rumen protozoan-mediated enhancement of invasion for multiple-antibiotic-resistant Salmonella enterica serovar Typhimurium

Multiple-antibiotic-resistant Salmonella enterica serotype Typhimurium is a food-borne pathogen that may be more virulent than related strains lacking the multiresistance phenotype. Salmonella enterica serotype Typhimurium phage type DT104 is the most prevalent of these multiresistant/hypervirulent strains. Multiresistance in DT104 is conferred by an integron structure, designated Salmonella genomic island 1 (SGI1), while we recently demonstrated DT104 hyperinvasion mediated by rumen protozoa (RPz) that are normal flora of cattle. Hyperinvasion was also observed in other Salmonella strains, i.e., other S. enterica serovar Typhimurium phage types and other S. enterica serovars, like S. enterica serovar Infantis, possessing SGI1, while DT104 strains lacking SGI1 were not hyperinvasive. Herein we attempted to identify SGI1 genes involved in the RPz-mediated hyperinvasion of Salmonella strains bearing SGI1. Transposon mutagenesis, coupled with a novel reporter system, revealed the involvement of an SGI1 gene previously designated SO13. Disruption of SO13 expression led to an abrogation of hyperinvasion as assessed by tissue culture invasion assays and by bovine challenge experiments. However, hyperinvasion was not observed in non-SGI1-bearing strains of Salmonella engineered to express SO13. That is, SO13 and another SGI1 gene(s) may coordinately upregulate invasion in DT104 exposed to RPz.     

Characterization of Salmonella enterica serovar Typhimurium and monophasic Salmonella serovar 1,4,[5],12:i:- isolates in Thailand

Duplex PCR was developed to screen Salmonella enterica serovar Typhimurium phage type DT104 and related strains in Thailand because a phage typing laboratory of serovar Typhimurium is not available. Of 46 isolates of serovar Typhimurium and 32 isolates of S. enterica serovar 1,4,[5],12:i:-, 15 (33%) and 30 (94%) were duplex PCR positive, respectively. All isolates were submitted for phage typing to analyze the specificity of the PCR assay. Among serovar Typhimurium isolates that yielded positive duplex PCRs, only seven isolates were phage types DT104 or U302, and eight isolates were undefined types, whereas the negative PCR isolates were either other phage types, including DT7, DT12, DT66, DT79, DT166, DT170, DT193, and DT208 or an undefined type. The serovar Typhimurium and serovar 1,4,[5],12:i:- isolates that were duplex PCR positive were further subtyped by using XbaI PFGE to reveal their genetic relatedness. All serovar Typhimurium phage type DT104 strains had indistinguishable chromosomal patterns. The isolates of phage type U302 and most of the serovar 1,4,[5],12:i:- isolates that were duplex PCR positive yielded similar pulsed-field gel electrophoresis patterns. The patterns of PCR-negative isolates distinctly differed from the patterns of PCR-positive isolates. A total of 26% of all isolates had a dominant R-type ACSSuTG that was not found in the isolates of phage type DT104.