Genomic diversity within phage types of Salmonella enterica ssp. enterica serotypes Enteritidis and Typhimurium

The diversity among 1354 strains of Salmonella enterica subsp. enterica, serotype Enteritidis (n = 847) and Typhimurium (n = 507) isolated in Finland in 1991-2002 (n = 608) and in 2003 (n = 746) were studied. The former strains were studied retrospectively by phage typing and pulsed-field gel electrophoresis (PFGE) harmonized in the European Salm-gene project. The latter strains were studied prospectively, and the results correlated to their antimicrobial susceptibility and association with travel to popular tourist destinations. During both periods, S. Enteritidis phage types (PTs) PT1 and PT4, and S. Typhimurium definite types (DTs) DT1 and DT104 were the major phenotypes. SENTXB.0001 was the dominating single PFGE type among S. Enteritidis strains (40% in 1991-2002; 57% in 2003), and accounted correspondingly for 23% and 63% of the PT1 strains, and 81% and 88% of the PT4 strains. No PFGE types dominated among the S. Typhimurium strains but a correlation was found between certain phage and PFGE types: among DT1 strains, STYMXB.0098 accounted for 66% (1991-2002) and 98% (2003) and among the DT104 strains STYMXB.0001 accounted for 84% and 97% in the two time periods, respectively. Of the S. Enteritidis strains isolated in 2003, 91% were associated with travel, most commonly to Spain, Greece, and Bulgaria. SENTXB.0001 was the major Salmonella PFGE type in these countries. In contrast, most (55%) S. Typhimurium strains were of domestic origin. While only 1.3% of the S. Enteritidis strains were multiresistant and 24% were resistant to nalidixic acid only, 30% of the S. Typhimurium strains were multiresistant. Among the multiresistant S. Typhimurium strains, R-type ACSSuT and PFGE type STYMXB.0001 of the DT104 complex dominated.     

Antimicrobial susceptibility and phage types of Salmonella enteritidis strains isolated in the Lublin area

The study aimed at specifying the value of two traditional methods of typing, using the collection of 241 strains of Salmonella Enteritidis isolated from people and animals in the Lublin area in 1994-1995 from the occasional cases of infections. There were 8 phage types identified among the examined strains. Phage types PT 6 (40.24% of strains) and PT 7 (29.46%) were the most numerous ones. Salmonella Enteritidis was numbered among 38 profiles on the basis of the analysis of resistance to 15 antimicrobial agents. It was found that nitrofurantoin had the lowest efficacy in vitro in relation to the examined collection of Salmonella isolates. High percentage of strains were characterized by lack of susceptibility to streptomycin, neomycin, cefixime, tetracycline and canamycine. While analyzing the profiles of resistance to chemiotherapeutics and the expression of phage type of the examined strains it was observed that only 14.1% of all strains showing resistance to nitrofurantoin represented at the same time phage type PT 6, whereas 10.8% of strains belonging to the same resistance profile indicated PT 7 phage type. In correlated analysis of resistance to chemiotherapeutics and phage type expression of the examined collection of 241 isolates of Salmonella Enteritidis, 18.3% of strains showed distinct and unique set of these two features, which can be used in epidemiological investigations for the preliminary characterization of the bacterial population.     

2011-2012年杭州市肠道沙门菌临床分离株的分子型别分析

目的研究2011-2012年杭州市肠道沙门菌临床分离株的型别,了解本地菌株分子流行病学特征。方法对66株肠道沙门菌临床分离株进行血清分型和多位点序列分型(MLST)。对其中主要血清型:鼠伤寒、甲型副伤寒、萨雷甲尼和肠炎沙门菌菌株进行脉冲场凝胶电泳(PFGE)分型。结果分布于21个血清型的66株沙门菌分成26个ST型别。发现一株纽波特沙门菌为新型ST1690。菌株血清型与MLST型别数据库中所对应的血清型符合率为100.00%。9株甲型副伤寒沙门菌的PFGE带型完全一致(P7型),与先前杭州流行菌株有差异(P1-P6型)。6株肠炎沙门菌分成4个PFGE型,型间最小相似性为92.70%。13株鼠伤寒沙门菌分为11个PFGE型,型间最小相似性为71.70%。7株萨雷甲尼沙门菌分成4个PFGE型别,型间最小相似性为91.00%。结论近年杭州腹泻病人中流行的肠道沙门菌菌株主要血清型为鼠伤寒、甲型副伤寒、萨雷甲尼和肠炎等。甲型副伤寒沙门菌菌株在杭州出现了新PFGE型别。MLST数据可以对沙门菌血清学鉴定提供一定的帮助。     

An outbreak of human salmonellosis caused by ampicillin-resistant Salmonella enterica serovar Enteritidis PT13 in the Czech Republic

In summer 2004, an outbreak caused by Salmonella enterica serovar Enteritidis phage type 13 (S. Enteritidis PT13) was recorded in the Czech Republic. As well being a relatively rare phage type the strain was also ampicillin resistant. Outbreak (n=39) and pre-outbreak isolates (n=13) were characterized by pulsed-field gel electrophoresis (PFGE), beta-lactamase gene polymerase chain reaction and plasmid profile. The majority of outbreak isolates (n=37) were identical in XbaI PFGE profile, and two other outbreak isolates each differed from this profile by one or two fragments respectively. The pre-outbreak isolates were uniform in PFGE profile but distinct from the outbreak strain. Ampicillin resistance was confirmed to be encoded by the blaTEM gene located on the TnA transposon. This gene was readily transferable to a S. Enteritidis recipient strain and was associated with the transfer of a 200-kb plasmid. Our results indicate that all S. Enteritidis PT13 tested from 2004 belonged to a single outbreak strain which prior to 2004 had not been recognized in the Czech Republic.     

Relationship of pulsed-field profiles with key phage types of Salmonella enterica serotype Enteritidis in Europe: results of an international multi-centre study

Salmonella is one of the most common causes of foodborne infection in Europe with Salmonella enterica serovar Enteritidis (S. Enteritidis) being the most commonly identified serovar. The predominant phage type for S. Enteritidis is phage type (PT) 4, although PT 8 has increased in incidence. Within these phage types, pulsed-field gel electrophoresis (PFGE) provides a method of further subdivision. The international project, Salm-gene, was established in 2001 to develop a database of PFGE profiles within nine European countries and to establish criteria for real-time pattern recognition. It uses DNA fingerprints of salmonellas to investigate outbreaks and to evaluate trends and emerging issues of foodborne infection within Europe. The Salm-gene database contains details of about 11 700 S. Enteritidis isolates, demonstrating more than 65 unique PFGE profiles. The clonal nature of S. Enteritidis is evidenced by the high similarity and distribution of PFGE profiles. Over 56% (6603/11 716) of the submitted isolates of several different phage types were profile SENTXB.0001, although this profile is most closely associated with PT 4. The next most common profiles, SENTXB.0002 and SENTXB.0005, were closely associated with PT 8 and PT 21 respectively. Studies to investigate the relationship of profile types with outbreaks and possible vehicles of infection suggest that the incidence of PFGE profile SENTXB.0002, and thus PT 8, in some countries may be due to importation of foods or food production animals from Eastern Europe, where PT 8 is amongst the most frequently identified phage types. Collation of subtyping data, especially in the commonly recognized phage types, is necessary in order to evaluate trends and emerging issues in salmonella infection.     

Differentiation of Salmonella enteritidis isolates by fluorescent amplified fragment length polymorphism

Salmonella Enteritidis is responsible for human gastroenteritis outbreaks worldwide, and the molecular characterization of isolates is an important tool for epidemiological studies. Fluorescent amplified fragment length polymorphism (FAFLP) analysis was performed on 31 Salmonella Enteritidis strains from South Brazil isolated from human, foods, swine, broiler carcasses, and other poultry-related samples to subtype isolates in comparison to pulsed-field gel electrophoresis (PFGE) analysis. Five strains of Salmonella Enteritidis from different geographical regions, Salmonella Enteritidis ATCC 13076, and four isolates of different Salmonella serovars were also tested. Among the 41 isolates tested, 96 polymorphic AFs and 40 distinct profiles were obtained, displaying a Simpson's index of diversity of 0.99; whereas the PFGE analysis presented 13 patterns and the resulting Simpson's index was 0.55. Nine FAFLP and seven PFGE clusters could be inferred based in Dice similarity coefficient. FAFLP clustering readily identified different serotypes of Salmonella but did not distinguish isolates epidemiologically nonrelated or distinct phage types. Therefore, these results indicate that FAFLP is a rapid method for epidemiological investigations of Salmonella outbreaks, presenting a high discriminatory power for subtyping of Salmonella Enteritidis.     

Molecular epidemiology of Salmonella enteritidis phage type 1b and 6a isolates in Portugal

Salmonella enterica serotype Enteritidis is an important serovar comprising 76 % of Salmonella isolates in Portugal in 2001. For better understand the epidemiology of salmonellosis, a total of 47 isolates of S. Enteritidis phage type (PT) 1b and 6a were analysed by pulsed-field gel electrophoresis (PFGE) and genomic DNA was subjected to macro restriction with XbaI. For PT 1b isolates, only three different patterns were observed, and PT6a showed a total of 10 digestion patterns. Curiously, the main pattern among PT1b isolates seams quite similar to main pattern of PT6a isolates, but when the two patterns were analysed with Bionumerics, we observed that they exhibited some differences. It was concluded that, in 2001, there was one predominant pattern for PT1b and PT6a and, possibly, we were in presence of clonal strains that exists all over the country.     

Phage type conversion in Salmonella enterica serotype Enteritidis caused by the introduction of a resistance plasmid of incompatibility group X (IncX)

The plasmid pOG670, a 54 kb, conjugative plasmid that specifies resistance to ampicillin and kanamycin and belonging to the incompatibility group X (IncX), was transferred into 10 isolates of Salmonella enterica serotype Enteritidis belonging to 10 different phage types (PT1, 2, 3, 4, 8, 9, 9b, 10, 11 and 13). Acquisition of the plasmid by these strains did not result in the loss of any resident plasmids but resulted in phage type conversion in 8 of the 10 strains (PT1, 2, 4, 8, 9, 9b, 10 and 11). The observed changes in phage type were found to result from the loss of sensitivity to 3 of the 10 typing phages used (phages 3, 5 and 7). Where the conversion resulted in a change to a defined phage type, both the new and original PTs belonged to the same, previously described, evolutionary lines. Enteritidis PTs 1, 4 and 8, commonly associated with poultry world-wide, were converted to PTs 21, 6 and 13a respectively. The results indicate a different route for phage type conversion Enteritidis from others reported in the literature and, although IncX plasmids are not normally present in PT8 or PT13a, may suggest a possible mechanism/link connecting these phage types.     

Epidemiological analysis of Salmonella enterica Enteritidis isolates in Japan by phage-typing and pulsed-field gel electrophoresis.

Salmonella enterica serotype Enteritidis isolates of phage types (PTs) PT1, PT4, PT13a and PT22 derived from sporadic cases and outbreaks of food poisoning in Japan during 1994 and 1995 were analysed by pulsed-field gel electrophoresis (PFGE). While PT1 strains from 5 different outbreaks showed 14 PFGE patterns, 5 PFGE patterns were observed among PT4 isolates from 5 different outbreaks and 6 independent isolates from imported chicken. Interestingly, 8 out of 9 PT4 strains associated with foreign travel to Southeast Asia were indistinguishable in PFGE pattern from 5 independent isolates of imported chicken from England. Although both PT13a and PT22 were first reported in Japan in 1994, PT22 showed various PFGE patterns compared to PT13a which had the same pattern within an outbreak, unlike PT1. These results could indicate that multiple clonal lines of PT1 and PT22 had already spread while relatively fewer clonal lines of PT4 and PT13a might exist in Japan.     

Occurrence of Salmonella enterica serotype Enteritidis phage types in the Slovak Republic

Phage typing of 741 isolates of Salmonella enterica serotype Enteritidis from the slovak Republic in 1995 has been carried out using the scheme of Ward and colleagues (1987). 202 strains (51 isolated from food) were from 9 outbreaks, 536 isolates were from sporadic cases and 3 isolates were from nosocomial infections of new-born babies. 704 isolates (95%) from all sources were typeable and belonged to 7 different phage types (PTs). PT8 was the phage type most frequently identified (72.6%). Other epidemiologically relevant phage types were PT2 (8.5%), 4 (7.9%) and 1 (4.2%). With an incidence of 1.3--0.1% isolates of PTs 21, 6, 9 were found. 26 (3.5%) isolates were designated RDNC and 11 (1.5%) were untypeable.     

Subdivision of Salmonella enteritidis phage types by plasmid profile typing

Differentiation of Salmonella enteritidis by plasmid profile typing has been compared to differentiation by phage typing. Examination of the type strains of the 27 S. enteritidis phage types showed that only 11 profile patterns could be identified. Moreover, two profile patterns were found in 15 of the type strains, including those of the two most common phage types in Britain, types 4 and 8. On this basis, plasmid profile typing is not as sensitive as phage typing for the primary subdivision of S. enteritidis. When differentiation of 534 strains of the 27 phage types was attempted using plasmid profiles, variation in pattern suitable for epidemiological subdivision was found in 13 phage types and there were 9 profile patterns in strains of phage type 4. Plasmid profile typing can, therefore, be regarded as an effective adjunct to phage typing for the subdivision of S. enteritidis.     

Tandem repeat analysis for surveillance of human Salmonella Typhimurium infections

In Denmark, as part of the national laboratory-based surveillance system of human enteric infections, all Salmonella enterica serotype Typhimurium isolates are currently subtyped by using phage typing, antimicrobial resistance profiles, and pulsed-field gel electrophoresis (PFGE). We evaluated the value of real-time typing that uses multiple-locus variable-number tandem-repeats analysis (MLVA) of S. Typhimurium to detect possible outbreaks. Because only a few subtypes identified by PFGE and phage typing account for most infections, we included MLVA typing in the routine surveillance in a 2-year period beginning December 2003. The 1,019 typed isolates were separated into 148 PFGE types and 373 MLVA types. Several possible outbreaks were detected and confirmed. MLVA was particularly valuable for discriminating within the most common phage types. MLVA was superior to PFGE for both surveillance and outbreak investigations of S. Typhimurium.     

Simultaneous infection with three different S. enteritidis strains in a nursing home resident

A culture taken from a nursing home resident as part of a S. enteritidis outbreak was found to have a mixed infection due to three different strains of S. enteritidis. One of the three strains belonged to phage type (PT) 4, one to PT6 and one reacted with phages but did not conform to any typing scheme (RDNC). All three strains had the 38.9 megadaltons (MDa) plasmid found in the isolates from the outbreak-related cases, in addition the PT6 and RDNC strains harboured a 69.9 MDa plasmid. The importance of phage typing and plasmid analysis for S. enteritidis strain characterization and their epidemiologic and bacterial significance are discussed.     

Characterization and epidemiologic subtyping of Shiga toxin-producing Escherichia coli strains isolated from hemolytic uremic syndrome and diarrhea cases in Argentina

Argentina has a high incidence of hemolytic uremic syndrome (HUS); 12.2 cases per 100,000 children younger than 5 years old were reported in 2002. Shiga toxin (Stx)-producing Escherichia coli (STEC) is the primary etiologic agent of HUS, and STEC O157 is the predominant serogroup isolated. The main objective of the present work was to establish the phenotypic and genotypic characteristics of the STEC strains in general isolated from Argentine children during a prospective study and the clonal relatedness of STEC O157:H7 strains using subtyping techniques. One hundred and three STEC strains isolated from 99 children were included. The phenotypic and genotypic features were established, and a polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) was performed to determine stx2 variants. The clonal relatedness of E. coli O157 isolates was established by phage typing and pulsed-field gel electrophoresis (PFGE). The 103 STEC strains belonged to 18 different serotypes, and 59% were of serotype O157:H7. Stx2 was identified in 90.3%, and stx1 in 9.7%. Among the 61 STEC O157 strains, 93.4% harbored the stx2/stx2vh-a genes; PT4 (39.3%) and PT2 (29.5%) were the predominant phage types. Using PFGE with the enzyme XbaI, a total of 41 patterns with at least 80% similarity were identified, and seven clusters with identical profiles were established. Some of the clusters were further split by PFGE using BlnI as the second enzyme. Isolates with indistinguishable PFGE patterns were with one exception also indistinguishable by phage typing and stx genotyping. These findings confirmed that some isolates were genetically related. However, no epidemiological linkages were identified. STEC strains with different genotypes and belonging to diverse serotypes were isolated in Argentina. Some STEC O157 strains could not be distinguished by applying subtyping techniques such as PFGE and phage typing.     

Phage conversion in Salmonella enterica serotype Enteritidis: implications for epidemiology.

A model system for the study of phage conversion of Salmonella enterica serotype Enteritidis is reported. Temperate phages 1,2,3 and 6 from the phage typing scheme were used to convert several individually recognized phage types into others. Phage type 4 was converted to PT8, PT6a to PT4, PT6a to PT7, PT13 to PT13a and PT15 to PT11; some new phage lysis patterns were also detected. This model was used to examine the relationships between phage types within a previously defined clonal lineage, SECLIII, to establish whether or not Enteritidis like Salmonella enterica serotype Typhi and Salmonella enterica serotype Paratyphi B possessed type determining phages. We were able to convert PT1 to PT20, and PT15 to PT11.     

Molecular epidemiology of Salmonella enterica serovar Agona: characterization of a diffuse outbreak caused by aniseed-fennel-caraway infusion

During 2002-2003 increased numbers of notified salmonellosis due to S. enterica serovar Agona were observed in Germany. In order to understand the recent spread of this serovar and to trace the route of infection to its source, a new phage-typing scheme and pulsed field gel electrophoresis (PFGE) were used to analyse these isolates. By using 14 bacteriophages, 52 phage types were distinguished among the S. Agona strains. PFGE also differentiated 52 different patterns. A combination of both methods generated 94 clonal types among 165 S. Agona strains originating from Germany and other countries including the United States, United Arab Emirates, Turkey, India, Austria and Finland, indicating a great biological diversity within this serovar. However, 36 recent S. Agona isolates from infantile gastroenteritis in Germany, from an untreated batch of aniseed imported from Turkey and from fennel-aniseed-caraway infusion (packed in tea bags) revealed clonal identity indicating their epidemiological relatedness as a new source of infection. It is suggested that strains of S. Agona will continue to be of public health concern, and that phage typing together with PFGE typing should be applied as reliable and rapid tools for epidemiological subtyping and future monitoring.     

Molecular characterization of Salmonella enterica serotype Enteritidis isolates from humans by antimicrobial resistance, virulence genes, and pulsed-field gel electrophoresis

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major serovar associated with human salmonellosis. A total of 425 clinical S. Enteritidis isolates of human origin were collected between June 2009 and September 2010 from North Carolina. The isolates were further characterized for antimicrobial susceptibility, antimicrobial resistance coding determinants, virulence genes, and fingerprint profiles to determine whether they were similar or different to the S. Enteritidis strain responsible for the human outbreak due to consumption of contaminated eggs. Ten different antimicrobial resistance phenotypes were observed with the highest frequency of resistance exhibited to ampicillin (n=10; 2.35%). The isolates were predominantly pansusceptible (n=409; 96.23%); however, seven isolates were multidrug resistant (MDR; i.e., resistant to three or more antimicrobials). Extended spectrum β-lactamase (ESBL) coding genes (bla(TEM) and bla(PSE)) were detected in the ampicillin-resistant isolates, whereas a single MDR isolate tested positive for class 1 integron (1 kb). The majority of the isolates (n=422; 99.3%) carried the invA, mgtC, stn, sopB, sopE1, and sefA virulence genes. However, 37 (8.7%) and 46 (10.82%) S. Enteritidis isolates tested negative for the plasmid encoded genes spvC and rck, respectively. Pulsed-field gel electrophoresis (PFGE) typing of 118 S. Enteritidis isolates by restriction enzymes XbaI and BlnI resulted in seven clusters, each with a discriminatory index (DI) of 0.715 and 0.785, respectively. The combination of XbaI-BlnI patterns generated a dendrogram with 14 clusters and a higher DI of 0.914. The PFGE profile of 80 isolates matched 100% with the S. Enteritidis strain that has been cited for the recent outbreak in the United States due to consumption of contaminated eggs. In conclusion, we identified a genotypic similar S. Enteritidis population in our study based on antimicrobial susceptibility, virulence gene, and PFGE fingerprint profiles.     

Molecular typing of Salmonella enterica serovars Enteritidis, Corvallis, Anatum and Typhimurium from food and human stool samples in Tunisia, 2001-2004

During the period from 2001 to 2004, a total of 72 isolates of Salmonella enterica serovars: Anatum (n=40), Enteritidis (n=18), Corvallis (n=8), and Typhimurium (n=6), of various origins (mainly food and diarrhoeagenic stool samples), were collected and further characterized by antibiotic resistance, plasmid analysis, and pulsed-field gel electrophoresis (PFGE). Forty-five isolates presented multidrug resistance to antibiotics. Among which one S. enterica serovar Anatum isolate was resistant to 11 antibiotics, and one S. enterica serovar Typhimurium DT104 isolate was resistant to eight antibiotics. Plasmid profiling identified eight plasmid profiles (with 1-5 plasmids) among the isolates, of which one plasmid profile (P01) was predominant. XbaI PFGE analysis revealed the presence of a predominant clone of the four studied Salmonella serovars circulating in Tunisia throughout the years 2001-2004.     

Genome fingerprinting of Salmonella typhi by pulsed-field gel electrophoresis for subtyping common phage types.

The genomic DNA of 39 strains of Salmonella typhi isolated from local residents and patients who had visited countries in the Asian region was analysed for restriction fragment length polymorphisms (RFLP). Pulsed-field gel electrophoretic (PFGE) analysis of Xba I- and Spe I-generated genomic restriction fragments established 22 PFGE types whereas phage typing differentiated the 39 isolates into 9 distinct phage types. This study showed that PFGE is more discriminatory than phage typing as it is capable of subtyping S. typhi strains of the same phage types. Genetic relatedness among the isolates was determined. Seven major clusters were identified at SABs of > 0.80 and the remaining 13 isolates were distributed into minor clusters which were related at SABs of less than 0.80. In conclusion, PFGE analysis in conjunction with distance matrix analysis served as a useful tool for delineating common S. typhi phage types of diverse origins from different geographical locales and separated in time.     

Phage typing of Campylobacter jejuni and Campylobacter coli and its use as an adjunct to serotyping.

Campylobacter is the most commonly reported cause of gastro-intestinal infection in England and Wales, with over 50,000 reported cases in 1997. The majority of human campylobacter isolates in England and Wales are C. jejuni (c. 90%) with most of the remainder being C. coli. We describe the use of phage typing as an extension to serotyping for more detailed characterization within these two species. The scheme was piloted during a study of 2407 C. jejuni and 182 C. coli strains isolated in Wales between April 1996 and March 1997. Fifty-seven C. jejuni phage types were identified, with the ten most prevalent phage types accounting for 60% of isolates tested; 16% of isolates were untypable. The most common phage type was PT 1 which represented c. 20% of isolates. A further 7% of isolates reacted with the phages but did not conform to a designated type (RDNC). Only 12 phage types were identified among C. coli, with the two most common types, PT 2 and PT 7 accounting for 75.2% of isolates. When used in conjunction with serotyping, the ability of phage typing to identify between 6 and 29 subtypes within each of the predominant HS types has enabled a further level of discrimination to be achieved that enhances the epidemiological typing of C. jejuni and C. coli.