Application of phage typing and pulsed-field gel electrophoresis to analyse Salmonella enterica isolates from a suspected outbreak in Lagos , Nigeria

Introduction: Inadequate potable water supply and poor sanitation predispose to foodand water-borne diseases associated with Salmonella enterica serovars in developing countries. In this study the possible source of an unprecedented upsurge of Salmonella-associated community gastroenteritis was traced using both phage-typing and pulsed-field gel electrophoresis (PFGE). Methodology: Nineteen Salmonella Typhimurium (three sporadic isolates included) and 13 Salmonella Enteritidis isolates from clinical, animal, and environmental samples were subjected to antimicrobial susceptibility testing, phage-typing, and PFGE analysis using standard procedures. Results: Eleven (68.8%) of the 16 outbreak-related multidrug resistant S. Typhimurium belonged to DT 71 phage type with cluster PFGE type X3, representing the most prevalent strain identified among human, animal, and environmental isolates. The remaining five (31.2%) outbreak-related strains reacted but did not conform with clear phage types (RDNC) with cluster PFGE types X1 and X2 (96.8% similarity). Sporadic strains were untypable and belonged to X4 PFGE type. However, the evaluated S. Enteritidis strains that were multidrug resistant without a definite phage type belonged to PFGE cluster type X1e and were identified among the water and human strains. None of the Typhimurium and Enteritdis isolates was resistant to the fluoroquinolone antibiotics that were evaluated. Conclusion: This study emphasizes the epidemiological usefulness of PFGE typing in the detection of emerging strains of multipledrug resistant Salmonella, particularly S. Typhimurium DT71, that pose serious health implications in our environment. The study provides epidemiological links between environmental reservoirs and human infection in this community.


Introduction
Non-typhoidal salmonellosis is a major foodborne infection with an estimated annual incidence of 1.3 billion cases and over 2 million deaths worldwide [1].Salmonella enterica serovars Typhimurium and Salmonella Enteritidis have now become the predominant serotypes in many countries over the past 10 years [2,3].Most cases are associated with outbreaks from meat, dairy and poultry products, or by cross-contamination; however, sporadic cases also occur [4,5].In Africa, well water and water from rivers or lakes are usually used for public consumption; therefore, contamination by raw sewage sometimes remains an important factor in the epidemiology of typhoidal and, in rare cases, nontyphoidal salmonellosis [6].
In Nigeria, salmonella-associated ailments have been documented in different geographical locations in clinical settings [7][8][9][10].However, there are few reports on the circulating strains of non-typhoidal salmonellae and their significance, particularly S. Typhimurium and S. Enteritidis, in this environment.Traditionally, phenotypic methods such as serotyping, antibiogram, and phage typing have been used to characterize Salmonella isolates.Phage typing has been used with great success to trace the source of human salmonellosis [11][12][13].A number of molecular typing techniques such as pulsed-field gel electrophoresis (PFGE), ribotyping, and restriction fragment length polymorphism (RFLP) have been used to identify and differentiate strains below the level of serotypes because of the reliability and high discriminating power of these methods, particularly during outbreaks [14].
Random amplified polymorphic DNA (RAPD) has high discriminatory powers; however, it has poor inter-and intralaboratory reproducibility [15].PFGE has become the method of choice in the analyses of novel epidemic strains from salmonellosis outbreaks [16,17].Basically, PFGE targets the intact and large chromosomal DNA embedded in an agarose plug, and that is why PFGE is capable of producing a more highly reproducible and reliable result than other methods in tracing the source of outbreaks [14,16].In this study, we applied both phage typing and PFGE to investigate the diversity of S. Typhimurium and S. Enteritidis strains from humans, animals, and the environment in an upsurge of communityacquired Salmonella-associated illness in Lagos.

Bacterial agents
Thirty-two Salmonella isolates from human, animals and the environment were evaluated in this study.Out of these, 21 were human isolates, 18 of which were collected during from clinically diagnosed patients during an unprecedented upsurge of Salmonella-associated gastroenteritis in Okokomaiko, Ojo community, of Lagos State between December 2004 and January 2005.The three remaining samples were sporadic isolates, collected in 1997, 1999, and 2002.Eight environmental isolates were identified from sewage or well waters within a 500-metre radius of the cases.All the well waters (the major source of drinking water in this area) were owned by private individuals and are usually open to the general public.The remaining three Salmonella isolates were from fresh faeces of cows reared by nomadic Fulani people in this area.Preliminary identification of the isolates was conducted by standard methods [18].The isolates were maintained and stored at -4 °C in vials containing Muller-Hinton agar.The isolates were further identified biochemically using the API 20E System (BioMerieux, Marcy l'Etoil, France) and serotyped according to the latest versions of the Kauffmann-White scheme [19].

Antimicrobial susceptibility testing
All Salmonella isolates were investigated for their in vitro susceptibilities to 13 antibiotics by both disk diffusion and microdilution assay per Clinical and Laboratory Standard Institute (CLSI; formerly NCCLS) guidelines [20].

Phage typing
The bacteriophage typing was performed at the Robert Koch Institute, Wernigerode, Germany, using the system of Ward et al. [11] as applied in the Central Public Health Laboratory, London, United Kingdom, for S. Enteritidis, and the scheme of Anderson et al. [21] for S. Typhimurium, with slight modifications based on over five decades of laboratory procedure adopted by the Koch Institute.Isolates were subjected to purity control by incubation on Endo agar plates [22].Strains showing a pattern that did not conform to any recognized phage patterns were designated as "reacted but did not conform" (RDNC).Strains that did not react with any of the typing phages were designated as "untypeable" (UT).

Pulsed-field gel electrophoresis
PFGE was performed according to the standardized laboratory protocol for molecular subtyping of non-typhoidal Salmonella of Pulse-Net [23], with minor modifications as follows: 300 l of adjusted cell suspension was transferred to 1.5-ml micro-centrifuge tubes with 15 l of proteinase K (20mg/ml), and was subsequently mixed with 300 l of melted 1% SeaKem Gold (Cambrex, East Rutherford, N.J.) : 1% sodium dodecyl sulphate agarose prepared with TE buffer (10mM Tris, 1mM EDTA, pH 8.0), and pipetted into disposable plug moulds.Digested DNA plugs were loaded into the wells containing 1% pulsed-field agarose certified (Bio-Rad, Dusseldorf, Germany) in 0.5 X TBE (0.9 M Tris base, 0.9 M boric acid, 0.02 M EDTA, pH 8.0) extended range buffer (Bio-Rad, Dusseldolf, Germany).
DNA from Salmonella Braenderup H9812 restricted with Xbal was used as a size marker.PFGE was performed on CHEF DR-II and CHEF DR-III (Bio-Rad, Dusseldorf, Germany), and running conditions were 6 V/cm at 120 °C for 19.5 and 19 hours for S. Enteritidis and S. Typhimurium respectively, and switching times ranged from 2.2 sec to 63.8 sec.Gels were stained with 0.5 l/ml ethidium bromide for one hour, destained in water, observed under UV light, and photographed.Macrorestriction patterns were compared with the use of GelComparII (Applied Maths, Sint-Martens-Latem, Belgium) software.
The PFGE of Xbal-digested chromosomal DNA from 19 S. Typhimurium strains gave stable and reproducible patterns.The dendrogram analysis of the isolates showed three clusters of 4 PFGE (Figure 1).The first cluster consisted of PFGE types X1 and X2 with 96.8% similarity.The second comprised PFGE type X3, and the third was made up of PFGE type X4.The overall level of genetic relatedness for the strains was 71.0% similarity.PFGE type X3 (57.9%) was the most prevalent, identified among five (41.7%) human, two (100%) water, two (66.7%)sewage and two (100%) cattle isolates, and they were resistant to different antibiotics One strain from a water sample, which shared the same resistance pattern with a strain from a sewage sample, belonged to DT71/X3 phage/PFGE type (Table ).Five RDNC phage type strains (four from humans and one from sewage) exhibited A, Am, G, T, C, Sf, Tp, and Ce resistance patterns with or without Ct, and belonged to PFGE types X1 and X2 (Table and Figure1).The three sporadic strains which belonged to UT phage type presented PFGE type X4 and were resistant to ampicillin and/ orgentamicin.Five of the 13 S. Enteritidis strains were untypeable (UT), seven belonged to RDNC, and a strain which belonged to PT29 was isolated from water.Digestion of the 13 isolates with Xbal restriction enzymes revealed 11 to 16 fragments.The dendrogram of the isolates revealed three clusters of four PFGE (Figure 2).The first consisted X1e, the most prevalent type (61.5%), identified in seven of the eight (87.5%)human strains and a strain from sewage.The second comprised PFGE types X2e and X3e with 80% similarity, identified in two human isolates.The third PFGE type X4e was identified in one and two isolates of animal and water origins respectively.The level of genetic relatedness recorded for all the strains was 64% similarity (Figure 2).Most S. Enteritidis strains evaluated in this study were multidrug resistant and belonged to PFGE type X1e (Table ).

Discussion
Epidemiological investigations for routine surveillance and for examining the strains that cause disease outbreaks are important in medical settings.Such data allow public health officials the ability to determine if the causative organisms are part of transient or resident populations.The results of phage typing of 16 S.Typhimurium outbreak-related strains revealed that 68.8 % belonged to DT71, identifying it as the most prevalent phage type in this study.DT71 Typhimurium is a rarely reported phage type in literature and this is the first of its kind in Nigeria, as reported in our previous publication [24].In an African country such as Kenya, genetic variations among different phage types in S. Typhimurium strains have been documented, thus suggesting diversification of serovar Typhimurium [25].
PFGE analysis of 19 S. Typhimurium isolates (including three sporadic isolates) revealed a high degree of genetic homogeneity among isolates of the same phage type, with PFGE type X3 predominating, and featuring in 11 (57.9%) of the isolates.Interestingly, 11 (68.8%) of the 16 suspected outbreak-related strains belonged to DT71 phage type and were genetically related to common PFGE type X3.However, five of the 11 strains with the DT71/X3 combination were isolated from patients.Two DT71 strains each isolated from well water, cattle, and sewage were found to share the same PFGE type X3 with these humans strains (Table ).This observation was an indication of sewage as a possible source and well water as a route of transmission of infection in five of the patients with gastroenteritis the community affected in the suspected outbreak.None of the strains in this study was resistant to ciprofloxacin, ofloxacin and perfloxacin antibiotics, but a major concern of S.
Typhimurium DT71 is the multiple drug resistance exhibited, particularly among the human strains (Table ).Like S. Typhimurium DT104, which is chromosomally encoded penta-drug resistant (26), the infection with DT 71 seems to follow a severe clinical course because all five patients involved in the suspected outbreak suffered acute gastroenteritis that lasted between five and eight days, an indication of a threat posed by S. Typhimurium DT71 to public health.Furthermore, a strain (NST-10) isolated from another sewage source within a 500 m radius of the cases shared common phage type RNDC with four strains isolated from the patients with gastroenteritis in this community who were affected in the suspected outbreak.However, RDNC phage type has been reported among S. Typhimurium isolates in a study conducted by Onyango et al. (25) in Maseno, Kenya.All the RDNC strains in this study belonged to two closely related PFGE types X1 and X2 (96.8% similarity) with a difference of one fragment in their binding patterns, and they were resistant to seven or more antibiotics (Table ).The slight difference observed in the two PFGE types among the five strains may be due to a small genetic element (e.g., plasmid) possibly acquired by the strains, or perhaps to small insertion or deletion events that had taken place within a short period.The appearance of different genovars within Salmonella serovars due to insertion and deletion events have been reported to occur within a relatively short time; at the same time, factors such as (i) serogroup, (ii) host specificity, and (iii) disease characteristics are known to influence the clustering of isolates [27].In fact, fingerprinting by PFGE indicated that all three sporadic strains (NST03, NST04 and NST14) belonged to the same cluster of PFGE type X4 (Figure 1).Since these strains were isolated from cases of human gastroenteritis at distant periods, this result may further attest to the sensitivity of PFGE in discriminating between epidemiologically unrelated and outbreak-related S. Typhimurium strains [13].
Xba1 analyses of 13 suspected outbreak-related S. Enteritidis strains revealed three clusters of four PFGE types.PFGE X1e was the predominant type, identified in eight of 13 S. Enteritidis strains, seven of which were from human origins and one strain (NST17) from a water sample.The eight S. Enteritidis strains were found to be identical by PFGE and were believed to have derived from a common source, an indication that the well water, where the NST17 strain was isolated, served as a possible source of the S. Enteritidis-associated gastroenteritis.Also, a strain (NST08) from cattle within the same sampling zone was genetically different but highly related to two strains (NST07 and NST21) isolated from other well water samples in the same area.It has been documented that indistinguishable strains could be recovered from a large number of sources [28].What is interesting is that most of the S. Enteritidis strains are either RDNC or untypeable by phage, which could be a new phage type causing a micro-epidemic in this community.Only one of the strains of S. Enteritidis studied belonged to a defined phage type PT29 and was isolated from well water within a 500 m radius of the cases.The fact that PT29 was not isolated from any of the patients affected in the supposed outbreak did not rule out the health hazard associated with this strain in this community.
The present study was hospital-based and close observations showed that most of the patients had been on antibiotic self-medication.In other parts of the world, different phage types appear to predominate in S. Enteritidis.For example, PT4 is known to be the most common in England [11], Germany [29], and Italy [30], while PT6 is most common in Denmark, PT8 in Poland and Slovak Republic, PT1 in Russia and Hungary, and PT8 and PT 13a in the United States of America [31,32].Generally, lack of data on Salmonella phage types in Nigeria make the basis for comparison difficult and thus one could underestimate the prevalence and risk associated with these strains of Salmonella isolates; however, phage profiles categorized as types 1, 3 and 5 in S. Typhimurium have been documented in Kenya [25].
In conclusion, despite the small sample size, our finding revealed that PFGE to a certain extent was able to trace and link the upsurge in the Salmonella-associated gastroenteritis in this community to well waters and a dirty environment.With regard to future works, it is important to confirm the true extent of genetic diversity among human, animal and environmental isolates of Salmonella serotypes circulating in Nigeria and other parts of the world with a view to identifying emerging strains such as S. Typhimurium DT71 and P29 S. Enteritidis that are becoming more relevant in public health.

Figure 1 .
Figure 1.Dendrogram (Left panel) and DNA fingerprints (Right panel) of 19 S. Typhimorium strains generated by the GelCompar II software

Table 1 .
PFGE patterns, phage types and resistance profiles of Salmonella enterica serovars Typhimurium and Enteritidis from humans, animals and environment.