Serotyping and molecular typing of Salmonella species isolated from wastewater in Nsukka , Nigeria

The objective of this investigation was to isolate and identify Salmonella serovars present in wastewater from the University of Nigeria, Nsukka (UNN) wastewater treatment plant and to evaluate the sensitivity and precision of different microbial typing methods (conventional and molecular), in identifying and characterizing Salmonella species. A total of 100 suspected Salmonella colonies on selective media (Salmonella-Shigella agar and MacConkey Agar) were subjected to biochemical testing. A total of 12 biochemically typical Salmonella isolates were identified and further characterized. Serotyping analysis further identified 3 (25%) of the isolates as Salmonella enterica serovar Limete. Salmonella specific (16S) polymerase chain reaction (PCR) assay validated the result obtained by serotyping, although 2 of the isolates could not be serotyped and were identified as rough strains. PCR assay produced positive amplifications of 574 bp of the 16S rRNA gene specific for Salmonella, while non-Salmonella serovars were negative (100%). Random amplified polymorphic DNA (RAPD-PCR) analysis revealed the genetic relatedness of Salmonella serovars isolated from wastewater. Primers 787 and RAPD2 identified 4 RAPD-binding patterns, while primer 1254 did not give any discriminatory pattern. Molecular analyses (16S PCR and RAPD) showed discriminatory power, reproducibility, easy interpretation and performance. It is therefore a promising alternative method for typing Salmonella species.


INTRODUCTION
Wastewater may contain millions of bacteria per milliliter including coliforms, Streptococci, Staphylococci, anaerobic spore forming bacilli, Proteus and many other types of organisms.Wastewater is also a potential source of many human pathogens including bacteria, viruses and protozoa (Younis et al., 2003).The presence of microbial pathogens in polluted, untreated and treated water presents a considerable health risk to both humans and animals with far reaching socio-economic implications.Salmonella species have been frequently isolated from wastewater and are known to cause severe disease symptoms that range from self-limiting diarrhoea to bacteremia.They are the etiological agents of a wide range of diseases such as salmonellosis and typhoid *Corresponding author.E-mail: anthonyc.ike@unn.edu.ng.Tel: +234 706 631 7800.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License fever and are among the leading causes of gastroenteritis worldwide.There are 16 million annual cases of typhoid fever, 1.3 billion cases of gastroenteritis and 3 million deaths worldwide due to Salmonella (CDC, 2005;Bhunia, 2008).
Traditionally, detection and quantification of Salmonella species have been largely based on the use of selective culture and standard biochemical methods.This approach requires confirmatory test of all typical and atypical colonies and can be very cumbersome and time consuming (Whyte et al., 2002;Kumar et al., 2009).Since Salmonella is closely related to both human and animal health, more rapid and sensitive methods for the identification of this bacterium are required (Schrank et al., 2001).The use of DNA-based typing methods is becoming increasingly important in epidemiological survey and differentiation of Salmonella species.Some of these methods include pulsed field gel electrophoresis (PFGE) (Mohand et al., 1999;Kubota et al., 2005), PCR ribotyping (Lagatolla et al., 1996), automated nuclease PCR assay (Hoorfar et al., 2000) and random amplification of polymorphic DNA (RAPD) (Shangkuan and Lin, 1998;Smith et al., 2011).RAPD-PCR does not require any specific knowledge of the target DNA sequence, making it a flexible and powerful tool with general applicability.Furthermore, many oligonucleotide primer sets have been described for the detection of Salmonella using the PCR technique (Stone et al., 1994;Guo et al., 2000;Liebana et al., 2001).
In this study, the conventional system for typing Salmomella species (biotyping and serotyping) was evaluated and its performance was compared to molecular typing methods (16S and RAPD-PCR analysis).

Sample collection
A total of 40 wastewater samples were collected from the Imhoff tanks (A and B) and waste stabilization ponds (WSPs) (A and B) of the wastewater treatment plant of the University of Nigeria, Nsukka (UNN), over a period of six months (October 2012 -March 2013).Wastewater samples were collected in sterile bottles and immediately transported to the Microbiology Laboratory, UNN and examined for Salmonella.

Isolation and identification of Salmonella
Salmonella species were isolated and identified according to the standard methods for the examination of water and wastewater described by ISO (2002) andAPHA (2005).Forty milliliters (40 ml) of wastewater was centrifuged at 2000 rpm for 10 min, and then 10 ml of the supernatant was pre-enriched in 100 ml buffered peptone water (BPW) in a 250 ml Erlenmeyer flask and incubated at 37°C for 24 h.Pre-enrichment was followed by selective enrichment in Rappaport Vassilidiadis (RV) broth.Subsequently, selectively enriched samples from RV broth were streaked onto Salmonella-Shigella Agar (SSA) and MacConkey Agar.Plates were incubated at 37°C for 24 h.Presumptive Salmonella colonies were subjected to a set of biochemical tests for confirmation.

Serotyping
All biochemically typical Salmonella isolates were serotyped based on reaction with somatic (O), flagellar (H) and capsular (Vi) antisera (Difco, USA).Salmonella O and Vi antigens were identified by slide agglutination test procedure.After the confirmation of the individual Salmonella O antisera, cultures were further characterized for H (phase I and II) antisera based on Spicer-Edwards antisera by tube test procedure, whereas, L, EN and I complex antigens were identified separately.The antigenic formulae of Salmonella serovars as listed by Popoff and Le Minor (2005) were used to name the serovars.Serotyping was carried out at the WHO Collaborating Centre for Reference and Research on Salmonella at Pasteur Institut, Paris, France.

Preparation of genomic DNA
Bacterial DNA was extracted by boiling according to the method described by Medici et al. (2003).A single colony of a pure nutrient agar culture was grown overnight at 37°C in 1 ml Luria Bertani broth.Bacterial cells were pelleted by centrifugation at 13,000 rpm for 5 min in a microcentrifuge (Eppendorf, Germany).The supernatant was discarded and the pellet was re-suspended in 500 μl deionized distilled water.The suspension was boiled for 10 min in a heat block (Techne, Barloworld, UK) then immediately cooled on ice.Extracted DNA was then stored at 4°C until used.
The extracted chromosomal DNA was amplified by an established PCR technique (Sambrook et al., 1989).Bacterial DNA amplification was carried out in a 25 μl total volume of PCR mixture containing 2 µl of template DNA, 4 µl of the PCR Master Mix (Solis BioDyne, Estonia) (1X PCR buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 1U Taq Polymerase), 20 pmol of each primer (Table 1), and ddH2O to the total volume of 25µl.DNA amplification was done in an Eppendorf vapo protect thermocycler (Hamburg, Germany) following standardized cycling conditions (Table 2).
PCR products were resolved on 1.5% agarose gel, stained with ethidium bromide (0.5 µg/ml) and visualized with a photogel documentation system (Cjinx Science Instrument, USA).For RAPD-PCR, parameters in each reaction were optimized in order to maximize discriminatory power of the reaction.Also, for each PCR assay, a positive control (DNA from S. Typhi) and a negative control (sterile distilled water) were included.Appearance of the target band specified for each primer set under specified gel electrophoresis conditions was considered as a positive result.

Identification of Salmonella species from wastewater
The result showed that out of 100 Salmonella-like colonies on SSA and MacConkey agar, only 65 (65%) were found to be negative for urea utilization.Similarly, of the 65 urea negative isolates subjected to a set of biochemical reactions, 12 (18.4%)isolates were found to be consistent with results expected for Salmonella strains (result not shown).

Serotyping of Salmonella isolates from wastewater
Serotyping of Salmonella isolates from wastewater  revealed that 3/12 (25%) isolates were identified as Salmonella enterica belonging to subspecies 1, serogroup B, named S. enterica serovar Limete (Table 3).The result further demonstrated that 2 of the isolates could not be serotyped and were identified as rough strains.

Number and percentage of Salmonella isolated from wastewater
The result demonstrated that 7.5% of wastewater tested was contaminated with Salmonella during the period of study.A total of 3 Salmonella isolates were detected and identified, 2 from Imhoff tank A and 1 from WSP B as shown in Table 4.

Salmonella specific PCR (16S rRNA) assay
The specificity and sensitivity of the 16S rRNA PCR assay revealed that 3/12 (25%) of the standard microbiological tests confirmed isolates from wastewater had the desired amplification of 574 bp fragment, whereas no amplicons were observed for non-Salmonella serovars (Figure 1).

RAPD-PCR of Salmonella isolates from wastewater
The discriminatory power of the RAPD-PCR assay was tested by considering the number of profiles (RAPD binding patterns) generated using a set of 3 primers.The result of the RAPD-PCR revealed that primers 787 produced 3 similar bands and RAPD 2 produced 1 uniform band for the 3 isolates (Figure 2), making a total of 4 RAPD binding patterns on the basis of genetic relatedness.Primer 1254 did not produce any discriminatory pattern amongst the Salmonella isolates.

DISCUSSION
The result of this study revealed a low occurrence (7.5%) of Salmonella spp. in wastewater collected from the University of Nigeria, Nsukka wastewater treatment plant (WWTP).This is in consonance with the work done by El Hussein et al. (2012) in Khartoum State, Sudan, who reported an   showed that municipal wastewater having undergone an activated sludge process continued to bear Salmonella at high concentration; the treated water yielded a most probable number (MPN) of 45/100 ml.Also, El-Taweel (1994) found that Salmonella spp.were detected in raw wastewater samples at oxidation pond in Mit-Mzah treatment plant in Dakahlia governorate, Egypt, in numbers ranging from 10 2 -10 5 cfu/100 ml.The result of serotyping was in tandem with the result of the 16S rRNA-PCR assay, although 2 isolates could not be serotyped and were identified as rough strains.Kumar et al. (2009) reported that some of the Salmonella serovars isolated from seafood could not be serotyped and were identified as rough strains, lacking O-antigen.This reveals a major limitation of the serotyping technique in subtyping Salmonella spp.; as serogrouping is not possible when Salmonella isolates lack O-antigen (rough strain) or lack both O and H antigens (Hoorfar et al., 1999).This highlighted the necessity of more discriminatory methods to compliment traditional typing methods.However, since the PCR method could not also identify these as Salmonella, it is inferred that this may not have been a limitation in this study.
The specificity and sensitivity of the PCR assay to detect Salmonella spp.recovered from wastewater was investigated.The PCR produced positive amplifications of 574 bp of the 16S rRNA gene specific for Salmonella serovars, while non-Salmonella serovars were negative (100%).The results obtained were similar to those reported by Lin and Tsen (1996) and Chiu and Ou (1996).These investigators reported that 16S PCR technique using Salm 16S primer was able to identify all the examined Salmonella serovars, while all non-Salmonella serovars gave negative results.
There was no diversity among the strains analyzed by RAPD-PCR.The discriminatory power of the RAPD-PCR was tested by considering the number of profiles (RAPD binding patterns) generated using a set of 3 primers.The analysis of the RAPD-PCR revealed that primers 787 and RAPD 2 were found useful in typing Salmonella isolates and 4 RAPD patterns were observed among the isolates on the basis of shared amplified product showing relatedness of the isolates.Primer 1254 did not produce any discriminatory pattern amongst the Salmonella isolates and obviously no typing was possible.This result is in consonance with studies conducted in Lagos, Nigeria by Smith et al. (2011) and Akinyemi et al. (2014).These investigators demonstrated that RAPD-PCR using primer 1254 did not discriminate among Salmonella isolates.However, this is in contrast with the report of Quintanes et al. (2004) which recorded highest discriminatory power amongst clinical Salmonella isolates using Primers 784 and 1254 in Brazil.
Standardization of PCR mixtures and conditions are very important for reproducibility of RAPD-PCR results.It was found that it was necessary to perform RAPD-PCR in duplicates to obtain valid result.It is important to note that the interpretation of DNA fragment patterns generated by RAPD-PCR requires a good understanding of the occurrence of random genetic events, including point mutations, insertion and deletions of DNA, which can alter the RAPD fingerprinting pattern (Tenover et al., 1997).For this reason, it was presumed that the differentiating bands in the profiles could be due to one or more genetic events.These findings show that RAPD-PCR yields reliable and reproducible results under precise assay conditions.
The traditional phenotypic typing methods used (biotyping) showed low discriminatory power, while serotyping yielded inconclusive results in few samples.RAPD-PCR analysis showed the potential to provide a discriminatory, reproducible, low cost, easy to perform and interpret method to type Salmonella strains.Molecular typing or fingerprinting is an invaluable epidemiological tool that can be used to track the source of infection and to determine the epidemiological link between isolates from different sources.The combination of traditional and molecular typing methods may be the best approach to characterize Salmonella isolates.Younis M, Soleiman HA, Abou Elmagd K (2003).Microbiological and chemical evaluation of bentonite as a new technique for sewage water treatment, Aswan city, Egypt.7 th International Water Technology Conference, Egypt, 1-3 April.
from Imhoff tank A; B = Samples from Imhoff tank B; C = Samples from Waste stabilization pond A; D = Samples from waste stabilization pond B.Table 4. Number and percentage of Salmonella isolated from the wastewater treatment plant, UNN.

Table 1 .
Primer sequence and reaction parameters.

Table 3 .
Serotypes of Salmonella isolates from wastewater.