Abstract
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. Key words: Wastewater, Salmonella, serovars, serotyping, polymerase chain reaction (PCR), 16S rRNA, random amplified polymorphic DNA.
Highlights
Wastewater may contain millions of bacteria per milliliter including coliforms, Streptococci, Staphylococci, anaerobic spore forming bacilli, Proteus and many other types of organisms
Random amplified polymorphic DNA (RAPD-polymerase chain reaction (PCR)) analysis revealed the genetic relatedness of Salmonella serovars isolated from wastewater
Forty milliliters (40 ml) of wastewater was centrifuged at 2000 rpm for 10 min, and 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
Summary
Wastewater may contain millions of bacteria per milliliter including coliforms, Streptococci, Staphylococci, anaerobic spore forming bacilli, Proteus and many other types of organisms. 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). The use of DNA-based typing methods is becoming increasingly important in epidemiological survey and differentiation of Salmonella species. 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)
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