Detection , Identification and Characterization of Staphylococci in Street Vend Foods — Characterization of Staphylococcus Isolates

In the present investigation the diversity of the Staphylococcus species in different street vend food samples was studied. A total of 35 staphylococcal food isolates comprising of various species from 14 different street vend food samples were identified and characterized phenotypically. Staphylococcus aureus was found to be the most prevalent species in these foods. A PCR-RFLP analysis based on 16S rRNA gene was used for identification of Staphylococcus species. Isolates showing distinct RFLP pattern for AluI restriction digestion were selected for nucleotide sequence analysis. Phylogenetic tree constructed using the multiple alignments of 16S rRNA gene sequences of isolates showed a hotspot region of 169 bp and the relationship among species was evaluated by bootstrap values generated in phylogenetic analysis. 16S rRNA gene sequences allowed bacterial identification that was reproducible and more accurate than that obtained by phenotypic testing. 16S rRNA gene sequence analysis would be helpful in timely and correct identification of pathogens.


Introduction
Members of Staphylococci are wide spread in nature and have been isolated sporadically from a wide range of environmental sources such as air, water, soil and plant surfaces, meat, poultry and dairy products [1].They are capable of causing mild to life threatening diseases, which also includes food borne illnesses.Several species in this genus are having capability to produce a wide range of heat stable enterotoxins, and the main members of this genus, S. aureus is considered the third most important cause of foodborne diseases in the world among the reported foodborne pathogens [2][3][4].
Several Staphylococcus species other than S. aureus are reported to produce enterotoxins [5].The coagulase positive S. hyicus and S. intermedius are the predominant non-S.aureus species, which have been shown to produce staphylococcal enterotoxins (SEs) and are involved in staphylococcal food poisoning outbreaks [6][7][8][9].Among the coagulase negative species, S. cohnii, S. epidermidis, S. xylosus and S. haemolyticus have been isolated from ewe's milk and were found to produce one or the other SEs [10].Other non Staphylococcus aureus species such as S. saprophyticus, S. warneri, S. chromogenes and S. lentus isolated from healthy goat milk and dry-cured hams were found to have the enterotoxigenic potential [11][12][13][14].
Screening for Staphylococcus species among various ready to serve food products available in the street vend shops is important for epidemiological reasons.As most of these products are sold in open conditions, this screening will also indicate the level of hygiene followed by these vendors.Therefore, the aim of the present investigation was to study the distribution of various Staphylococcus species in street vend food.NaCl concentration [15].On the basis of biochemical test results, isolates were identified to species level with the aid of Bergey's Manual of Systematic Bacteriology [1].

DNA Extraction
Genomic DNA was isolated as described previously (A.

PCR Amplification of 16S rRNA and Purification
16S rRNA Universal primer forward 5' AGA GTT TGA TCC TGG CTC AG 3' and reverse 5' AAG GAG GTG ATC CAG CCG CA 3' were synthesized from Sigma Aldrich (Bangalore, India).A reaction mixture containing approximately 1 ng of template DNA, 2.5 μl of 10X buffer, 20 pM concentration of each PCR primer, 10 mM dNTP mix and 1 U of Taq polymerase (Bangalore Genei, Bangalore, India) in a total of 25 μl was prepared by adding sterile milli Q water for all the reactions.The PCR was carried out in a Thermal Cycler (Mastercycler, Eppendorf, Hamburg, Germany).The amplified product of 16S rRNA gene was purified with the PCR purification kit following the standard protocol as supplied by the manufacturer (HiMedia, Mumbai, India).The purification involved adding binding buffer to the PCR mix and centrifuging through filter tubes.The unincorporated nucleotides were removed by adding wash buffer and centrifugation at 10,000 rpm for 2 min.The PCR products were eluted using elution buffer by centrifugation at 10,000 rpm for 1 min.

Restriction Endonuclease Digestion
The 16S rRNA amplified products were digested with BamHI, HindIII, EcoRI, AluI and HaeIII restriction enzymes (Bangalore Genei, Bangalore, India).PCR product was digested separately with 10 U of each enzyme for 3 h at 37˚C in a circulating water bath (Julabo, Germany).Reaction was stopped by incubating the samples at 65˚C for 5 min [16].Digested samples were electrophoresed on 2.5% agarose gel and photographed by gel documentation system (Vibler Lourmat, Marne-la-Vallee, France).

Cluster Analysis
Different fragments on the gel were numbered sequentially, followed by scoring the samples based on presence and absence of fragments (present 1, absent 0) and compared according to the genetic distance method.The strains were clustered by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) with the aid of NTSYS software (Applied Biostatistics Inc V.1.07).

Sequence Analysis of 16S rRNA Gene
The purified product was sequenced in an automated DNA sequencing facility (Bangalore Genei, Bangalore, India).A single sequence analysis data obtained was blasted in NCBI database for the identification of isolates.These sequences were compared with the reported Staphylococcus species 16S rRNA gene sequences available in the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/index.html).Based on the highest percentage homology to the reported sequences, the food isolates were identified.Multiple sequence alignments were performed using clustalW (Kyoto University, Bioinformatics Center; http://align.genome.jp/).Phylogenetic tree and Bootstrapping were constructed based on the 16S rRNA gene sequences using CLC Main workbench software (version 5.6.1,CLC bio, www.clcbio.com).

Nucleotide Sequence Accession Numbers
The 16S rRNA sequences determined for the food isolates were submitted to the eMBL database (http://www.ebi.ac.uk/embl/Submission/).

Results
Based on differences in morphological characteristics on BPA such as big to pin point colonies showing grayish black to black colour with lecithinase positive as well as negative activity, a total of 35 native isolates were selected from 14 street vend food samples in Mysore city area.The results of biochemical characterization of these isolates showed that S. aureus was the most frequently isolated species among different food samples accounting for 20.0% the population studied.1).
To confirm the phenotypic identification of the food isolates, definitive species identification was done on the basis of PCR-RFLP and sequence data analysis for the 16S rRNA gene.All Staphylococcal food isolates and reference strains generated a single PCR product of about 1500 bp length for 16S rRNA gene.The PCR products were then digested with BamHI, HindIII, EcoRI, HaeIII and AluI for RFLP analysis.It was observed that majority of the isolates and reference standards namely S. aureus (FRI 722), S. epidermidis (ATCC 12228), S. saprophyticus (ATCC 15305), S. haemolyticus (ATCC 29978) and S. xylosus (ATCC 35033) had identical restriction profiles with BamHI, HaeIII, HindIII and EcoRI, but these strains yielded different digestion patterns when subjected to AluI restriction digestion.On the basis of scoring, presence and absence of fragments (present 1, absent 0), phylogenetic tree was constructed with the help of NTYSIS software and isolates found at different branching nodes were selected for further sequence analysis.When comparing with the reference standards to the food isolates using biochemical tests it was observed that even the isolates of same species were forming in different nodes (Figure 1) this indicates that either there is a difference among the Staphylococcus species or biochemical test is not so accurate for identification at species level.Therefore, 14 isolates that were found at different nodes were selected for sequence analysis and the data were compared with NCBI database (Table 2).The highest similarity with a best match to the reported sequence data were S. cohnii (99%); S. sciuri (99%); S. saprophyticus (97%); S. saprophyticus subsp saprophyticus (98%) and S. gallinarum (96%) indicating high degree of similarity between the food isolates and the reported species.These sequences were deposited in EMBL database under accession No FN646065 -FN646078.
Multiple alignments of 16S rRNA gene data sequences of Staphylococcus food isolates were obtained using ClustalW programme.Sequence similarity of 16S rRNA gene among 14 different Staphylococcus species ranged from 30% -97% with a mean similarity of 63.5% (Table 3).These values are consistently lower and indicate high discrimination among the different Staphylococcus species and the most variability among all the food isolates was observed at 595 to 763 bp region of 169 bp (Table 4).The pair wise comparisons among different Staphylococcal species showed a wide variability (3 to 70%).The highest percentage similarity was observed between the species S. cohnii and S. saprophyticus isolates (96.5%), S. cohnii and S. sciuri isolates (92%) and S. saprophyticus and S. sciuri isolates (91.5%), and the least similarity was observed between S. saprophyticus subsp saprophyticus and S. simulans (30%).
The relationships among species of the genus Staphylococcus were confirmed by phylogenetic analysis based on the 16S rRNA gene sequencing, and the topology of the tree was evaluated by bootstrap values.The phylogenetic tree (Figure 2) showed that Staphylococcus species were divided in distinct subgroups.The group consisting of S. cohnii, S. saprophyticus subsp saprophyticus and S. xylosus was characterized as being novobiocin resistant, and it formed a monophyletic clade in the phylogenetic tree with 89% of the bootstrap value.S. cohnii has a relatively deep subline with in a cluster group of S. saprophyticus subsp saprophyticus.According to our phylogenetic tree another closely related group consists of S. aureus, S. simulans, S. felis and S. sciuri,  *+: Positive, -: Negative, R: Resistant, S: Sensitive, d+: Delayed Positive; **Food source of isolates a-Agra Peda, b-Badam milk, c-Bhelpuri, d-Boiled milk, f- Churmuri, h-Curd rice, i-Dosa batter, j-Icecream, l-Laddu, m-Namkeen, o-Raw milk, q-Sevpuri, r-Sweet, s-Voda.Highlighted Staphylococcus species were taken for molecular studies.
Copyright © 2011 SciRes.FNS nation at the sub species level, but results presented here show that 16S rRNA might be more discriminative target sequence to differentiate among subspecies.Pair wise comparison of the 16S rRNA gene sequences of 14 Staphylococcus food isolates ranged from 30% -97% with mean similarity value of 63.5% (Table 3) indicating suitability of 16S rRNA gene for discrimination among the Staphylococcus food isolates, as the higher % similarity is less discriminatory [24].While comparing relatedness among the food isolates, the phylogenetic tree (Figure 2) also showed distant groups indicating suitability of the method used for differentiation.

Conclusions
In present study, S. aureus was found to be the most prevalent species among the species of Staphylococcus identified in the street vend foods.Although pathogenicity of these species needs to be studied, their accurate identification is important, as many staphylococcal species are known for their enterotoxigenic potential.The 16S rRNA sequencing employed in this study for identification of Staphylococcus species may be useful for precise and timely identification and prevention of any untoward staphylococcal food poisoning incidences.

Figure 1 .
Figure 1.Unrooted Neighbour-joining phylogenetic tree of Staphylococcus food isolates constructed on the basis of digestion of 16S rRNA gene product with AluI restriction enzyme.The scale bar indicates the evolutionary distance value between the species.

Figure 2 .
Figure 2. Neighbour-joining phylogenetic tree of Staphylococcus food isolates constructed on the basis of 16S rRNA gene sequence.