Genetic Identification of Predominant Bacteria Isolated from Student Kitchen

The domestic kitchen represents one of the most significant areas associated with the prevalence of foodborne disease. Literature related to exploring and discussing bacterial contamination and sanitation practices in shared kitchens is scarce. The aim of this study is to carry out genetic identification of 2 of the predominant bacterial isolates previously obtained from a shared student kitchen. Partial 16S rDNA gene sequencing was used to further identify two selected isolates. Gene sequencing revealed the 2 selected isolates to be closely related to Enterobacter cloacae (99% sequence similarity) and Pseudomonas sp. (100% sequence similarity); this is in agreement with the previous presumptive identifications.

Foodborne disease leads to substantial morbidity and mortality all over the world 1 , and is thus considered a severe public health problem 2 . In 2010, there were 56,991 recorded cases of food poisoning illness in England and Wales 3 . However, it is generally accepted that actual figures may be orders of magnitude higher than those recorded due to significant under-reporting amongst other factors 2,4 .
Campylobacter jejuni and Salmonella spp. are the main culprits responsible for a significant proportion of reported cases of foodborne disease in the UK 2,[5][6][7] . This is explained by the close association of these species with chicken, a common component of the UK diet. Numerous surveys have been conducted to assess the extent of contamination of raw poultry sold in the UK, and they have revealed that up to 30% is contaminated with salmonellae and up to 90% with campylobacters 7 .
Salmonella spp. and Campylobacter spp. are widely recognized as the most common pathogens isolated in human cases of bacterial gastroenteritis 2, 8 although it has been reported that Campylobacter is more often implicated in gastrointestinal infection than Salmonella 4 . Campylobacter is also considered to be the leading bacterial cause of diarrheal disease/infectious intestinal disease (IID) in the developed world 8 . In addition to Salmonella and Campylobacter, it has been reported that Shigella and Shiga toxin and Escherichia coli (STEC) O157 significantly contribute to the incidence of foodborne disease in the United States 9 . Other foodborne pathogens include Staphylococcus aureus, rotavirus and hepatitis A virus 1 .
Foodborne outbreaks of Listeria monocytogenes, responsible for septicaemia and meningoencephalitis in the immunocompromised and the elderly, have been reported 10 . L. monocytogenes is common in the environment and has caused several cases of hospital-acquired/ nosocomial listeriosis, including in neonates, in the UK. Nosocomial outbreaks of Salmonella infection, particulary Salmonella enteritidis, have also been recorded 11 .
Bacterial contamination in the domestic environment was first comprehensively investigated by Finch et al. in (1978) 12 . In this study, samples were isolated from various sites in the kitchen, bathroom, living room, toilet and hall of 21 homes and the bacterial flora therein examined. Several studies followed assessing the levels of microbial contamination in the domestic kitchen and the home environment 6, 13-16 highlighting the growing significance of the home, and the kitchen in particular, as locations for harbouring pathogens responsible for foodborne disease. Scott et al.'s study in 1982 15 was similar to that of Finch et al. 12 but on a much larger scale, looking at microbial contamination in 251 domestic houses by sampling 60 sites in the bathroom, toilet and kitchen. Speirs et al. 16 presented findings from the investigation of a wider range of sites (76 in total) in 13 domestic kitchens together with focusing on specific sites with potential for cross-contamination in a further 33 kitchens. Josephson et al. 6 looked at the effectiveness of different cleaning regimes through a two year study that involved the quantification of specific bacterial pathogens and indicator organisms in 10 household kitchens in the United States in the presence, both 'casual' and 'targeted', and absence of disinfectant cleaner use. Rusin et al. 14 examined fourteen sites in the kitchen and bathroom of 15 households over an extended period of time to determine which of these had the highest levels of contamination with faecal coliform, coliform and heterotrophic plate count (HPC) bacteria. Their study also included an investigation of the effectiveness of various cleaning and disinfection regimes using hypochlorite cleaners. Finally, Haysom 13 recognised that previous kitchen studies only looked at samples taken from one point in time and decided to investigate the change of microbial load in domestic kitchens over a 24-hour period.
The above studies provide substantial evidence confirming the presence of viable microorganisms across various sites in domestic kitchens. However, the type of organism together with its likelihood of contaminating food very much determine how significant this microbial contamination is in relation to human health 17 .
The present study was carried out to provide genetic identification of 2 of the predominant bacterial isolates previously obtained from a shared student kitchen.

Selected Isolates
Isolate D5, previously identified as a member of the Enterobacteriaceae family. Isolate S2, previously identified as Pseudomonas spp.

Bacterial DNA Extraction
Two of the bacterial isolates obtained from the student kitchen were chosen for sequencing and were sub-cultured on nutrient agar. A few bacterial colonies were aseptically removed from each plate using a sterile wire loop and homogenised in 100µl of nanopure water in a reaction tube. The reaction tubes containing the bacterial suspensions were then heated in a boiling water bath to 100°C for 10 min and then centrifuged for 10 min (10,000 x g). The resulting supernatants were used as templates for PCR (Polymerase Chain Reaction).

DNA amplification using PCR
The primers 8FPL1 (52 -GAG TTT GAT CCT GGC TCA G-32 ) and 806R (52 -GGA CTA CCA GGG TAT CTA AT-32 ) at a concentration of 5µM each were used to amplify the partial 16s rRNA gene sequences. Each PCR consisted of the following: • 25µl Red Taq DNA polymerase ready mix.

Agarose gel electrophoresis
The quality of the extracted DNA was assessed using 0.8% agarose gel electrophoresis. 0.4g of agarose powder was dissolved in 50ml

Sequencing and identification of isolates
The method used for partial 16S ribosomal DNA gene sequencing of isolates was adapted from McBain et al. 18 . Following purification, the amplification products were sequenced using the primers described above. FinchTV (available from http://www.geospiza.com/Products/finchtv. shtml) was used to read the partial 16S rDNA gene sequences, and both compiled sequences were subjected to a BLAST search against those in the EMBL nucleotide database.

RESULTS
Partial 16S ribosomal DNA gene sequencing results, based on PCR of the selected isolates D5 and S2 (previously identified), are presented in Table 1.
Gene sequencing revealed isolates D5 and S2 to be closely related to Enterobacter cloacae (99% sequence similarity) and Pseudomonas sp. (100% sequence similarity) respectively; this is in agreement with the previous identifications.

DISCUSSION
Partial 16S ribosomal DNA gene sequencing of two selected predominant isolates was conducted in order to confirm their identities (results shown in Table. 1). The first selected isolate (D5) that was obtained from the sink drain sample, and previously identified as a species of Enterobacteriaceae by means of morphological and biochemical testing, was further identified as Enterobacter cloacae. This finding concurs with several other bacteriological studies of the domestic kitchen which also reported the isolation of Enterobacter cloacae from sink sites 12, 15, 16 . The second isolate (S2) that was an oxidase positive, gram-negative rod, obtained from the sponge sample, and previously identified as Pseudomonas sp., was confirmed as Pseudomonas sp.
These gene sequencing results are therefore in agreement with the previous presumptive identifications made by means of phenotypic methods for the samples tested, and may imply that the remaining presumptive identifications were fairly accurate. This is supported by a study in 1991 by Garaizar et al. 19 who found that there was a good agreement between genotypic and the more conventional phenotypic methods when discriminating among Enterobacter cloacae isolates. Despite this, the identifications made by phenotypic methods should still be interpreted with caution due to the reliance of these methods on more subjective determination in relation to cellular morphology, Gram-reaction and biochemical tests, which poses problems in terms of consistent identification 20 .