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103-115 Lani MN.Pmd Tropical Biomedicine 37(1): 103–115 (2020) Survival of antibiotic resistant Escherichia coli in vacuum- packed keropok lekor: Food safety alert among SME keropok lekor producers Wan-Hamat, H.1,2, Lani, M.N.2*, Hamzah, Y.2, Alias, R.3, Hassan, Z.4 and Mahat, N.A.5,6,7,8 1Food Safety and Quality Laboratory, Terengganu Health State Department, Kg. Bukit Tunggal, 21200 Kuala Terengganu, Terengganu, Malaysia 2Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia 3Institute of Halal International, Universiti Selangor, Jalan Zirkon A 7/A, Seksyen 7, 40000 Shah Alam, Selangor, Malaysia 4Faculty of Science and Technology, Universiti Sains Islam Malaysia, Bandar Baru Nilai, 71800 Nilai, Negeri Sembilan, Malaysia 5Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 Johor Bahru, Malaysia 6Centre for Sustainable Nanomaterials, Ibnu Sina Institute for Scientific and Industrial Research, Universiti Teknologi Malaysia, 81310 Johor Bahru, Malaysia 7Enzyme Technology and Green Synthesis Group, Universiti Teknologi Malaysia, 81310 Johor Bahru, Malaysia 8Centre of Research for Fiqh Forensics and Judiciary (CFORSJ), Islamic Science Institute, Universiti Sains Islam Malaysia, Bandar Baru Nilai, 71800 Nilai, Negeri Sembilan, Malaysia *Corresponding author email: [email protected] Received 27 February 2019; received in revised form 8 December 2019; accepted 9 December 2019 Abstract. The microbiological quality of thirty ready-to-eat (RTE) keropok lekor (a sausage shape Malaysian fish product) was evaluated in comparison to microbiological guidelines for ready to eat foods. The two E. coli isolates were subjected to DNA sequencing, identified and tested for their resistance towards fifteen different antibiotics. The survival and growth of the isolated E. coli strains inoculated in keropok lekor at atmospheric air and vacuum packaging were also evaluated. Results revealed that four samples (13.33%) contained Enterobacteriaceae counts that exceeded the recommended allowable counts of 4.0 log10 CFU/g. Unsatisfactory level of coliforms (> 1.7 log10 CFU/g) was also observed in ten of the samples; two of which contained E. coli (2.1 ± 0.17 and 3.7 ± 0.02 log10 CFU/g), suggesting of poor hygiene and sanitation practices. While the ‘Possible E10’ E. coli strain was observably resistant towards Nalidixic acid (30µg) alone, B10 E. coli isolate was worryingly resistant towards Ampicillin (10µg), Ceftazidime (30µg), Ciprofloxacin (5µg), Ceftriaxone (30µg), Nalidixic acid (30µg) and Tetracycline (30µg). This study also revealed that the growth and survival of the ‘Possible E10’ and B10 E. coli strains were not significantly affected by vacuum packaging when stored at both 4°C and 28°C. Therefore, intervention programmes to alert and educate small- medium enterprisers (SMEs) of keropok lekor producers on food safety as well as potential health risks that can be associated due to inappropriate handling procedures of such product, merits consideration. INTRODUCTION is prepared by mixing fish flesh with sago starch, ice, salt and monosodium glutamate, Keropok lekor is one of the famous which then rolled into sausage-like shape traditional Malaysian cuisine, widely sold and boiled, prior to serving. Keropok lekor at hawker stalls, night markets, canteens is primarily produced by small- or medium- and restaurants, and it is considered as scale (SME) enterprises, and remains as the local heritage and tourism icon for the the main economic activity for local people state of Terengganu, Malaysia. The dough as well as it is known as one of tourism 103 attraction (Noraien, 2015). Despite the especially E. coli have acquired con- high demand throughout Malaysia, the siderable attention (Kirbis & Krizman, 2015). distribution process can be challenging Based on previous studies (Lani et al., as keropok lekor is an easily perishable 2017; Wan-Hamat et al., 2019), it has been product that may become organoleptically found that, source of contamination of unacceptable after keeping at room keropok lekor production was due to post- temperature for more than a day (Tang boiling contamination. Keropok lekor is et al., 2014). Recently, prevalence of normally boiled at 100°C for 10 min and this microbiologically assessment of keropok process is sufficient to kill Gram-negative lekor in Kuala Nerus (Lani et al., 2017), bacteria (Lani et al., 2017). However, there Kuala Terengganu and Marang (Wan-Hamat is no apparent study that focused on the et al., 2019) have been reported. In order survival of E. coli in vacuum packed keropok to overcome this barrier, the use of lekor. Vacuum packaging has lack of oxygen vacuum packaging (VP) has been acquiring that is normally used by SME keropok lekor popularity among keropok lekor pro- for extending the shelf life of the product, ducers, attributable to its efficiency during however, study on the safety of vacuum- transportation. In general, utilization of VP packed keropok lekor is not seemingly followed by storage at a strictly controlled visible. Therefore, this study was carried out temperature of -1.5°C can significantly to determine the microbiological quality reduce the growth of aerobic bacteria; and antibiotic resistance as well as to however, this approach may also unwantedly evaluate the survival of E. coli in vacuum- support the growth of pathogenic anaerobic packed keropok lekor. and microaerophilic organisms (Bala- murugan et al., 2013). Antimicrobial resistance has become a MATERIALS AND METHODS serious global public health and food safety concern. Previous researchers (Kirbis & Sampling and general microbiological Krizman, 2015) have reported that multidrug quality assessment resistance (MDR) bacteria (e.g. Escherichia Thirty samples (250 g each) of ready-to-eat coli) may spread from hospitals, nursing (RTE) keropok lekor purchased from the homes and livestock animals to healthy different production premises in Kuala humans, foods as well as pets animals, Terengganu, Malaysia during December leading to serious infections and providing 2017 were transported separately to the treatment can be an uphill challenge. The laboratory in a sterile cold box that contained situation is further exacerbated by the fact packaged ice cubes and immediately that antimicrobial resistant genes can analysed. About 25 g of each sample placed rapidly move through bacterial populations, into a sterile stomacher bag (225 mL of and emerge in pathogenic bacteria via 0.1% peptone water, Oxoid CM0009) was horizontal gene transfer (Wright, 2012). Being homogenized in a paddled laboratory blender a common inhabitant of gastrointestinal (BagMixer® 400, Interscience) for 1 min. tract of humans and animals (Costa et al., Following the Silbernagel and Lindberg 2009), E. coli is easily disseminated through (2003), Silbernagel (2003) and AOAC Official the food chain and water (Ryu et al., 2012). Methods 991.14 (AOAC, 2002), an aliquot It is known to transfer its resistant genes to of the individual homogenate (1 mL) was transient bacterial pathogens that can cause inoculated onto a 3MTM PetrifilmTM Count diseases in humans (Alexander et al., 2010; Plate and incubated at 37°C (Memmert INE Kirbis & Krizman, 2015). Since foods derived 400) for 24 h for Enterobacteriaceae, Staph from animals may become potential sources Express as well as E. coli and coliform of antimicrobial resistant and virulent assessments, respectively. The detection bacteria, studies focusing on the antibiotic of Salmonella, Vibrio cholerae and Vibrio resistance of non-pathogenic bacteria, parahaemolyticus was also performed for 104 each sample using the International Standard described by the guidelines of the Clinical Protocol ISO 6579 (2002) and ISO/TS 21872- and Laboratory Standards Institute (CLSI, 1 (2007), respectively. The E. coli isolates 2012) with E. coli ATCC 25922 as the were identified via DNA sequencing analysis, reference strain. and their survivals at atmospheric air packed (AP) and vacuum-packed (VP) Survival and growth of E. coli isolates keropok lekor were evaluated. at atmospheric air and vacuum packed keropok lekor Identification of E. coli isolates and The survival rate of E. coli was evaluated their antimicrobial resistance by performing inoculation in the rolled of Prior in evaluating antimicrobial resistance, keropok lekor dough which was bought from genomic DNA of both the E. coli isolates a production premise in Kuala Terengganu. from two separate samples were extracted Prior to the inoculation, the dough was boiled using the QIAamp DNA Mini kit (Qiagen in sterile distilled water for 10 min and Inc., Valencia, CA). The purity of extracted aseptically cooled to 28°C in an incubator DNA was verified by comparing the SYBR (Memmert IPP 400). Then, the absences of (Invitrogen, USA) stained agarose gel aerobic plate count, coliforms and E. coli electrophoresis (0.8% agarose in 1x Tris- in the prepared dough were ascertained borate-EDTA buffer) visualised under Blue- using the 3MTM PetrifilmTM Aerobic Count light LED Transilluminator (JUNYI, JY-ERV- (AC) based on AOAC Official Method 01) with that of 1 kilobase DNA ladder 990.12 (AOAC, 2002) and 3MTM PetrifilmTM (Promega, USA). Upon completion, the E. coli and Coliform Count (ECC) plates, purified samples were outsourced to Apical respectively as stated in (mention the part Scientific Sdn. Bhd for DNA sequencing. To here). The prepared dough (25 g) was placed identify the strains of the E. coli isolates, the into a sterile stomacher
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