Prevalence of Microbiological Contaminants in Milk Samples in Kathmandu, Nepal

Introduction

Milk is an excellent source of nutritional supplements such as protein, lactose, fat, minerals and vitamins. According to survey of the Food and Agriculture Organization of the United Nations in 2020/21, annually 75 litres of milk is consumed by a single person in Nepal [1, 2]. Generally, people consume either unpas¬teurized milk directly from farm and dairy shops or pasteurized packaged milk from different outlets. Naturally milk contains beneficial lactic acid bacteria such as Streptococcus, Lactococ-cus, Lactobacilli, Bifidobacteria, Enterococcus, and Pediococci that produce several secondary metabolites which are advanta¬geous to human. In addition to these useful bacteria, milk serves as a rich source of nutrients for growth of pathogenic bacteria, which is influenced by several other factors such as temperature, pH, humidity, etc. [3]. It was observed that dairy products were found to be contaminated with pathogens such as Mycobacteri-um bovis, Campylobacter spp., non-typhoidal Salmonella enter-ica and Brucella spp., Cryptosporidium spp., Shiga-toxin pro¬ducing Escherichia coli and Toxoplasma gondii.

In developing countries, food-borne diseases were associated with disability adjusted life years that was 20 per 100,000 in-dividuals [4]. A study from Iran revealed that E. coli, S. aureus, L. monocytogenes, and Salmonella spp. isolated from raw milk were resistant to drugs. Simultaneously, poor sanitation and hy- giene, lack of advance facilities in farming industry, and inade¬quate training are responsible for the microbiological contami¬nation of milk. Furthermore, irrational use of antibiotics in dairy industry is escalating drug resistant microorganisms which are responsible for public health burden. Therefore, this study is de¬signed to investigate the prevalence of microbial contaminants in the milk samples collected from different sites in Kathmandu

Materials and Methods

A cross-sectional study was conducted between January and July 2019 on 90 milk samples (30 farm milk, 30 dairy milk and 30 pasteurized packaged milk) collected from different farms, dairy shops and local stores in Kathmandu. Exactly, 5ml of milk samples were collected in a sterile leak-proof screw-capped container and transported to Department of Medical Laboratory Technology, Janamaitri Foundation Institute of Health Sciences, Lalitpur in an icebox. Collected samples were subjected to ten-fold serial dilution in sterile peptone water. Then, pour plate cul¬ture was performed in Violet Red Bile Lactose Agar (VRBA) and Plate Count Agar (PCA) and incubated for 24 hours at 37°C to estimate total coliform count and total bacteria count respective¬ly. Also, undiluted milk samples were streaked on Mannitol Salt Agar (Hi-Media), MacConkeyAgar (Hi-Media) and Sabouraud Dextrose Agar with Chloramphenicol (Hi-Media) for bacteri¬al and fungal isolation at standard in-vitro conditions. Further, gram staining, biochemical tests and Lactophenol cotton blue staining were performed for identification of the isolates. Anti-microbial sensitivity test was then performed via Kirby-Bauer method using the following antibiotic discs: Ampicillin(10µg), Amikacin(30µg), Gentamicin(10µg), Chloramphenicol(30µg), Cotrimoxazole(2µg), Levofloxacin(5µg),Imipenem(10µg),Pip-eracillin-Tazobactam(100/10µg), Cefotaxime(30µg), Cefoxit-in(30µg), Erythromycin(15µg). Data were entered in Statistical Package for Social Sciences version 20 for further analysis and interpretation. Ethical approval was taken from Nepal Health Research Council, Kathmandu, Nepal with reference number 3339.

Results

In overall, out of 90 milk samples, microbiological prevalence was found to be 79 (87.7%), where 37 (41.1%) samples were positive for bacterial growth and 42 (46.6%) samples were pos¬itive for fungal growth. S.aureus was the most commonly found bacteria in farm milk (50.0%) followed by dairy milk (37.5%). However, E. coli was mainly prevalent in pasteurized packaged milk (46.1%). Aspergillus spp. and Penicillium spp. were mainly present in dairy milk samples, details are shown in Table 1.

                                                 Table 1: Prevalence of selected microbial contaminants

                                              Keys: FM- Farm milk; DM-Dairy milk; PPM-Pasteurized packaged milk.

The mean bacterial and mean coliform count significantly re-duced with dilution in pasteurized packaged milk samples compared to farm and dairy milk samples. Also, polymicrobial growth was observed in six samples, abundantly in pasteurized packaged milk sample. The mean bacterial count was highest in dairy milk (70.13 × 106 ± 118.08) followed by farm milk (39.83 × 106 ± 77.39) in contrast to pasteurized packaged milk (3.30 × 106 ± 10.84). Also, the mean total coliform count was compara¬tively higher in dairy milk (13.13 × 106 ± 54.90) than pasteurized packaged milk (2.67× 106 ± 10.87) while farm milk had 2.20× 106± 5.26, details are shown in table 2.

                                                    Table 2: Mean bacterial and coliform count in milk samples

                                  Notes: FM: Farm milk, DM: Dairy milk, PPM: Pasteurized packaged milk.

Out of 24 S. aureus isolates, 25.0% isolates were methicillin-re-sistant Staphylococcus aureus (MRSA). Also, S.aureus isolates from farm milk and dairy milk were mainly resistant to Am-picillin and Chloramphenicol. Interestingly, out of three S.au- reus isolates from packaged pasteurized packaged milk, one was MRSA. Besides, E.coli was mainly resistant to Cefotaxime and Ampicillin in pasteurized packaged milk as shown in table 3.

                                                           Table 3: Antimicrobial resistance prevalence

Keys: NT- Not tested

Discussion

In this study, 87.7% milk samples were contaminated with mi-croorganisms where 41.1% samples were positive for bacteri¬al growth. Similarly, a study from Northern Ethiopia showed 52% prevalence of microbial contaminants in milk samples [5]. Milk nutrients act as an enriched medium for microbial growth. Usually milk is free from pathogenic bacteria. However, during handling, processing, transportation and storage as well as non-compliance to the hygiene practices lead to microbial de-terioration of milk. S.aureus was the most commonly isolated species in our study with a prevalence rate of 26.6% (24/90), which was similar to a study done in Eastern Ethiopia where the prevalence was 24.2%. However, E.coli was the most commonly isolated species 58.0% which is in contrast to our study (14.4%) [6]. In an effort to measure the level of microbiological quality and practices associated with handling, coliform counts are con¬sidered to be important indicators. It has been indicated in many studies that coliform and Staphylococcus are most commonly isolated species because of their sufficient attainability in the an¬imal body as well as the environment. Since S.aureus is a habit¬ual bacteria which can arise from various parts of warm blooded animals, as well as from faeces, soil and fresh water, this might have contributed to higher count in our study. In Greece, a study revealed 47.8% prevalence of S. aureus of which 4.1% was MRSA and had ability to produce enterotoxin and biofilm [7]. In our study MRSA prevalence was 25.0%. Likewise, the high coliform count can be attributed to the use of substandard water quality at their source, detrimental milking practice, unsanitary utensils or milking equipment as well as poor conditions of stor¬age. In this study, Aspergillus spp. (26.6%) was most commonly isolated followed by Penicillium spp.(20.0%). A similar study from Egypt has found the opposite result, where Penicillium spp. were most commonly isolated [8]. Also, Cladosporium spp. were isolated from milk products of China [9]. Besides this, Mucor spp. was also found in milk products [10]. It was also noted that the presence of pathogenic fungus was found mostly in the raw milk of cow, ewe and goat [11]. Hence, the fungal contamination in milk and its products may be due to inadequate cleaning practices, improper sanitation and lack of proper stor¬age, transportation and packaging techniques.

In a similar study conducted in Kathmandu valley, the mean total bacterial count of pasteurized, unpasteurized and raw milk was 1.2X106cfu/ml, 2.3 X 107 cfu/ml and 2.0 X 107 cfu/ml respec¬tively which was similar to our study [12]. Similarly, the mean total coliform count of pasteurized, unpasteurized and raw milk was higher in our study. A study conducted in farm milk and its products in Poland found that the isolated bacteria were main¬ly resistant to penicillin (50.8%), chloramphenicol (5.7%) and tetracycline ( 4.1%) [13]. In our study, bacterial isolates were mainly resistant to ampicillin and chloramphenicol. A study from Connecticut has found that raw milk bacterial isolates were resistant to streptomycin, penicillin, ampicillin, neomy-cin, chloramphenicol, polymyxin, and tetracycline. In addition to this, bacterial isolates from pasteurized dairy products were resistant to polymyxin, penicillin and ampicillin [14]. Howev¬er, in our study, isolates from pasteurized packaged milk were resistant to ampicillin, cefoxitin, erythromycin and cefotaxime. In our study 25.0% S. aureus were MRSA. A similar study have found that 33.2% of S. aureus isolates were MRSA [12]. The high numbers of drug resistant bacterial isolates were due to the irrational use of antibiotics in livestocks [15]. Furthermore, milk contains many beneficial probiotic bacteria. They produce bene¬ficial secondary metabolites which exhibit antimicrobial activity against several human pathogens [16]. In addition, molecules from probiotic bacteria have shown anti-tumorigenic activity [17, 18]. The limitations of this study are: only selected bacteria were processed, anti-fungal sensitivity test was not performed due to limited laboratory facilities and genomic analysis was not done.

Conclusion

Funding

Conflict of interest

References

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