1. INTRODUCTION

Milk is a food commodity valued for its nutrition besides providing regular income for the dairy farmers [1]. In Malaysia, high local demand for dairy products resulted in the country having to rely substantially on imports [2]. The development of the local dairy production capacity began since 1974 when the Malaysian government initiated the National Dairy Development Program to reduce dependency on milk imports [2]. However, up to date, the local dairy industry still is unable to meet the demand of the growing population. Efforts are also made by local extension personnel on ensuring that while milk production capacity increases, milk quality is also maintained so as to ensure that food safety is not compromised [2]. The decrease in milk quality can be attributed to the presence of microorganisms and problems with hygiene practices [3]. Improvement in farm hygiene practices has been demonstrated to reduce bacterial count and mold in raw milk [4], elevating milk quality by 2.4 times reduction of risk exposure to milk pathogens and its potential toxins. Members of the genus Corynebacterium include species that occasionally cause infection in humans and some species having been recovered solely from animals, the environment, food, water, and synthetic materials [5]. C. ulcerans, for instance, was thought to cause disease in farm workers after contact with contaminated milk or farm animals [5]. Here, we performed a microbiological analysis on raw milk after the implementation of improved farm hygiene practices to assess the impact of the initiatives. We report recovery of a rare Corynebacterium species from raw milk after the implementation of improved farm hygiene practices.

2. MATERIALS AND METHODS

Before the start of milking, the udders and teats of healthy dairy cows and the hands of farm workers were rinsed with 1.0% diversol and wiped dry with clean towels. Milking equipments were washed with sanitizing solution comprising of 5.4 g iodophor diluted in 10 L of water and rinsed with clean water before the milking process. Foremilk specimen was collected directly from the teat after discarding the first milk. All milk specimens were collected from mastitis-free cows and inoculated onto the respective Petrifilm™ (Escherichia coli and coliform, yeast and mold, total aerobic count, Staphylococcus aureus, and Enterobacteriaceae) (3M Corporation, St. Paul, MN, United States) and enumerated [4]. Colonies on the films were then plated onto nutrient agar and incubated under aerobic conditions at 37°C for 24–48 h.

Table 1: Biochemical and antimicrobial susceptibility profiles of Corynebacterium lipophiloflavum isolated from raw milk [Click here to view]

3. RESULTS AND DISCUSSION

One colony originating from the Enterobacteriaceae film grew unique yellow-pigmented colonies between the sizes of 0.5 and 1.0 mm after 48 h incubation. Molecular identification using 16S rDNA sequencing [6] showed that the isolate shared 99% sequence similarity to Corynebacterium lipophiloflavum DMMZ 1944 (accession no. Y09045) [Figure. 1]. Due to the close 16S rDNA similarity between different Corynebacterium strains, selected differentiating biochemical tests were performed. The Corynebacterium isolate was able to hydrolyze urea but could not ferment glucose, maltose, and sucrose [Table 1] similar to C. lipophiloflavum [7]. The exhibited biochemical test results were different to those displayed by its closest phylogenetic relative, C. mycetoides [8] [Figure. 1], as such corroborating the 16S rDNA sequencing results, confirming the isolate’s identity as C. lipophiloflavum. Antimicrobial susceptibility assays performed according to the guidelines by the Clinical and Laboratory Standards Institute [9] demonstrated that the C. lipophiloflavum isolate was sensitive to cefotaxime, ceftriaxone, ciprofloxacin, erythromycin, gentamicin, meropenem, penicillin, tetracycline, and vancomycin [Table 1]. The minimum inhibitory concentration assays were repeated twice to verify the results. Visual inspection of the bacterial isolate after incubation in a 96-well plate at 37°C for 48 h [10] noted that the C. lipophiloflavum isolate did not produce biofilm. Morphological characteristics of the bacterium unveiled by transmission electron microscopy [11] displayed structures similar to C. phoceense [12] and C. lactis [13] [Figure. 2]. The bacterium showed characteristic club-shaped cell and the presence of an external lipid layer.

Figure 1: Neighbor-joining tree based on 16S rDNA gene of representative Corynebacterium species. Their respective accession numbers are listed before the species names. The Corynebacterium lipophiloflavum isolate in this study is indicated in bold. Number at nodes indicates bootstrap values (%) for 1000 replicates. [Click here to view]

Even though other dairy-spoilage Corynebacterium species have been found in raw milk [3], this is the description of the first C. lipophiloflavum milk isolate. It was doubtful that C. lipophiloflavum was derived from the environment since the sanitization protocols have been proven to be effective in reducing bacterial counts [4]. This bacterium first described from a woman with bacterial vaginosis [7], could probably resist the high hydrogen peroxide concentration environment of the vagina, akin to C. aurimucosum [5], and, hence, could possibly be part of the cow’s vaginal flora too. There is strong possibility that C. lipophiloflavum was transferred from the mother to the calf during birth, comparable to the transfer of vaginal flora from mother to child during delivery [14]. This suggestion was substantiated by the antimicrobial susceptibility profile of the C. lipophiloflavum isolate, indicating that it may have emerged from an antibiotic-free environment [15]. Recovery of C. lipophiloflavum even after sanitization can be explained by the transfer of endogenous bacteria during milking, which resembled the human milk bacteria colonization of breastfed neonates [16]. Although the C. lipophiloflavum isolate did not produce biofilm, it may switch from the dormant state to biofilm producer [17] on leaving the normal flora, as a result of selection and environmental pressures, further suggesting the undesirable influence of corynebacteria in dairy contamination. While C. lactis may seem harmless when it was first isolated from the cow’s milk, recent studies have shown that it was also found in ticks and can cause infection in companion animals [13]. Hence, treating C. lipophiloflavum as a harmless normal flora of the cow could have disastrous aftermath to the entire dairy supply chain. Besides, the finding of C. lipophiloflavum in the milk carries significant interest for the dairy industry. It may induce milk spoilage by expressing lipolytic and proteolytic enzymes resembling C. variabilis [18], besides potentially causing mastitis akin to C. bovis [19]. Furthermore, there are risks to the human health since C. lipophiloflavum possesses the ability to cause human infection [7].

Figure 2: Morphological characteristics of Corynebacterium lipophiloflavum as displayed by transmission electron microscopy at 16,300 times magnification. Arrows show the presence of an external lipid layer. [Click here to view]

4. CONCLUSION

Taken together, the findings highlight the increasing spectrum of Corynebacterium species with potential adverse impact to the dairy industry. Previous milk-associated Corynebacterium strains identified mostly to the genus level could perhaps be C. lipophiloflavum if further characterization was performed. It is recommended to screen for C. lipophiloflavum in all milk processing facility to ensure that milk is safe for consumption and its products prepared to the highest quality and safety standards.

5. CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

6. ACKNOWLEDGMENTS

This study was funded in parts by the University of Malaya Research University Grants (RU005-2017 and RU008-2018) and the Indian Graduates Association of Universiti Putra Malaysia.

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