Case Report

Molecular Detection of Methicillin-resistant Staphylococcus aureus (MRSA) from a Clinical Case of Myiasis Wound

Pravin Mishra1, Md. Muket Mahmud2, Md. Ahosanul Haque Shahid2, Alamgir Hasan2, Vivek Kumar Yadav3 and Moinul Hasan1*

1Department of Surgery and Obstetrics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh; 2Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh; 3Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

Editor | Muhammad Abubakar, National Veterinary Laboratories, Park Road, Islamabad, Pakistan.

Received | May 09, 2020; Accepted | June 12, 2020; Published | July 15, 2020

*Correspondence | Moinul Hasan, Department of Surgery and Obstetrics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh; Email: moinul.vet@bau.edu.bd

Citation | P. Mishra, M.M. Mahmud, M.A.H. Shahid, A. Hasan, V.K. Yadav and M. Hasan. 2020. Molecular detection of methicillin-resistant Staphylococcus aureus (MRSA) from a clinical case of myiasis wound. Veterinary Sciences: Research and Reviews, 6(2): 96-99.

DOI | http://dx.doi.org/10.17582/journal.vsrr/2020/6.2.96.99

Keywords | Calf, mecA gene, MRSA, myiasis, nuc gene

Introduction

Myiasis refers to the infestation of humans and/or animals by dipterous larvae (Maggot) (Juyena et al., 2013). Maggot is the larva of a dipterous insect with legless soft-body. Overall, more than 100 species of dipteran flies are present to cause Myiasis (Penner, 1958). Myiasis wound mostly occurs when fly larvae infest open wounds but not limited to larvae only, this can be infested by all dipteran life stages (Pezzi et al., 2019). Major predisposing factors of this sort of myiasis are poor socioeconomic conditions, extremes of age, and negligence (Huntington et al., 2008). Myiasis is a well-known condition to veterinarians or practitioner from underdeveloped regions and causes severe economic losses globally (Otranto, 2001). The prevalence of myiasis are reported both in human and animals mostly in rural, tropical and subtropical regions but now commonly seen in the temperate zone also (Noutsis and Millikan, 1994; Lwanga et al., 2018). 

As with any sort of infestation of parasites, myiasis causes concerns for the possibility of secondary bacterial infection, since certain species of fly and their larvae harbor associated bacteria (Islam et al., 2015). Different gram-positive (Corynebacterium spp.) and gram-negative (Proteus spp., Stenotrophomonas spp., Brevundimonas spp.) bacteria are isolated in the case of maggot infestations (Toth et al., 2006). Of particular concern in our case, methicillin-resistant Staphylococcus aureus (MRSA) is a gram-positive round shaped, anaerobic bacteria which are genetically distinct from other strains of Staphylococcus aureus. MRSA is usually a bacterium of both humans and animals and often described as community-associated, healthcare-associated, or livestock- associated. Around 10% of the sporadic infections are due to livestock-associated MRSA (Cuny et al., 2015). The MRSA is only susceptible to Vancomycin but it has a carcinogenic effect. Therefore, we report a case of methicillin-resistant Staphylococcus aureus isolated from a calf with myiasis wound in Bangladesh, as this is of global concern.

Case presentation

Two and the half-year-old indigenous male bovine calf was brought to Veterinary Teaching Hospital, Bangladesh Agricultural University, Mymensingh having body weight 158 kg with the complaint of a wound. Visual examination revealed the case of myiasis leading to wound.

Materials and Methods

The dressing was performed, swab sample from the wound before dressing was collected using sterile cotton bud both from the outer and inner part of the wound and transferred to the nutrient broth for molecular study (Figure 1). The collected broth samples were incubated at 37 ºC for 2 hours for the enrichment and inoculated into selective media i.e. Mannitol salt agar (MSA) and incubated at 37 ºC for overnight. The next day the growth of bacteria was observed and the pure culture of each bacteria was obtained by repeated culture of a single colony. The pure cultures of isolated bacteria were subjected to Gram´s staining for observation of bacterial morphology, arrangement, and staining characteristics under the light microscope at 10X magnification, as per the method described by Jaman et al. (2018). Bacteria from pure culture subjected to DNA extraction by boiling method, as per described by Hussain et al. (2016) and PCR was done using S. aureus specific nuc gene and MRSA specific mecA gene primers, with expected product size 279 bp and 533 bp respectively. PCR amplification was done by initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 1 min, annealing temperature of primers was 55°C for 45 sec and extension at 72 °C for 1 min. The final extension was conducted at 72 °C for 10 min. Forward and Reverse primers used to detect the nuc gene and mecA gene were mentioned in Table 1.

Table 1: Primers used in PCR for nuc gene and mecA gene.

Based on cultural characteristics and staining properties Staphylococcus aureus was identified from the outer part of the wound as on Mannitol Salt Agar (MSA), Staphylococcus aureus produces smooth and convex golden yellow colony (Figure 2) and on Gram´s staining, it shows grape-like cluster which is the characteristics of Gram-positive bacteria (Figure 3).

Based on the PCR amplification result, it was further confirmed that the isolated bacteria are Staphylococcus aureus and methicillin-resistant Staphylococcus aureus by nuc gene and mecA gene respectively (Figures 4 and 5).

S. aureus is well known causing agents of skin and soft tissue infections as well as food poisoning (Hussain et al., 2016). Methicillin-resistant Staphylococcus aureus (MRSA) has emerged worldwide as a significant public health problem both in humans and animals and got zoonotic importance when scientists suggested the possibility of animals serving as reservoirs for human MRSA infection (Garoy et al., 2019).

In our study we reported that MRSA was detected from the outer part of the wound but not from the inner part, this might be because of the unhygienic environmental conditions and untreated condition of myiasis wound. This result is also supported by Islam et al. (2015). The reason for no detection of bacteria from the inner part is maggot itself because maggot does not allow to grow any bacteria and eat dead tissues (Huntington et al., 2008; Arnold, 2013).

Arnold, C., 2013. New science shows how maggots heal wounds. Sci. Am., 308(4). https://doi.org/10.1038/scientificamerican0413-19a

Cuny, C., Wieler, L.H. and Witte, W., 2015. Livestock-associated MRSA: The impact on humans. Antibiotics, 4(4): 521-543. https://doi.org/10.3390/antibiotics4040521

Garoy, E.Y., Gebreab, Y.B., Achila, O.O., Tekeste, D.G., Kesete, R., Ghirmay, R., Kiflay, R. and Tesfu, T., 2019. Methicillin-resistant Staphylococcus aureus (MRSA): prevalence and antimicrobial sensitivity pattern among patients, a multicenter study in Asmara, Eritrea. J. Can. J. Infect. Dis. Med. Microbiol., https://doi.org/10.1155/2019/8321834

Huntington, T.E., Voigt, D.W. and Higley, L., 2008. Not the usual suspects: human wound myiasis by phorids. J. Med. Entomol., 45(1): 157-159. https://doi.org/10.1093/jmedent/45.1.157

Hussain, K., Rahman, M., Nazir, K.H.M.N.H., Rahman, H. and Khair, A., 2016. Methicillin resistant Staphylococcus aureus (MRSA) in patients of community based medical College Hospital, Mymensingh, Bangladesh. Am. J. Biomed. Life Sci., 4(3): 26-29. https://doi.org/10.11648/j.ajbls.20160403.11

Islam, M.T., Al-Maruf, A., Mannan, M.A., Rahman, H.M.R., Tarafder, M.M., Samad, M.A., Al Noman, A., Hossain, M.B. and Rahman, M.M., 2015. Isolation and identification of associated bacteria and maggots from myiasis affected wounds of cattle and goats in Bangladesh. J. Adv. Vet. Anim. Res., 2(2): 95-100. https://doi.org/10.5455/javar.2015.b55

Jaman, M.M., Mishra, P., Rahman, M. and Alam, M.M., 2018. Clinical and laboratory investigation on the recurrence of the umbilical hernia after herniorrhaphy in bovine calves. J. Bangladesh Agric. Univ., 16(3): 464-470. https://doi.org/10.3329/jbau.v16i3.39418

Juyena, N., Tapon, M., Ferdousy, R., Paul, S. and Alam, M., 2013. A Retrospective study on occurrence of myiasis in ruminants. Progr. Agric., 24(1-2): 101-106. https://doi.org/10.3329/pa.v24i1-2.19110

Kalorey, D.R., Shanmugam, Y.K., Nitin V.C., Kapil K.B. and Sukhadeo B., 2007. PCR-based detection of genes encoding virulence determinants in Staphylococcus aureus from bovine subclinical mastitis cases. J. Vet. Sci., 8(2): 151-154. https://doi.org/10.4142/jvs.2007.8.2.151

Lwanga, A., Anis, M., Ayoubi, M., Sharma, J. and Khosla, P., 2018. Two cases of myiasis associated with malignancies in patients living in the continental United States. Cureus, 10(1): e2049. https://doi.org/10.7759/cureus.2049

Noutsis, C. and Millikan, L.E., 1994. Myiasis. Dermatol. Clin., 12(4): 729-736. https://doi.org/10.1016/S0733-8635(18)30136-0

Otranto, D., 2001. Myiasis: Devils in the flesh of humans and animals. Hosp. Pract., 53: 98-102.

Penner, L.R., 1958. Concerning a rabbit cuterebrid, the larvae of which may penetrate the human skin (Diptera, Cuterebridae). J. Kansas Entomol. Soc., 31(2): 67-71.

Pezzi, M., Bonacci, T., Leis, M., Mamolini, E., Marchetti, M.G., Krčmar, S., Chicca, M., Del Zingaro, C.N.F., Faucheux, M.J. and Scapoli, C., 2019. Myiasis in domestic cats: A global review. Parasites Vectors, 12(1). https://doi.org/10.1186/s13071-019-3618-1

Toth, E., Hell, E., Kovacs, G., Borsodi, A. and Marialigeti, K., 2006. Bacteria isolated from the different developmental stages and larval organs of the obligate parasitic fly, Wohlfahrtia magnifica (Diptera: Sarcophagidae). Microb. Ecol., 51(1): 13-21. https://doi.org/10.1007/s00248-005-0090-6