ISOLATION OF BURKHOLDERIA CEPACIA COMPLEX FROM RAW MILK OF DIFFERENT SPECIES OF DAIRY ANIMALS IN ASSIUT GOVERNORATE

ISOLATION OF BURKHOLDERIA CEPACIA COMPLEX FROM RAW MILK OF DIFFERENT SPECIES OF DAIRY ANIMALS IN ASSIUT GOVERNORATE

NAGAH M. SAAD and WALLAA F. AMIN

Department Food Hygiene, Faculty of Veterinary Medicine, Assiut University.

__________________________________________________________________________________________

                                         ABSTRACT

___________________________________________________________________________

___________________________________________________________________________

Key words: Burkholderia cepacia complex, raw milk, opportunistic, antibiotic.

__________________________________________________________________________________________

INTRODUCTION

Burkholderia are gram negative non spore forming aerobic bacilli. They are versatile microorganisms that inhabit a wide variety of ecological niches as soil, water, animals and human respiratory tract (Coenye and Vandamme, 2003). Burkholderia is an important bacterial genuswith a complex taxonomy that contains species of both ecological and pathogenic importance, including nine phenotypically similar species collectively termed Burkholderia cepacia complex (Bcc) (Luvizotto and Marcon, 2010). The genus Burkholderia was created by Yabuuchi (Yabuuchi et al., 1992) and named after Burkholder who had discovered the genus but classified it as Pseudomonas (Burkholder, 1950).

Because the pseudomonads are commonly associated with the spoilage microflora of foods, various studies have previously identified B. cepacia complex as a spoilage organism in food. Moreover, Bcc have emerged as important life threatening opportunistic pathogen for human particularly in individuals with cystic fibrosis (LiPuma, 1998a) and patients with chronic granulomatous disease (Speert et al., 1994). B. cepacia has also been identified as the causative agent in some cases of endocarditis (Hirose et al., 1998) and nosocomial infection outbreaks (Kaitwatcharachai et al., 2000).

Animal infections caused by Bcc have been reported (Berriatua et al., 2001), but in general, its distribution in animal species and infection are not well documented.

There is a general consensus that the widespread use of antimicrobial agents has imposed a strong selective pressure that contributed to the emergence of multidrug resistant microorganisms (Levy, 2002). Since Bcc are resistant to most antimicrobial agents, effective therapies are not straightforward and management efforts are therefore aimed at prevention of infection (LiPuma, 1998b). Therefore, the aim of this study was to detect the incidence of B. cepacia in raw milk of different species of dairy animals, in order to assess its potentiality as a source of infection. Also, to study the resistance of Bcc to antibiotics, since they display high levels of resistance, therefore, infections with these microorganisms are difficult to treat and in some cases result in death.

MATERIALS and METHODS

A total of 120 raw milk samples of cow’s, buffalo’s, sheep’s and goat’s milk (30 samples each) were collected from different localities in Assiut Governorate, Egypt. The samples were transported to the laboratory at 4ºC with a minimum of delay to be microbiologically examined.

Enrichment procedures: (Moore et al., 2001)

Ten milliliters of samples were aseptically inoculated into 225 ml of nutrient broth and incubated at 30 ºC for 24 hours.

Selective plating and identification of isolates:

Incubated broth cultures were streaked onto plates of B. cepacia selective agar (BCSA) as described by Henry et al. (1997). Plates were incubated at 30 ºC for 48 hours followed by a further incubation at ambient temperature for 5 days. The isolates were identified by oxidase test, nitrate reduction test and glucose, lactose, sucrose fermentation (Cowan and Steel, 1974; Harrigan and McCance, 1976; A.P.H.A., 1992; Henry et al., 2001).

Antimicrobial sensitivity test:

The antimicrobial sensitivity test and its interpretation were done using the disc diffusion method following the NCCLS standards (1997) for 10 Bcc isolates selected randomly. The following antimicrobial agents discs (Oxoid) were used to determine the pattern of resistance; ampicillin 10 µg, amoxycillin 10 µg, streptomycin 10 µg, gentamicin 10 µg and chloramphenical 30 μg.

RESULTS

Table 1: Incidence of Burkholderia cepacia complex in raw milk samples of different dairy animals species

Table 2: Antibiotic sensitivity of B. cepacia complex isolates

DISCUSSION

Presence of Burkholderia cepacia complex is not well documented in milk, so this study was carried out to investigate its presence in milk. In addition, this study aimed to detect Bcc in milk of different dairy animal species to have an overall prospective on its prevalence.

The detection of B. cepacia in raw milk (Uraz and Citak,1998) and cheese (Smith et al., 1987) were previously reported, indicating the possibility of food-borne spread to susceptible humans. The used selective agar medium, B. cepacia selective agar (BCSA) has proved to be the most effective selective agar for B. cepacia complex as it actually suppresses the growth of non B. cepacia bacteria (Henry et al., 1999).

The recorded results in Table 1, show that Bcc was detected in 31 out of 120 raw milk samples (25.83%); representing 5 (16.66%) of buffalo’s milk, 7 (23.33%) of cow’s milk, 10 (33.33%) of sheep’s milk and 9 (30%) of goat’s milk. Higher incidence was reported by Moore et al. (2001), who reported that 14 of 26 (53.8%) samples of raw bovine milk were positive for Burkholderia cepacia complex. Its presence in raw milk could be attributed to the use of contaminated water or from animals suffering from mastitis. Berriatua et al. (2001) isolated Bcc from ewe’s milk suffering from subclinical mastitis.    

Moreover, it could contaminate the milk from the environment, and this could explain the higher incidence in sheep’s and goat’s milk than that of buffalo’s and cow’s. Since the samples of sheep’s and goat’s milk were collected from farmers’ houses who most likely don’t follow hygienic measures during milking and handling of milk. The detection of Bcc in raw milk samples is of great concern as it can survive in milk for a long time (Mohan Nair et al., 2002).  

Studying the antibiotic sensitivity of Bcc in Table 2, revealed that all isolates exhibited resistance to more than one antibiotic. All isolates were resistant to ampicillin and amoxicillin (100%), while 70%, 80%, 20% of the tested isolates were susceptible to streptomycin, gentamicin and chloramphenical, respectively. Isles et al. (1984) studied the antimicrobial sensitivity of Bcc isolates and found that they were resistant to ampicillin (97%), gentamicin (97%) and chloramphenicol (45%). Further surveys on Bcc species also observed this type of broad resistance (Nzula et al., 2002 and Zhou et al., 2007). Moreover, Mahenthiralingam et al. (2005) reported that Bcc is characterized by innate resistance to antibiotics.

CONCLUSION

Results of the present study indicated that Burkholderia cepacia complex were detected in raw milk samples of different dairy animals species. Therefore, raw milk is a potential source of infection. The presence of Bcc in milk is of great concern because of their capability to grow at low temperature and because Bcc have emerged as an opportunistic pathogen especially for patients suffering from cystic fibrosis that may lead to life-threatening infections. Bcc exhibit resistance to many antibiotics, and infected patients don’t seem to respond to treatment. The eradication of Bcc as a human pathogen will become increasingly important.

REFERENCES

A.P.H.A. (American Public Health Association) (1992): Standard Methods for the Examination of Dairy Products. 16 ^th Ed., American Public Health Association.

Berriatua, E.; Ziluaga, I.; Miguel-Virto, C.; Uribarren, P.; Juste, R.; Laevens, S.; Vandamme, P. and Govan, J.R.W. (2001): Outbreak of subclinical mastitis in a flock of dairy sheep associated with Burkholderia cepacia complex infection. J. Clin. Microbiol, 39 (3): 990–994.

Burkholder, W. (1950): Sour skin, a bacterial rot of onion bulbs. Phytopath., 40: 115-117.

Coenye, T. and Vandamme, P. (2003): Diversity and significance of Burkholderia species occupying diverse ecological niches. Enviro. Microbiol., 5(9): 719-729.

Cowan, S.T. and Steel, K.J. (1974): Manual for the Identification of Medical Bacteria. 2 ^nd Ed., Cambridge Univ. Press, England.

Harrigan, W.E. and McCance, M.E. (1976): Laboratory Methods in Food and Dairy Microbiology. Academic Press, London.

Henry, D.A.; Campbell, M.E.; LiPuma, J.J. and Speert, D.P. (1997): Identification of Burkholderia cepacia isolates from patients with cystic fibrosis and use of a simple new selective medium. J. Clin. Microbiol., 35: 614- 619.

Henry, D.A.; Campbell, M.E.; Mcgimpsey, C.; Clarke, A.; Louden, L.; Burns, J.; Roe, M.; Vandamme, P. and Speert, D.P. (1999): Comparison of isolation media for recovery of Burkholderia cepacia complex from respiratory secretions of patients with cystic fibrosis. J. Clin. Microbiol., 37: 1004-1007.

Henry, D.A.; Mahenthiralingam, E.; Vandamme, P.; Coenye, T. and Speert, D.P. (2001): Biochemical and molecular approaches for determining genomovar status of the Burkholderia cepacia complex. J. Clin. Microbiol., 39: 1073- 1078.

Hirose, S.; Nakano, K.; Kosakai, Y.; Sasaki, T.; Kobayashi, J.; Sasako, Y.; Yamamoto, E.; Ueda, H.; Yutani, C. and Kitamura, S. (1998): Surgical treatment for prosthetic valve endocarditis. J. Cardiol., 31:85- 89.

Isles, A.; Maclusky, I.; Corey, M.; Gold, R.; Prober, C.; Fleming, P. and Levison, H. (1984): Pseudomonas cepacia infection in cystic fibrosis: an emerging problem. J. Pediatr., 104 (2): 206–210.

Kaitwatcharachai, C.; Silpapojakul, K.; Jitsurong, S. and Kalnauwakul, S. (2000): An outbreak of Burkholderia cepacia bacteremia in hemodialysis patients: An epidemiologic and molecular study. Am. J. Kidney Dis., 36: 199- 204.

Levy, S.B. (2002): Factors impacting on the problem of antibiotic resistance. J. Antimicrob. Chemother., 49: 25-30.

LiPuma, J.J. (1998a): Burkholderia cepacia epidemiology and pathogenesis: implications for infection control. Curr. Opin. Pulm. Med., 4: 337- 441.

LiPuma, J.J. (1998b): Burkholderia cepacia: management issues and new insights. Clin. Chest Med., 19: 473- 486.

Luvizotto, D. and Marcon, J. (2010): Genetic diversity and plant-growth related features of Burkholderia spp. from sugarcane roots. World J. Microbiol. Biotechnol., 26: 1829-1836.

Mahenthiralingam, E.; Urban, T.A. and Goldberg J.B. (2005): The multifarious, multireplicon Burkholderia cepacia complex. Nat. Rev. Microbiol., 3: 144- 156.

Mohan Nair, M.K.; Vasudevan, P.; Hoagland, T. and Venkitanarayanan, K. (2002): Survivability of Burkholderia cepacia in pasteurized and unpasteurized bovine milk stored at 4ºC and 8ºC. Annual Meeting and Food Expo, Food Microbiology, General I, 61 C, Anaheim California, USA.

Moore, J.E.; McIlhatton, B.; Shaw, A.; Murphy, P.G. and Elborn, J.S. (2001): Occurrence of Burkholderia cepacia in foods and waters: Clinical implications for patients with cystic fibrosis. J. Food Prot., 64 (7): 1076- 1078.

NCCLS (National Committee for Clinical Laboratory Standards) (1997): Approved standard M2-A6. Performance standards for antimicrobial disc susceptibility tests. 6^th Ed. NCCLS, Wayne, PA.

Nzula, S.; Vandamme, P. and Govan, J. (2002): Influence of taxonomic status on the in vitro antimicrobial susceptibility of the Burkholderia cepacia complex. J. Antimicrob. Chemother., 50 (2): 265–269.

Smith, D.; Mikolajcik, E. and Lindamood, J. (1987): Causative organisms and chemical nature of the Swiss cheese rind rot defect. Cult. Dairy Prod. J., 22: 9–12.

Speert, D.P.; Bond, M.; Woodman, R.C. and Curnette, J.T. (1994): Infection with Pseudomonas cepacia in chronic granulomatous disease: role of nonoxidative killing by neutrophils in host defence. J. Infec. Dis., 170: 1523- 1531.

Uraz, G. and Citak, S. (1998): An investigation on the distribution and isolation of Pseudomonas from raw milk samples obtained from different areas. Turk. J. Agric. For. 22: 469–474.

Yabuuchi, E.; Kosako, Y.; Oyaizu, H.; Yano, I.; Hotta, H.; Hashimoto, Y.; Ezaki, T. and Arakawa, M. (1992): Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia comb. nov. Microbiol Immunol., 36: 1251-1275.

Zhou, J.; Chen, Y.; Tabibi, S.; Alba, L.; Garber, E. and Saiman, L. (2007): Antimicrobial susceptibility and synergy studies of Burkholderia cepacia complex isolated from patients with cystic fibrosis. Antimicrob. Agents Chemother., 51 (3): 1085–1088.