AssiutUniversity web-site: www.aun.edu.eg

DETECTION OF ACINETOBACTER SPECIES IN MILK AND SOME DAIRY PRODUCTS

SAAD, N.M. ¹; AMIN, W.F. ¹ and MOSTAFA, S.M. ²

¹ Department of Food Hygiene, Faculty of Vet. Med., AssiutUniversity

² Department of Food Hygiene, Animal Health Institute, Assiut. Corresponding author: wallaa_800@yahoo.com

Received: 16 November 2017;       Accepted: 3 December 2017

INTRODUCTION

The importance of milk in diet is explained by its essential nutritive constituents. However, milk obtained from healthy udder contains numbers of bacteria that may proliferate depending on the handling and processing of milk. Acinetobacter species could be one of these bacteria. They are recognized as food borne pathogens and act as a potential human pathogen.

Bacteria of the genus Acinetobacter have gained increasing attention in recent years for their potential to cause severe nosocomial infections (Towner, 2006), their profundity in developing multidrug (MDR) and extreme drug resistance (XDR) (Prashanth and Badrinath, 2005)and for the ability of some strains to produce verotoxins (VA) (Grupper et al., 2007).The genus Acinetobacter comprises 38 species which had been recognized (Visca et al., 2011).

Acinetobacter spp. has long been recognized as a part of normal flora of the skin of animals and humans besides being present in soil, water, sewage, food and milk (Wani et al., 2006). The gastrointestinal tract of humans is the most important reservoir of resistant strains. As a result of the food colonization  studies, it

Corresponding author: Dr. AMIN, W.F.

E-mail address:wallaa_800@yahoo.com

Present address: Department of Food Hygiene, Faculty of Vet. Med., AssiutUniversity

has been suggested that transmission may be through food. Digestive tract colonization accounts for 41% of Acinetobacter baumannii cases (Corbella et al., 1996).

Acinetobacter has emerged as an important microorganism (Peleg et al., 2008).They are often associated with nosocomial infections, community acquired diarrhea outbreaks and pneumonia in tropical regions of the world especially during summer months (Chen et al., 2001). Infrequent manifestations of Acinetobacter are meningitis, bacteremia, urinary tract infection and ophthalmic infections (Jain and Danziger, 2004). 

Acinetobacter baumannii is an important emerging nosocomial pathogen worldwide. Itis a frequent cause of skin and wound infections, septicemia, peritonitis, cholangitis, osteomyelitis and endocarditis (Dent et al., 2010).Acinetobacter haemolyticus isassociated with endocarditis and verotoxin production, and hence bloody diarrhea (Grotiuz et al., 2006).  

The most common sources of Acinetobacter in milk are residual water in milking machines, milk pipelines or coolers, inadequate cleaning of dairy equipment, dirty udders and teats, transport and storage of milk, and biofilm (Santana et al., 2004).

Acinetobacter show lipolytic activity (Hantsis-Zacharov and Halpern, 2007). As a result of milk fat hydrolysis by bacterial lipases of Acinetobacter, free fatty acids are released causing changes in the product flavor. The lipolytic flavor defects are noticeable in cream, cheese and sterilized milk (Champagne et al., 1994).

Since, Acinetobacter species continue to be an important pathogen particularly A. baumannii, therefore, this work was planned to estimate the incidence of Acinetobacter spp. in milk and some dairy products and to study the lipolytic activity of the isolated strains.

MATERIALS AND METHODS

I. Sampling:

A total of 240 random samples of milk and some milk products were divided as 120 raw milk samples, 30 pasteurized milk samples, 60 Domiati and Kareish cheese (30 each) and 30 cream samples. All the samples were collected from different localities in Assiut city, Egypt. Samples were transferred to the laboratory as soon as possible to be examined for the incidence of Acinetobacter spp. Each milk sample supposed to be raw was tested for heat treatment using Storch test according to Lampert (1975)to exclude heat treated milk. The samples were prepared according to the technique recommended by A.P.H.A (1992).

II. Isolation and identification of Acinetobacter species:

One milliliter of each well mixed milk sample or 1 g of each prepared milk product was inoculated into nutrient broth. The broth was incubated at 37°C for 24-48 hours. Aliquots of the broth cultures were streaked onto plates of Leeds Acinetobacter agar medium and incubated at 37°C for 24-48 h. Acinetobacter spp. colonies appeared pink with mauve background (Jawad et al., 1994). Gram negative bacilli were identified biochemically according to Procop et al. (2017). Acinetobacter isolates were non motile, oxidase negative, catalase positive, failed to decarboxylate lysine. Acinetobacter species differentiation was done by biochemical reactions including growth at 42°C, gelatin hydrolysis, citrate utilization and growth at 37°C according to Bouvet and Grimont (1987).

III. Detection of Acinetobacter baumannii by multiplex PCR for the identification of blaOXA-23-like,blaOXA-51-like and class 1 integrase genes:

DNA Extraction

Bacterial DNA was extracted following overnight culture on nutrient agar plates using QIAamp kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions.  

Amplification of the genes of Acinetobacter baumannii according to Turton et al. (2006)

The extracted DNA was subjected to PCR amplification in a thermal cycler (Master cycler, Eppendorf, Hamburg, Germany) using 25 μl reaction volumes with 3 μl of extracted DNA, 12.5 pmol of each primer (blaOXA-23-like, blaOXA-51-like & Int 1) (Pharmacia Biotech, USA) and 1.5 U of Taq DNA polymerase (Biotools, Madrid, Spain) in 1x PCR buffer containing 1.5 mM MgCl₂ (QIAGEN) and 200 µM of each deoxynucleoside triphosphate. The amplification conditions were denaturation 94°C for 3 min, followed by 35 cycles at 94°C for 45 sec, annealing at 57°C for 45 sec, elongation at 72°C for 1 min and extension at 72°C for 5 min. Amplified products were analyzed by 1.5% of agarose gel electrophoresis stained with ethidium bromide and visualized and captured on UV transilluminator.  

The primers used are shown in the following table:

IV. Detection of the lipolytic activity of the isolated Acinetobacter spp.  

Overnight cultures were streaked onto tributyrin agar and incubated at 30°C for 3 days. The medium appeared opaque, but a clear zone surrounded lipolytic colonies(Harrigan and McCance, 1976).

RESULTS

Table 1: The Incidence of Acinetobacter species in the examined raw milk samples.

Table 2: The Incidence of Acinetobacter species in the examined milk and dairy products samples. 

Figure 1. Detection of Acinetobacter baumannii by using blaOXA-23-like, blaOXA-51-like and Class 1 integrase (Int 1) genes.

Lane M: 100 bp ladder as molecular size DNA marker.

Lane 1: Control positive A. baumannii for blaOXA-23-like gene (501 bp), blaOXA-51-like gene (353 pb) and Int 1 gene (160 bp)

Lane 2: Control negative

Lanes 3- 13: Positive examined A.baumanniistrains

Table 3: The lipolytic activity of the isolated Acinetobacter species.

Table 4: The lipolytic activity of the identified species of Acinetobacter organisms in the examined samples.

DISCUSSION

Acinetobacter species are of major concern because of their rapid development of resistance to a wide range of antimicrobials and their long persistence in the environment. In addition, their transmission is via person-to-person contact, water and food contamination(Doughari et al., 2011).

This study took in consideration to cover most of the customers’ purchasing habits by examining the four types of raw milk sources; street vendors, dairy shops, dairy farms, and farmers’ houses.

The recorded results in Table 1showed that 20% of the examined street vendors’ raw milk samples were contaminated with Acinetobacter spp.; they included A. baumannii and A. junii (3.3% each), A. haemolyticus and A. calcoaceticus (6.7% each). The high percentage obtained in street vendors’ raw milk samples may be due to the natural habitats of Acinetobacter in water, soil and environmentand may be due to contaminated tanks (Fournier et al., 2006). Moreover, Acinetobacter species were detected in 13.3% of dairy shops raw milk samples, only 3.3% of dairy farms milk samples were contaminated with Acinetobacter spp. In either type of samples A. baumannii was the only detected species. Higher incidence was reported by Nam et al. (2009)who isolated Acinetobacter spp. in a percentage of 8.2%. Also, Acinetobacter spp. was isolated from 23.3% of the examined farmers’ houses raw milk samples; out of which, A. baumannii was detected in20% of the samples and A. haemolyticus in 3.3%.The obtained results showed that the highest percentage of Acinetobacter was in farmers’ houses raw milk samples. This could be explained by Wani et al. (2006) who mentioned that Acinetobacter spp. is recognized as a normal flora of skin of animals and humans besides being present in soil, water and sewage.

It was clear from Table 2 that Acinetobacter spp. was detected in 15% of the raw milk samples; A. baumannii (10%), A. haemolyticus(2.5%),A. calcoaseticus(1.7%) andA. junii(0.8%).Other investigators could isolate Acinetobacter spp. from raw milk in lower incidences;Uraz and Çitak (1998)could isolate Acinetobacter spp. in a percentage of 4.5%, whileGurung et al. (2013) isolated Acinetobacter spp. (7.7%) and A. baumannii (2.5%) from milk samples andRafei et al. (2015)could isolate A. baumannii in 2.7% of raw milk samples.

Not surprisingly, Acinetobacter spp. was not detected in the examined pasteurized milk samples and it was because of the high pasteurization temperature. This was confirmed by Wang et al. (2006) who demonstrated that pasteurization was highly effective against Acinetobacter.

 Acinetobacter spp. was isolated from 3.3% of the examined Domiati cheese samples (Table 2) and A. baumannii was the only recovered species. The presence of Acinetobacter in Domiati cheese may be due to the use of raw milk or inefficient heat treated milk for the production of the cheese or contamination after pasteurization during the production and the handling of the cheese. Furthermore, the way of selling of Domiati cheese (not properly packed) could be a cause of contamination in the refrigerator of the groceries where various types of foods are put closely to each other.

The popularity of traditional cheese varieties is increasing progressively all over the world, and some are still being traditionally produced. Kareish cheese is a traditional cheese produced in Egypt from raw milk. Acinetobacter was detected in 13.3% of the examined Kareish cheese samples (Table 2); A. baumannii was detected in 10% of the Kareish cheese samples while A. calcoaceticus in 3.3%. It is worth mentioning that Rafei et al. (2015) isolated A. baumannii from raw cheese samples in a percentage of 14.3%. The presence of Acinetobacter in the Kareish cheese samples may be due to the bad handling and cutting during processing in farmers houses and contamination from soil, the primitive way of production and selling and the use of unpasteurized milk.

For the examined cream samples, Acinetobacter spp. was isolated (13.3%). A. baumannii (10%) and A. haemolyticus (3.3%) were identified.

The Multiplex PCR was used to identify the genes of A. baumannii; blaOXA-23-like, blaOXA-51-like and class 1 integrase genes (Figure 1). Turton et al. (2006)identified blaOXA-51-like gene in each of 141 isolates of Acinetobacter baumannii, and they confirmed that blaOXA-51-like is ubiquitous in A. baumannii.  

Many investigators have reported the role of extracellular enzymes in virulence. Lipases constitute virulence factors by interacting with human leukocytes or by affecting several immune system functions by free fatty acids generated by lipolytic activity (Oliver et al., 1997).Moreover,degeneration of milk components through various enzymatic activities can reduce the shelf life of the processed milk. Lipases hydrolyze tributyrin and milk fat and yield free fatty acids, which produce a range of flavor defects (Shah, 1994).

Out of the 27 tested Acinetobacter strains from the examined milk and dairy products, 23 (85.2%) showed lipolytic activity (Table 3). The results presented in Table 4 showed that out of the 15 strains showing lipolytic activity from the raw milk samples, 60% were A. baumannii, 20% A. haemolyticus, 13.3% A. calcoaceticus and 6.7% A. junii. Additionally, the only A. baumannii that was isolated from Domiati cheese showed lipolytic activity. The 4 strains obtained from the Kareish cheese samples possessing lipolytic activities were A. baumannii (75%) and A. calcoaceticus (25%). Only 3 strains out of four from the cream samples showed lipolytic activity; A. baumannii (66.7%) and A. haemolyticus (33.3%). Gennari et al. (1992)could isolate Acinetobacter from dairy sources and reported that only 3.7% of Acinetobacter was able to produce ropy milk due to their lipolytic activity.

Therefore, the aforementioned data proved that attention must be paid to the problems of this pathogen in food. Consequently, more restriction and preventive measures should be taken to improve the quality of milk and dairy products to protect consumers from being infected by the discussed microrganism. Also, a precautionary approach is advisable for the reduction or eradication of Acinetobacter from the human food chain should be encouraged.

REFERENCES

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

Bouvet, P.J. and Grimont, P.A. (1987):“Identification and biotyping of clinical isolates of Acinetobacter”. Annales de l'Institut Pasteur/Microbiologie, Elsevier, 138: 569-578.

Champagne, C.P.; Laing, R.R.; Roy, D.; Mafu, A.A.; Griffiths, M.W. and White, C. (1994): “Psychrotrophs in dairy products: their effects and their control”. Critical Reviews in Food Science & Nutrition, 34(1): 1-30.

Chen, M.Z.; Hsueh, P.R.; Lee, L.N.; Yu, C.J.; Yang, P.C. and Luh, K.T. (2001): “Severe community- acquired pneumonia due to Acinetobacter baumannii”. Chest Journal, 120(4): 1072-1077.

Corbella, X.; Pujol, M.; Ayats, J.; Sendra, M.; Ardanuy, C.; Domínguez, M.A.; Liñares, J.; Ariza, J. and Gudiol, F. (1996): “Relevance of digestive tract colonization in the epidemiology of nosocomial infections due to multiresistant Acinetobacter baumannii”. Clinical Infectious Diseases, 23(2): 329-334.

Dent, L.L.; Marshall, D.R.; Pratap, S. and Hulette, R.B. (2010): “Multidrug resistant Acinetobacter baumannii: a descriptive study in a city hospital”. BMC Infectious Diseases, 10(1): 196.

Doughari, H.J.; Ndakidemi, P.A.; Human, I.S. and Benade, S. (2011): “The ecology, biology and pathogenesis of Acinetobacter spp.: an overview”. Microbes and Environments/JSME, 26(2): 101-112.

Fournier, P.E.; Richet, H. and Weinstein, R.A. (2006): “The epidemiology and control of Acinetobacter baumannii in health care facilities”. Clinical Infectious Diseases, 42(5): 692-699.

Gennari, M.; Parini, M.; Volpon, D. and Serio, M. (1992): “Isolation and characterization by conventional methods and genetic transformation of Psychrobacter and Acinetobacter from fresh and spoiled meat, milk and cheese”. International Journal of Food Microbiology, 15(1): 61-75.

Grotiuz, G.; Sirok, A.; Gadea, P.; Varela, G. and Schelotto, F. (2006): “Shiga toxin 2-producing Acinetobacter haemolyticus associated with a case of bloody diarrhea”. Journal of Clinical Microbiology, 44(10): 3838-3841.

Grupper, M.; Sprecher, H.; Mashiach, T. and Finkelstein, R. (2007): “Attributable mortality of nosocomial Acinetobacter bacteremia”. Infection Control and Hospital Epidemiology, 28(3): 293-298.

Gurung, M.; Nam, H.; Tamang, M.; Chae, M.; Jang, G.; Jung, S. and Lim, S. (2013): “Prevalence and antimicrobial susceptibility of Acinetobacter from raw bulk tank milk in Korea”. Journal of Dairy Science, 96(4): 1997-2002.

Hantsis-Zacharov, E. and Halpern, M. (2007): “Culturable psychrotrophic bacterial communities in raw milk and their proteolytic and lipolytic traits”. Applied and Environmental Microbiology, 73(22): 7162-7168.

Harrigan, W. and McCance, M.E. (1976): “Laboratory methods in food and dairy microbiology”. Academic Press, London. 7: 549-564.

Jain, R. and Danziger, L.H. (2004): “Multidrug-resistant Acinetobacter infections: an emerging challenge to clinicians”. Annals of Pharmacotherapy, 38(9): 1449-1459.

Jawad, A.; Hawkey, P.; Heritage, J. and Snelling, A. (1994): “Description of Leeds Acinetobacter Medium, a new selective and differential medium for isolation of clinically important Acinetobacter spp., and comparison with Herellea agar and Holton's agar”. Journal of Clinical Microbiology, 32(10): 2353-2358.

Koeleman, J.G.; Stoof, J.; Van Der Bijl, M.W.; Vandenbroucke-Grauls, C.M. and Savelkoul, P.H. (2001): “Identification of epidemic strains of Acinetobacter baumannii by integrase gene PCR”. Journal of Clinical Microbiology, 39(1): 8-13.

Lampert, L. (1975): “Modern Dairy Products”. 3^rd Ed. Chemical Publishing Company, New York, USA.

Nam, H.; Lim, S.; Kang, H.; Kim, J.; Moon, J.; Jang, K.; Joo, Y. and Jung, S. (2009): “Prevalence and antimicrobial susceptibility of gram-negative bacteria isolated from bovine mastitis between 2003 and 2008 in Korea”. Journal of Dairy Science, 92(5): 2020-2026.

Oliver, J.; Kaper, J.; Doyle, M.; Beuchat, L. and Montville, T. (1997): “Food microbiology: fundamentals and frontiers”. ASM Press, Washington, D.C., USA.

Peleg, A.Y.; Seifert, H. and Paterson, D.L. (2008): “Acinetobacter baumannii: emergence of a successful pathogen”. Clinical Microbiology Reviews, 21(3): 538-582.

Prashanth, K. and Badrinath, S. (2005): “Epidemiological investigation of nosocomial Acinetobacter infections using arbitrarily primed PCR & pulse field gel electrophoresis”. Indian Journal of Medical Research, 122(5): 408.

Procop, G.; Church, D.; Hall, G.; Janda, W.; Koneman, E.; Schreckenberger, P. and Woods, G. (2017): “Nonfermenting Gram negative bacilli. In Koneman's Color Atlas and textbook of Diagnostic Microbiology”. 7^th Ed. Lippincott Williams and Wilkins Company, Philadelphia, USA. 

Rafei, R.; Hamze, M.; Pailhoriès, H.; Eveillard, M.; Marsollier, L.; Joly-Guillou, M.L.; Dabboussi, F. and Kempf, M. (2015): “Extrahuman Epidemiology of Acinetobacter baumannii in Lebanon”. Applied and Environmental Microbiology, 81(7): 2359-2367.

Santana, E.; Beloti, V.; Müller, E.; Ferreira, M..; Moraes, L.; Pereira, M. and Gusmão, V. (2004): “Milk contamination in different points of the dairy process: mesophilic, psychrotrophic and proteolytic microorganisms”. Semina: Ciências Agrárias, Londrina, 25(4): 349-358.

Shah, N.P. (1994): “Psychrotrophs in milk: a review”. Milchwissenschaft, 49: 432-437.

Towner, K. (2006): “The genus Acinetobacter”. The Prokaryotes, Proteobacteria: Gamma Subclass, 6: 746-758.

Turton, J.F.; Woodford, N.; Glover, J.; Yarde, S.; Kaufmann, M.E. and Pitt, T.L. (2006): “Identification of Acinetobacter baumannii by detection of the blaOXA-51-like carbapenemase gene intrinsic to this species”. Journal of Clinical Microbiology, 44(8): 2974-2976.

Uraz, G. and Çitak, S. (1998): “The isolation of Pseudomonas and other Gram (—) psychrotrophic bacteria in raw milk”. Journal of Basic Microbiology, 38(2): 129-134.

Visca, P.; Seifert, H. and Towner, K.J. (2011): “Acinetobacter infection–an emerging threat to human health”. IUBMB Life, 63(12): 1048-1054.

Wang, C.Y; Wu, H.D.; Lee, L.N.; Chang, H.T.; Hsu, Y.L.; Yu, C.J.; Yang, P.C. and Hsueh, P.R. (2006): “Pasteurization is effective against multidrug-resistant bacteria”. American J. Infection Control, 34(5): 320-322.

Wani, S.; Samanta, I.; Bandey, M. and Bhat, M. (2006): “Isolation of Acinetobacter lwoffii from broiler chicken with septicaemia in Kashmir valley”. Indian Journal of Animal Research, 40(1): 61-63.

Woodford, N.; Ellington, M.J.; Coelho, J.M.; Turton, J.F.; Ward, M.E.; Brown, S.; Amyes, S.G. and Livermore, D.M. (2006): “Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp”. International Journal of Antimicrobial Agents, 27(4): 351-353.