DETECTION OF AEROMONAS HYDROPHILA IN RAW MILK AND SOME MILK PRODUCTS WITH REFERENCE TO ITS PUBLIC HEALTH HAZARD

Assiut University web-site: www.aun.edu.eg

DETECTION OF AEROMONAS HYDROPHILA IN RAW MILK AND SOME MILK PRODUCTS WITH REFERENCE TO ITS PUBLIC HEALTH HAZARD

O.A. SADEK¹, N.H. MAKAR² and S.M. EL BERBAWY²

¹ Food Hygiene Department, Animal Health Research Institute, Assiut Lab.

² Bacteriology Department, Animal Health Research Institute, Assiut Lab.

Received: 31 January 2017;       Accepted: 22 February2017

INTRODUCTION

Milk is an excellent medium for the growth of numerous microbes which produce consequential spoilage of the milk and various milk products or food-borne pathogens to the consumers (Oliver et al., 2005). Aeromonads are autochthonous to the aquatic environment (Aboulhamd, 2010) and are also common contaminants in diverse variety of foods like fish, sea foods, raw and cooked meats, poultry, milk and milk products, eggs and vegetables (Sharma and Kumar 2011). Scoaris et al. (2008) showed that, Aeromonads are efficient colonizers of surfaces and are an important constituent of bacterial biofilms in both water distribution systems and food processing environments.

Corresponding author: Dr.O.A. SADEK

E-mail address:onsi_2000@yahoo.com

Present address:Food Hygiene Department, Animal Health Research Institute, Assiut Lab

Isolation of Aeromonas hydrophila from water and food sources, and the increasing resistance of this organism to antibiotics and chlorination in water, presents a significant threat to public health (Chang et al., 2008). Also, Aeromonas species have also been linked to both food and water-borne diseases in different parts of the world especially developing countries due to poor personal hygiene and lack of quality water (Odeyemi and Ahmad, 2014). Moreover, Aeromonas hydrophila and Aeromonas caviae are considered major pathogens most commonly implicated in human intestinal infections (Van Gravenitz, 2007) and also account for more than 95% of all blood-borne infections (Ghenghesh et al., 2008). Furthermore, Five types of diarrhea of Aeromonas related gastroenteritis, secretory (acute watery diarrhea often with vomiting), dysenteric (accompanied by blood and mucus in the stool), chronic (lasting longer than 10 days), choleric (rice water stools) and travelers were reported (Janda and Duffey, 1988).

Extra-intestinal infections due to Aeromonas species as fatal bacteremic pneumonia, orbital cellulitis, fatal Aeromonas hydrophila infection of soft tissue in a cirrhotic patient, epidural abscess, wound infection, prostatitis, septic shock, ecthyma gangrenosum, diabetic foot, fatal Aeromonas hydrophila myonecrosis and sepsis were reported (Kao et al., 2003; Chou et al., 2004; Liu et al., 2005; Tsai et al., 2005; Easow and Tuladhar, 2007; Avolio et al., 2009; Talan et al., 2014  and Yumoto et al., 2014).

It has been observed that some strains of Aeromonas are enteropathogenic and possess virulence factors as enterotoxins, cytotoxins, haemolysins and invasins and this association between haemolysin production and enterotoxicity is none other than the Aeromonas sp. which can grow and release enterotoxin and haemolysin even at fridge temperature conditions (Sharma and Kumar, 2012). Antimicrobial resistance among enteric pathogens is a serious problem in developing countries where there is a high frequency of gastroenteric illness and many antibiotics fall routinely into inadequate use. Antibiotic resistance is particularly relevant in pathogenic Aeromonas species in which, besides the classical resistance to β-lactamic antibiotics, multiple-resistance has been frequently identified (Vila et al., 2002). These bacteria can receive and transfer antibiotic resistance genes to other Gram negative bacteria (Marchandin et al., 2003).

Due to the public health hazard of Aeromonas species, this study aimed to detect these microorganisms in raw milk and some milk products consumed in Assiut city, Egypt. Also, to determine the antimicrobial resistance, proteolytic, lioplytic, psychrotrophic and haemolytic activities of the isolated organisms. Moreover, confirmation of Aeromonas hydrophila strains by detection of 16S rRNA gene and determination of haemolysin (ahh1) and aerolysin (aerA) virulence genes of isolated strains by PCR assay were assessed.

MATERIALS AND METHODS

A total of 100 raw milk, kareish cheese (traditional Egyptian cheese made from raw skimmed milk), ice cream and baladi yoghurt (25 samples, each) were collected from dairies and street peddlers in Assiut city, Egypt. The samples were collected in clean and sterile plastic bags in an ice-box and transferred rapidly as soon as possible to the laboratory for bacteriological examination.

A)   Preparation of samples:

The apparently normal raw milk samples were mixed thoroughly and tested for heat treatment by Storch test according to Lampert (1975) before being subjected to examination. Ten ml from liquid samples and 10g from solid samples were added individually to 90 ml of 0.1% sterile peptone water. Ten-fold serial dilutions from each sample were done up to 10⁶ (A.P.H.A., 2001).

B)   Bacteriological examination of the prepared samples:

1)       Enumeration of Aeromonas hydrophila, using m-Aeromonas selective agar, according to Palumbo et al. (2001): From each dilutions, 0.1 ml was spread over m-Aeromonas selective agar containing 10 mg ampicillin/liter with sterile bent glass rod and incubated at 28 ^oC for 24 hours. Then the numbers of isolated Aeromonas were calculated and typical colonies were picked into nutrient agar slants for biochemical identification.

2)       Application of PCR for identification of 16S rRNA, aerolysin (aerA) and haemolysin (ahh1) virulence genes of Aeromonas hydrophila:-

a)       Primer sequences of Aeromonas hydrophila used for PCR identification system: Application of PCR for identification of 16S rRNA, aerolysin (aerA) and haemolysin (ahh1) virulence genes of Aeromonas hydrophila was performed essentially by using primers (Pharmacia Biotech) as shown in the following table:

b)       DNA Extraction using QIA amp kit (Shah et al., 2009): After sub-culturing of Aeromonas hydrophila on starch ampicilin agar, one or two colonies were suspended in 20 ml of sterile distilled water, and the suspension was then heated at 100 ºC for 20 minutes. Accurately, DNA was extracted from all isolates by using QIAamp kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions). All DNA extracts were stored at -20 ºC until used.

c)       Amplification reaction of Aeromonas hydrophila (Wang et al., 2003): The amplification was performed on a Thermal Cycler (Master cycler, Eppendorf, Hamburg, Germany). Multiplex PCR assays were adopted by using 25 μl of the reaction mixture contained 2X AmpliTaq DNA polymerase (Perkin-Elmer) – 12.5 μl, and 1.0 μl of each ahh1 and aerA primers, 0.2 μl of 16S rRNA primers and 1 μl extracted DNA. Amplification conditions consisted of an initial denaturation at 95 °C for 5 min., 50 cycles at 95 °C for 30 sec., 59 °C for 30 sec., 72° C for 30 sec., followed by final elongation at 72° C for 7 min. Amplified DNA fragments were analyzed by 2% of agarose gel electrophoresis (Applichem, Germany, GmbH) in 1X TAE buffer (0.04 M Tris, 0.02 M Acetic acid, 0.002 M Na2 EDTA) at 100 V for 45 min with 8 μl PCR product. Finally, the gel was stained with ethidium bromide and captured as well as visualized on UV transilluminator. A 100 bp plus DNA Ladder (Qiagen, Germany, GmbH) was used to determine the fragment sizes.

3)       Detection of proteolytic activity of Aeromonas spp. according Swift et al. (1999): This was performed by placing aseptically loopful of culture on the center of the 10 % of skim milk agar plate and spreading it in a circular fashion to cover an area about 5 to 18mm in diameter; then the plates were incubated in an inverted position at 37 ºC for 24 to 48 h. Clearing the cloudy agar (zone of proteolysis) indicated a positive result.

4)       Detection of lipolytic activity of Aeromonas spp. according to Harrigan (1998): The strains were subcultured in tributyrin agar (Plate Count Agar supplemented with 1% tributyrin) and then incubated at 37 °C for 48 h. The colonies were considered positive when a precipitation halo formed around the colony, indicating the release of enzymes into the growth medium.

5)       Determination of psychrotrophic activity of Aeromonas spp., using Standard Plate count Agar, according to A.P.H.A. (2001): The isolated Aeromonas spp. strains were inoculated on Standard Plate Count Agar and incubated at 7 ^oC for 10 days then examined for Aeromonas growth.

6)       Determination of haemolytic activity of Aeromonas spp. according to Swift et al. (1999): Haemolytic positive isolates were identified by the presence of clear (β-haemolysis) halos around the colonies grown at 37 °C for 24 h on TSA agar (Difco) containing 5 % rabbit blood.

7)       Antibiogram of Aeromonas species according to CLSI (2011): Aeromonas spp. strains isolated in the present study were subjected to susceptibility testing against 8 antimicrobials commonly used. Susceptibility was determined by the disk-diffusion technique of Kirby-Bauer on Mueller-Hinton agar plates with inocula adjusted to an optical density of 0.5 McFarland standard units. Disks containing Ampicillin (AMP 10μg), Amoxicillin (AML 10μg), Kanamycin (K 30μg), Ceftriaxone (CRO 30μg), tetracycline (TE 30μg), Erythromycin (E 15μg), Trimethoprim/Sulfamethoxazole (SXT 25μg) and ciprofloxacin (CIP 5μg) were used. After 24 h incubation at 30°C, organisms were classified as Sensitive (S), Intermediately resistant (I) or Resistant (R) on the basis of the size of the zone of bacteria growth inhibition.

RESULTS

Table 1: Aeromonas spp. count in raw milk, kareish cheese, ice-cream and yoghurt (n= 25).

< 100 means negative samples

Table 2: Incidence of countable Aeromonas spp. in raw milk, kariesh cheese and ice-cream samples (n= 25).

Table 3: Proteolytic and lipolytic activities of Aeromonas spp. detected in raw milk, kareish cheese and ice-cream samples.

Table 4: Psychrotrophic and haemolytic activities of Aeromonas spp. detected in raw milk, kareish cheese and ice-cream samples.

Table 5: Incidence of 16S rRNA, aerolysin (aerA) and haemolysin (ahh1) virulence genes in isolated Aeromonas  hydrophila organism (n= 9).

Photograph (1): Agarose gel electrophoresis of multiplex PCR of 16S rRNA (356 bp), aerA (309 bp) and ahhl (130 bp) genes for characterization of Aeromonas hydrophila.

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

Lane C+: Control positive Aeromonas hydrophila for 16S rRNA, aerA and ahhl genes.

Lane C-: Control negative.

Lanes 2, 3, 4, 6 & 9: Positive Aeromonas hydrophila for 16S rRNA, aerA and ahhl genes.

Lanes 1 & 7: Positive Aeromonas hydrophila strains for 16S rRNA and ahhl genes.

Lane 8: Positive Aeromonas hydrophila strain for 16S rRNA and aerA genes.

Lane 5: Positive Aeromonas hydrophila strain for 16S rRNA gene.

Table 6: Antibiogram of Aeromonas spp. isolated from raw milk and some milk products.

^*Trimethoprim-Sulfamethoxazole

DISCUSSION

The illustrated results in Table 1 revealed that, the incidence of counted Aeromonas spp. in the examined raw milk samples was 36%, with counts ranging from < 100 to 1.2×10⁶ and with an average count of 1.0×10⁵ cfu/ml. Lower incidences (8.7, 26 and 32%) were estimated by Amer et al. (2008); Enany et al. (2013) and Ahmed et al. (2014), respectively. On the other hand, higher incidences (40, 49.2, 86.7, 46.7 and 58%) were detected by El-Shorbagy and Al-Ganzoury (2002); Yucel et al. (2005); Korashy (2006); El-Taib and Mohamed (2010) and Eid et al. (2013), respectively. It is worth mentioning that, Aeromonas organisms can invade udder tissues; multiply in mammary tissues and subsequently discharge in milk. Also, the contaminated water used for washing milking equipments is considered as a significant source of contamination. Therefore, presence of Aeromonas in a high level in raw milk samples in this study is indicative to bad hygienic measures of milk production and distribution (Korashy, 2006).

The incidence of counted Aeromonas spp. in the examined kareish cheese samples was 32%, with a count ranging from < 100 to 6.0×10⁵ and with an average count of 3.2×10⁴cfu/g (Table 1). Lower incidence of 12% was reported by Enany et al. (2013), whereas, higher incidences (92, 58, 70 and 70%) were revealed by Al-Ashmawy (2000); Effat et al. (2000); Korashy (2006) and Eid et al. (2013), respectively. The presence of Aeromonas species in kareish cheese could be attributed to the bad quality of the raw milk used, the unsanitary manufacturing practices, improper handling and distribution. Also, the kareish cheese manufacturing process itself does not appear to be deleterious to Aeromonas spp.

Moreover, the incidence of counted Aeromonas spp. in ice cream samples was 24%, with a count ranging from < 100 to 7.0×10³ and with an average count of 5.0×10²cfu/g (Table 1). Lower incidence of 18% was estimated by Sharef et al. (2006). The presence of Aeromonas spp. in ice cream samples indicated the unhygienic measures during preparation and distribution of the products. Aeromonas spp. isolated from human stool samples in percentage of 3.4% (Aslani and Alikhani, 2004), therefore, presence of Aeromonas hydrophila in ice cream may indicate faecal contamination from workers. Furthermore, good hygienic practices and good personal hygiene must be applied to produce products save for human consumption.

Aeromonassp. was not detected in baladi yoghurt samples in this study (Table 1). Motlagh et al. (1991) reported that, starter culture bacteria in yoghurt can produce antimicrobial activity against Aeromonas hydrophila and they declared that, diacetyl had some bactericidal activity against the tested strains. This observation could explain why Aeromonas was not detected in yoghurt samples in this study. Moreover, low pH of yoghurt may have an inhibitory effect on Aeromonas organisms or other factors which need further investigation.

The incidence of Aeromonas hydrophila in the examined raw milk samples was 16% (Table 2). Similar incidences (15.9 and 17.1%) were found by Melas et al. (1999) and Subashkumar et al. (2006), respectively. Whereas, lower incidences (9 and 7%) were detected by El-Leboudy et al. (2014) and Alrazakkazal and Abdullah (2016), respectively. On the other hand, higher incidences (36 and 24%) were revealed by Abdel-Raouf and Naima (2011) and Zeinhom and Abdel-Latef (2014), respectively. Aeromonas hydrophila isolated from feaces of normal cow in percentage of 21.1% (Agarwal et al., 2000), therefore, presence of Aeromonas hydrophila in raw milk may indicate faecal contamination of milk.

Table 2 revealed that, the incidence of Aeromonas hydrophila in the examined kareish cheese samples was 12%. Higher incidence of 32.5% was obtained by Nazem et al. (2010). While for the examined ice cream samples, the incidence of Aeromonas hydrophila was 8%. Higher incidence of 40% was estimated by Nazem et al. (2010). From the aforementioned results, raw milk, kareish cheese and ice cream may cause risks for public health from Aeromonas hydrophila infection, especially for immune-compromised person, children and aged. Hence, there is need for public enlightenment, campaign and general education to assist in curtailing the outbreak of diseases in human through ingestion of the bacteria along with milk and milk products. Preventive method should be taken during food preparation; good personal hygiene and proper sanitation procedure should always be use to prevent human exposure to this disease.

The entire isolated Aeromonas hydrophila organism detected in raw milk, kareish cheese and ice cream (9 strains), in this study, were confirmed by PCR assay for detection of 16S rRNA gene and all the tested isolate were harboring this gene (Table 5 and Photograph 1). The 16S rRNA gene was included as an internal control and has become the gold standard method for definitive species identification (Wang et al., 2003 and Geetha and Michael, 2015).

Aeromonas caviae was isolated from raw milk samples in percentage of 12% (Table 2). Similar incidence of 13% was revealed by Melas et al. (1999). For kareish cheese samples, the incidence of Aeromonas caviae was 16% (Table 2) and this result was lower than that detected by Effat et al. (2000). Concerning ice cream samples, the incidence of Aeromonas caviae was 12% (Table 2).

The incidence of Aeromonas sobria in the examined raw milk samples was 8% (Table 2). Lower result (3.6%) was obtained by Melas et al. (1999), whereas higher incidence was estimated by Akan et al. (1996). In contrary, Seker et al. (2015) could not isolate the organism from milk samples. Regarding to kareish cheese samples, the incidence of Aeromonas sobria was 4% (Table 2). Higher incidences were detected by Effat et al. (2000) and Korashy (2006). With regard to ice cream samples, the incidence of Aeromonas sobria was 4% (Table 2). The aforementioned results revealed that, Aeromonas hydrophila and Aeromonas caviae organisms were the most predominant strains isolated from raw milk, kareish cheese and ice cream samples followed by Aeromonas sobria. 

The isolated Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria organisms in this study were tested for its virulence properties by proteolytic and lipolytic assay and all the tested isolates exhibited 100% proteolytic and lipolytic activities (Table 3). Similar results were observed by Castro-Escarpulli et al. (2003), Singh et al. (2010), Al-Fathawy and Al-Ammar (2013), Al-Oqaili et al. (2016) and Simon et al. (2016).

It is worth mentioning that, Proteases play an important role in gastroenteritis by proteolytic activation of the haemolysin and also, it plays an important role in pathogenesis by providing nutrients for the bacteria during the colonization and the invasion of the bacteria in the host (Santos et al., 1999 and Zhu et al., 2007). Furthermore, Lipases have been considered to be important for bacterial nutrition and also play role as virulence factors by interacting with human leukocytes or by affecting several immune system functions through free fatty acids generated by lipolytic activity. Extracellular lipases secreted by Aeromonas spp. actively participate in the alteration of the host plasma membrane and thus increase the severity of infection (Pemberton et al., 1997).

Nevertheless, proteolytic and lioplytic properties of the Aeromonads in this study may cause bitterness, rancidity and other defects in milk and milk products. Therefore, good hygiene masseurs and strict eradication programs must be applied in dairy plant to produce milk and milk products save for human consumption and of good keeping quality.

Results in Table 4 revealed that, all the tested Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria organisms had 100% psychrotrophic properties. Lower results were estimated by Ahmed et al. (2014). The psychrotrophic properties of Aeromonads are aiding factors for survival and multiplication of the organisms in foods even at refrigerator temperature consequence health risks to consumers.

Table 4 showed that, all the tested Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria had 100% β-haemolytic activities. Similar results were estimated by Subashkumar et al. (2006); Al-Fathawy and Al-Ammar (2013); Joseph et al. (2013); Taj-Aldeen et al. (2014); Al-Oqaili et al. (2016) and Simon et al. (2016). On the other hand, lower results were revealed by Majeed (2011); Manna et al. (2013); Amsaveni et al. (2014); Mansour et al. (2014) and Sharma et al. (2015).

Haemolytic activity is an index of pathogenicity and the relationship between the production of haemolysin and the enterotoxigenicity in Aeromonas spp. well documented (Obi et al., 2007). Nevertheless, all the isolated Aeromonads in this study were haemolytic on blood agar indicating their pathogenic nature and had potential public health significance in food of animal origin. Therefore it is concluded that, haemolytic activity on blood agar is simpler and an easier way than PCR assay for detection Aeromonads pathogenicity and also economical to use especially for developing countries.

The illustrated results in Table 5 and Photograph 1 revealed that, the incidences of aerA and ahh1 genes in the tested Aeromonas hydrophila were 66.67 and 77.78%, respectively. Similar incidence was detected by Al-Fathawy and Al-Ammar (2013), whereas lower result was estimated by Yousr et al. (2007). In contrary, higher incidences were recorded by Rather et al. (2014) and Simon et al. (2016). The aerA and ahh1 genes are cytotoxic and these two toxins contribute to development of severe diarrhoea in humans because of their synergistic effects (Guerra et al., 2007).

From Photograph 1 it is clear that, the strain of Aeromonas hydrophila in Lane 5 had neither aerA gene nor ahh1 gene while it exhibited haemolytic activity on blood agar (Table 4). Therefore one can easily conclude that, haemolytic activity of Aeromonas hydrophila may be expressed by genes other than aerA and ahh1 genes and this observation need further investigations.  

The tabulated results in Table 6 showed that, all the tested Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria organisms exhibited 100% resistance towards Ampicillin, Amoxicillin and Erythromycin antibiotics. Similar results were revealed by Kannan et al. (2001); Majeed et al. (2011); Enany et al. (2013); Furmank-Blaszk (2014) and Sharma et al. (2015). The tested strains of Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria exhibited sensitivity to Kanamycin in percentages of 22.22, 40 and 75%, respectively. Similar results were obtained by Subashkumar et al. (2006) and Didugu et al. (2016).

The isolated Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria organisms were sensitive towards Ceftriaxon in percentages of 55.56, 40 and 75%, respectively (Table 6). Similar results were observed by Ragunathan et al. (2012) and Eidet al. (2013). For Tetracycline, the Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria organisms exhibited 44.44, 50 and 25% sensitivity, respectively (Table 6) and these results simulated what obtained by Kaskhedikar and Chhabra (2010). While for Trimethoprim-Sulfamethoxazole, the tested organisms of Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria showed sensitivity percentages of 0.0, 20 and 50%, respectively and these were lower than what detected by Aboulhamd (2010). Concerning Ciprofloxacin, all the tested Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria strains were sensitive in percentage of 100%  and theses results go parallel with what estimated by Castro-Escarpulli et al. (2003); Yucel and Citak (2003); Guerra et al. (2007)  and Kaskhedikar and Chhabra (2010).

The problem of Aeromonads resistance towards multiple antibiotics causes an increase in the risk of treatment failure and cost for antimicrobial therapy and hospitalization, while the range of therapeutic options decreases (Wen-Chien et al., 1998). Moreover, the emergence of pathogenic bacteria resistant to most, if not all, currently available antimicrobial agents has become a critical problem in modern medicine, particularly because of the concomitant increase in immune-suppressed patients.

CONCLUSION

This study revealed that, raw milk, kareish cheese and ice cream that retailed in Assiut city, are largely contaminated with Aeromonads microorganisms. The isolated Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria exhibited virulence properties on the bases of proteolytic, lioplytic, psychrotrophic and haemolytic activities and in addition to the presence of aerA and ahh1 genes in Aeromonas hydrophila. Resistance of the recovered Aeromonads towards different varieties of antibiotics was observed and Ciprofloxacin was the most efficient antibiotic against Aeromonas species. Good hygienic control measures must be applied in dairy farms in addition to the use of raw milk of good bacteriological quality before manufacturing of different varieties of milk products.

Dedication: To the soul of Dr. N.H. Makar, Senior Researcher, Bacteriology Department, Animal Health Research Institute, Assiut Lab., who died before accomplishing this work.

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