Public health risks of Escherichia coli and Staphylococcus aureus in raw bovine milk sold in informal markets in Egypt

Introduction: Milk is an important food in Egypt and most of it is sold as raw milk in informal markets. Methodology: This study was conducted to investigate the public health risks of Escherichia coli and Staphylococcus aureus in milk sold in informal markets in Egypt. A total of 121 milk samples were analyzed for occurrence, virulence genes and antibiotic resistance of E. coli and S. aureus. Results: A total of 35/121 (28.9%) of milk samples were contaminated with 16/121 (13.2%) E. coli, 22/121 (18.2%) S. aureus, and 3/121 (2.5%) both isolates. Shiga-toxin producing E. coli (STEC), Enterotoxigenic E. coli (ETEC) and Enterotoxigenic S. aureus were detected in 5/121 (4.1%), 2/121 (1.7%) and 8/121 (6.6%) of the examined milk samples, respectively. Multiple drug resistances (MDRs) were showed by 14/16 (87.5%) and 21/22 (95.5%) of E. coli and S. aureus isolates, respectively. E. coli isolates showed high resistance for cephalothin (87.5%), ampicillin (68.8%) and tetracycline (68.8%), but were sensitive for gentamicin and chloramphenicol. Resistance phenotypes of E. coli were diverse; however, STEC isolates were significantly associated with co-resistance to cephalothin, ampicillin and tetracycline (P< 0.05). Two (9.1%) of S. aureus isolates were methicillin-resistant (MRSA) but sensitive to gentamicin (GS-MRSA). Five (22.7%) of S. aureus isolates were gentamicin-resistant methicillin-sensitive S. aureus (GR-MSSA). S. aureus isolates also showed high resistance for ampicillin (100%), tetracycline (90.1%) and sulfamethoxazole-trimethoprim (90.1%). Conclusion: These findings highlighted the potential public health hazards of E. coli and S. aureus pathogens in raw milk sold in informal


Introduction
Raw milk consumption by consumers may be attributed to the lack of awareness of foodborne pathogens in raw milk [1].Another segment of the population may consume dairy products manufactured from raw milk.In a study in Egypt, over 80% of the farmers would process dairy products from raw milk [2].In developed countries, up to 5% of food-borne infections were related to the consumption of milk and dairy products [3].The case scenario could be worse for developing countries where high rates of milk contamination associated with unhygienic milk production and lack of efficient preservation [4].
Escherichia coli pathogens are often used as indicator of fecal contamination of milk and may impose presence of pathogenic serotypes for humans [4,3].Whereas, Staphylococcus aureus contamination of milk either associated with milkers or milk handlers, especially those with poor hygienic habits as coughing or sneezing during milking or milk handling [5], or with infected cows as reservoirs of S. aureus infection [6].In addition, E. coli and S. aureus are major causes of subclinical and clinical mastitis [6,7].In subclinical mastitis, E. coli and S. aureus are shed in milk without abnormalities in milk consistency or udder shape; hence, humans may impact their health by consuming or processing milk from these cases.
Shiga-toxin producing E. coli (STEC) and Enterotoxigenic E. coli (ETEC) were associated with several life-threatening food-borne outbreaks worldwide [8,9].STEC produces cytotoxins encoded by stx1 and stx2 genes.These cytotoxins are associated with serious human illnesses as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) that usually end up with fatal consequences [9].STEC causes around 3 million cases of acute illness and over 200 deaths each year [9].ETEC causes diarrhea via production of heat-stable and heat-labile enterotoxins encoded by ST and LT genes, respectively.ETEC isolates are the most common cause of endemic diarrhea in children in developing countries [8,10].In addition, it is the most frequent etiology of travelers' diarrhea [8].
S. aureus is one of the most common pathogens of food poisoning.S. aureus pathogens in milk, under favorable conditions, produce heat-stable enterotoxins which cause vomiting, and abdominal cramping with or without diarrhea [5].The disease is self-limiting; however, few cases especially in infants and elderly may suffer acute illness and death [5].Staphylococcal enterotoxins (SEs) are encoded by several genes; however, sea, seb, sec, sed and see were the most common genes implicated in cases of food poisoning worldwide [5].The seriousness of E. coli and S. aureus milk-borne food poisoning was exaggerated with the emergence of Multi drug resistant (MDR) isolates worldwide, which may pose an additional threat to human health [11,12].
Egypt is considered the largest producer of bovine milk in Africa with an annual production of more than 5 million tons [13].However, around 70% of milk is produced by Smallholder and sold in informal markets in Egypt [2].Standard hygienic measures are rarely applied in this kind of production, therefore this study aimed to investigate the prevalence of E. coli and S. aureus as important food-borne pathogens in raw milk collected from informal markets in Kafrelsheikh governorate.The potential zoonotic hazards of these pathogens in term of virulence and antibiotic resistance were also studied.

Study area and design
Kafrelsheikh is the largest governorate located in the middle of Nile-Delta of Egypt (31°06′42′′N 30°56′45′′E).A cross-sectional study was conducted between June and December 2014 to determine the prevalence, virulence genes and antibiotic resistance of E. coli and S. aureus as food-borne pathogens in raw milk sold in informal market for residents of Kafrelsheikh governorate.In informal milk markets, consumers purchased raw milk directly from individual farmers or milk collectors.The official authorities do not supervise informal markets.Majority of farmers who sell their milk in informal markets are smallholders of 3 to 5 household reared animals [2].Farmers either sell their milk directly to consumers or sell it to milk collectors, who in turn sell the milk in the market.Milk in informal markets is sold without any heat treatment.

Sampling
A total of 121 raw milk samples were collected from informal markets in 10 villages in Kafrelsheikh governorate.Milk samples were collected from 10 to 15 individual farmers or milk collectors in each village.All collected milk samples were of normal colour, odour and without any flakes.Collected milk samples were immediately transported in icebox to lab for further analysis.On Arrival to Lab, milk samples were tested for possible heat treatment by peroxidase (storch) test [14].

Microbial isolation and identification
Pre-enrichment: all milk samples were diluted at rate of 1:9 in nutrient broth (LabM, Heywood, U.K.) and incubated at 37 o C for 6 hours.
Isolation and identification of E. coli [15]: 1 mL of broth culture was mixed with 9 mL of EC broth (Oxoid, Hampshire, U.K.) and incubated at 44 o C for 20 hours.An inoculum from EC broth with gas production was streaked on Levine's Eosin Methylene Blue agar (Oxoid, Hampshire, U.K.) and MacConkey agars (Oxoid) and incubated at 35 o C for 20 hours.Suspected colonies were purified by sub-culturing on new selective agar plates and preserved on in nutrient agar slants for further analysis.For isolation of E. coli O157, the milk samples were selectively enriched in Tryptone Soy broth supplemented with 20mg/L Novobiocin (Oxoid, Hampshire, U.K.) and incubated for 6 hours at 37 o C. A loopful from the broth was streaked on Sorbitol MacConkey agar (Oxoid, Hampshire, U.K.) supplemented with Cefixime -Tellurite supplement (Oxoid, Hampshire, U.K.) and incubated at 35 o C for 20 hours.All suspected E. coli colonies were confirmed by biochemical tests using API-20E (bioMérieux, Marcyl'Etoile, France) and PCR according to [16].Confirmed isolates were sent for serotyping at Animal Health Research institute, Ministry of Agriculture of Egypt.
Isolation and identification S. Aureus [17]: A loopful from each pre-enrichment culture was streaked on Baired Parker Agar (Oxoid, Hampshire, U.K.) supplemented with Egg Yolk Tellurite (50mL/L) (Oxoid, Hampshire, U.K.) and incubated at 37 o C for 24-48 hours.Suspected S. aureus colonies were identified by PCR detection of Staphylococcal 16s RNA gene [18], biochemical tests using API Staph system (bioMérieux, Marcy-l'Etoile, France) and tube coagulase test.Tube coagulase test was conducted according to the guidelines of FDA [17].In brief, 0.3 mL of overnight culture was transferred into a sterile tube containing 0.5 mL of rabbit plasma with EDTA.The tubes were incubated at 37°C and examined periodically for 6 hours.Only tubes with complete and firm coagulation were considered positive.

DNA extraction and Molecular analysis
DNA extraction: DNA extraction from overnight Broth culture was performed using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations.Briefly, 200 µL of the sample was incubated with 10 µL of proteinase K and 200 µL of lysis buffer at 56 o C for 10 minutes.After incubation, 200 µL of 100% ethanol was added to the lysate.The sample was then washed and centrifuged.Nucleic acid was eluted with 100 µL of elution buffer provided in the kit.
Molecular analysis: PCR amplification of the targeted virulence genes was conducted by serious of multiplex PCR reactions as follow; for Stx1 and Stx2 genes [19], for Sta and LT genes [20] and for SEs genes [21] using the following mixture; 25µLEmeraldAmp Max PCR Master mix (Takara Bio, Kusatsu, Japan), 1µL(20 pmol) of each primer (Metabion, Steinkirchen, Germany), 10µL of DNA template and water added up to 50µL reaction volume.For mecA gene [22],a 25-µL reaction volume containing 12.5 µL of EmeraldAmp Max PCR Master Mix (Takara Bio, Kusatsu, Japan), 1 µL(20 pmol) of each primer, 4.5 µL of water, and 6 µL of DNA template.The reaction was performed in an Applied biosystem 2720 thermal cycler and the PCR products were separated by electrophoresis on 1.5% agarose gel (Applichem, Darmstadt, Germany) before examination.Primers sequences, target genes, amplicon sizes and annealing temperature are listed in Table 1.

Antibiotic sensitivity test
Standard disk diffusion assay was conducted using Muller-Hinton agar and broth culture equivalent to 0.5 McFarland standards as recommended by the Clinical and Laboratory Standards Institute [23].Antibiotic disks were chosen based on commonly used antibiotics for animal and human therapy in the study region and the guidelines of CLSI [23].A total of 15 antibiotic disks (Oxoid, Hampshire, U.K.) were used (14 antibiotics for E. coli isolates and 10 antibiotics for S. aureus) as showed in Table 2. S. aureus isolates that showed phenotypic resistance for cefoxitin was confirmed as MRSA by molecular detection of mecA gene [22].

Statistical analysis
Association between virulence genes and antibiotic resistance profile in the isolated E. coli and S. aureus was calculated using Fisher's exact test on SPSS v19 (SPSS Inc. 2010).Significant association was considered at P<0.05.

Results
A cross-sectional study was conducted to assess the potential public health risks associated with consumption or dairy manufacture of informally marketed raw bovine milk in Kafrelsheikh governorate, Egypt.
Results in Table 3 and Figures (1,2) showed that investigated virulence genes were detected in 43.8% and 45.5% of the E. coli and S. aureus isolates, respectively.Stx2 (18.8%) and STa (12.5%) were the predominant virulence genes in STEC and ETEC isolates, respectively.In S. aureus, sec gene was the most prevalent gene (27.3%),while lowest detection rates were for sea and seb genes (4.5% for each).None of the E. coli and S. aureus isolates showed the existence of LT or Sed genes, respectively (Figures 1,  2).
MDR was also recorded by 95.5% of the S. aureus isolates.All S. aureus isolates were resistant to ampicillin (100%) and 90.1% of the isolates showed resistance for tetracycline and sulfamethoxazole-Trimethoprim (Table 2).Two MRSA isolates (9.1%) were identified by resistance to Cefoxitin and PCR detection of mecA gene in these isolates (Table 2, 4).
E. coli isolates with either Stx1 or Stx2 genes were significantly associated with co-resistance to cephalothin, ampicillin and tetracycline (P= 0.03),  however there was no clear association between virulence genes and resistance phenotype in the isolated S. aureus (Table 4).

Discussion
A cross-sectional study was conducted to assess the potential public health risks associated with consumption or dairy manufacture of informally marketed raw bovine milk in Kafrelsheikh governorate.
Almost one third (28.9%) of the examined bovine milk in this study showed the existence of either or both E. coli or S. aureus pathogens.E. coli was detected in 13.2% of the examined milk samples, which was comparable with previous report (15.9%) in Saudi Arabia [24].Higher prevalence was recorded in Ethiopia (29.6%) by Garedew et al. [4].
STEC were recorded in 4.1% of the milk samples, which was lower than another report (17.4%) in Iran Untypable O-serogroup; Bold: highlight the significant association between phenotypic co-resistance to cephalothin, ampicillin and tetracycline with Stx genes (P < 0.05).
[25] and higher than prevalence (2.4%) reported in USA [1].O157-STEC were detected in 0.8% of the milk samples which was lower than other studies in Egypt (2.3%) [3], and in Saudi Arabia (4.8%) [24].However, Selim et al. [11] failed to detect any O157-STEC in milk samples in another study in Egypt.Non O157-STEC isolates were predominant in this study (3.3%).Four different Non O157-STEC O serogroups were detected (O158, O146, O125 and O126), which indicates high diversity of E. coli serotypes circulation among cattle in the study regions.This diversity of STEC O serogroups may also implies high rate of Stx genes transfer between different serogroups.
Particularly, when the predominant O125 and O126 serogroups, classical enteropathogenic (EPEC), gained Stx genes probably from mixing with other STEC O serogroups in same host as previously reported [26].O158-STEC and O125-STEC were previously reported from cattle samples in Egypt [11] and elsewhere [27].
All detected STEC serogroups detected in this study were implicated in human diseases as follow; O157-STEC in cases of Diarrhea in Egypt [11], O126-STEC, O157-STEC and O158-STEC in urinary tract infection cases in Egypt [11,28], while O125-STEC and O146-STEC were detected in human diarrhea cases from elsewhere [29].ETEC were isolated from 1.7% of milk samples.O126-ETEC and O167-ETEC were the reported serogroups in this study.O126-ETEC and O167-ETEC were associated with cases of human diarrhea in Egypt [10] and worldwide [8].Moreover, O126-ETEC was isolated from more than 30% of human urine samples in Egypt [28].
The relatively high rate of E. prevalence in examined milk samples may indicate high rate of fecal contamination, which correlate with inadequate udder hygiene.Hygienic milking practices as the use of teat dipping, udder washing, use of disinfectant in animal's house cleaning are not applied by most of smallholders in many developing countries including Egypt [4].
Virulence genes were detected in 43.8% of E. coli isolates.In STEC, Stx2 was prevalent than Stx1, which agreed with several studies [24,25].LT gene was not detected in ETEC, yet both LT and ST genes were previously reported in human isolates from cases with diarrhea in Egypt [10].
S. aureus was recorded in 18.2% of the milk samples, which was higher than that reported in Brazil (7.3%) [30], and lower than another report in Czech Republic (32.2%) [31].Enterotoxigenic S. aureus isolates were detected in 6.6% of milk samples, which was lower than another study (25%) in Hungary [32].Raw milk sold in informal market usually combine unhygienic production with lack of any preservation before or during marketing, which could be the reasons for the relative high S. aureus prevalence in milk samples in this study.
SEs genes were detected in 45.5% of the isolated S. aureus, which was higher than those reported in Brazil (22.2%) [30], and in Hungary (27.1%) [32].The sec gene was the prevalent SE gene in this study.The sec gene was also the predominant SE gene in S. aureus isolated from cases of mastitis in Egypt [5].The sea, seb and see genes were also detected but sed gene was absent from all S. aureus isolates in this study.Enterotoxins of sea, sec and to lesser extent see genes were implicated in several Staphylococcal food poisoning outbreaks in USA, France, Japan, Taiwan and UK [5].
MDRs were reported at high rates for E. coli (87.5%) and S. aureus (95.5%) isolates in this study.In accordance with our findings, high MDR rates of these two pathogens in milk and dairy products were reported in several countries including Egypt [11,12,25,28,31].The highest resistance rates of E. coli isolates were for cephalothin, which agreed with findings of Paneto et al. [27].In addition, E. coli isolates showed high resistance for ampicillin and tetracycline, which was in line with several reports worldwide [11,25,27].Same high resistance against ampicillin and tetracycline were also reported in E. coli isolates from human cases in Egypt [11,33].This may indicate high rates of resistance transmission for these antibiotics between animal and human isolates in Egypt.Similar findings were reported by Cho et al. [34], who detected a significant association for cephalothin and gentamicin resistance between E. coli isolates from animals and humans in contact with them.
Despite diversity of phenotypic resistance patterns among E. coli isolates in this study, there was a significant association (P< 0.01) between isolates with Stx genes (STEC) and phenotypic co-resistance to cephalothin, ampicillin and tetracycline.This finding was in line with Rehman et al. [35], who observed significant positive associations between resistance phenotypes and some virulence genes in E. coli isolates from ruminants in China.
All S. aureus isolates showed high resistance for ampicillin (100%) and tetracycline (90.1%), which agreed with other reports in Czech Republic [31] and in China [12].MRSA isolates represented 9.1% of the S. aureus isolates in this study.This finding was lower than another report (51%) in Czech Republic [31].The MRSA isolates showed resistance to all antibiotics except for gentamicin (GS-MRSA), however five of the MSSA isolates were resistant to this antibiotic (GR-MSSA).Both GS-MRSA and GR-MSSA were implicated in nosocomial and community acquired human outbreaks worldwide [36,37].
Unlike high diversity of resistance profiles of E. coli isolates, 54.5% of the S. aureus isolates can be grouped in 5 resistance profiles.The major profile (18.2% of the isolates) showed co-resistance to 8 antibiotics including ampicillin, kanamycin, and tetracycline which agreed with Li et al. [12] findings, who reported the predominance of co-resistance to ampicillin, kanamycin and tetracycline in S. aureus isolates from milk samples in China.This high degree of similarities between S. aureus isolates in this study may indicate genetic relatedness and a common source of infection.

Conclusions
Around one third of the examined samples were contaminated with E. coli and /or S. aureus.Over 40% of these pathogens were toxigenic and some of these toxigenic isolates are highly pathogenic to human even in low infectious doses (i.e.STEC).MDRs was reported in a very high rate by majority of S. aureus and E. coli isolates and transmission of the antibiotic resistance of theses pathogens to humans via foods may complicate the treatment options for clinical cases.These findings indicate that raw milk sold in informal markets in Egypt may possess a potential public health hazards for consumers.Restriction of informal raw milk marketing and public awareness about its zoonotic hazards may be a good strategy for minimizing the risks of milk-borne food poisoning in Egypt.

Table 1 .
Primers used for detection of virulence genes in E. coli and S. aureus pathogens isolated from milk in this study.

Table 2 .
Antibiotic resistance phenotypes of E. coli and S. aureus pathogens isolated from milk in this study.

Table 3 .
Frequency distribution of virulence genes in E. coli and S. aureus isolated from milk in this study.

Table 4 .
Association between antibiotic resistance phenotypes and virulence genes in E. coli and S. aureus pathogens isolated from milk in this study.