African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5090

Full Length Research Paper

Effect of using treated wastewater on the bacteriological quality of raw cow's milk: A case of a farm in Northeastern Algeria

Keltoum Chorfi
  • Keltoum Chorfi
  • Department of Microbiology and Biochemistry, Faculty of Nature and Life Sciences, University of Batna 2, Batna, Algeria.
  • Google Scholar
Katia Bendjemana
  • Katia Bendjemana
  • Department of Molecular and Cellular Biology, Faculty of Nature and Life Sciences, Abbes Laghrour University, Khenchela, Algeria. BP: 1252 Route de Batna Khenchela, Algeria.
  • Google Scholar
Ammar Ayachi
  • Ammar Ayachi
  • Department of Veterinary Sciences, Institute of Veterinary and Agricultural Sciences, University of Batna 1, Batna, Algeria.
  • Google Scholar
Fatma Mahdi
  • Fatma Mahdi
  • Laboratory of Microbiology Applied to Agri-food, Biomedical and Environment (LAMAABE), University of Tlemcen, Algeria.
  • Google Scholar
Nour El Aimane Bouzidi
  • Nour El Aimane Bouzidi
  • Larbi Ben M'Hidi University, Oum El Bouaghi, Algeria.
  • Google Scholar

  •  Received: 20 March 2020
  •  Accepted: 27 April 2020
  •  Published: 31 August 2020


This study aims to assess the impact of the use of treated wastewater (without chlorination) in farming and dairy cattle breeding. Milk samples were collected from a farm in northeastern Algeria. The treated wastewater from the treatment plant is used on this farm for different activities. The results obtained show that the average contamination of milks with total flora is 3.7.105 CFU /ml. Fecal coliforms are present at an average value of 1.5.103 CFU/ml. All of the samples (100%)  were positive for the count of fecal enterococci with an average value of 2.5.10 CFU/ml. Fungal flora was present with an average value of 1.36,103 CFU/ml. Escherichia coli was isolated in 100% of the samples with high resistance rates for beta-lactam antibiotics. The results obtained for the search for pathogens belonging to the genus Staphylococcus show that 64% of the isolates were coagulase-negative Staphylococcus and 36% of the isolates were coagulase-positive. The study of Staphylococcus susceptibility/resistance to antibiotics revealed high frequencies of resistance, especially to beta-lactam antibiotics and macrolides. The bacteria tested show a majority resistance for Penicillin and Oxacillin (100%). These results reflect the microbiological risk that the consumption and marketing of this milk represents for the health of consumers and the need to implement preventive measures.


Key words: Irrigation, fecal coliforms, Escherichia coli, Staphylococcus sp, antibiotic resistance, microbiological risk.


The emergence and spread of antibiotic resistance genes among   pathogenic   and   non-pathogenic  bacteria  has  been a growing threat in recent decades and there is a rapid  lack  of  therapeutic options (Li and Webster, 2018;
Barancheshme and Munir, 2018). The emergence of this resistance in bacteria in animals and their products has attracted considerable interest due to the potential of transferring this resistance to the human population (Vásquez et al., 2017; McDermott et al., 2018). Suspected sites of resistance transmission include wastewater treatment plants where wastewater from various sources, including municipalities, sanitary wastewater, hospital effluents, storm water runoff and industries, is mixed and treated using a multi-stage purification process (Mohammadali and Davies, 2017; Hultman et al., 2018).
The use of wastewater for irrigation is observed as a way to address the imbalance between demand and supply of water. However, the literature shows that irrigation with treated wastewater is not without implications, some of which are negative (Gatto D'Andrea et al., 2015; Becerra-Castro et al., 2015).
Inadequately treated water from sewage systems represents both a risk to human and animal health if it is used to pasture or fodder crops grazed by livestock or otherwise consumed (Cass and Lowe, 2014). As a result, the water may contain bacteria, viruses, protozoa and helminth eggs that would be a risk to the livestock, or to humans who have contact with or consume livestock products (meat, milk, eggs, etc.) (Drechsel et al., 2010). The wastewater treatment plant on the wilaya of Khenchela (Northeastern Algeria) is a low load activated sludge   with  a  capacity  of  23,000  m3/day  for  192,000 equivalent/inhabitant. A mixture of urban, industrial, agricultural, storm water runoff and hospital wastewater from the city of Khenchela is discharged to this treatment plant, only to be finally discharged without tertiary treatment and disinfection into Baghai wadi.
The owner of traditional farm uses treated water at the outlet of a wastewater treatment plant to irrigate his pasture field and breed his cows (Figure 1). On the farm, this water is used for three main activities: pasture cultivation, dairy barn farming and cleaning, and for dairy cow consumption.
The main objective of this study is to assess the risks associated with the reuse of treated wastewater in agriculture. The dairy industry is particularly concerned about the potential effect on dairy cattle and milk quality following pasture irrigation with waste water. Therefore, monitoring bacterial pathogens, their survival and transfer is of the utmost importance to ensure that milk quality is not compromised.


Raw milk samples were obtained after a manual milking of the four healthy cows, from the lactating udder, just before the first morning milking. Milk sample (100 ml) was collected in a sterile bottle after washing and disinfecting the teats and removing the first draft. The milk samples were immediately placed at a temperature of +4°C and then quickly sent to the laboratories for an analysis of its biochemical and microbiological composition.
FT-IR instrumental analysis
The biochemical composition of raw milk samples was performed by Fourier Transform Infrared Spectroscopy (FTIR). It is a rapid biochemical fingerprinting technique (Nicolaou et al., 2010). It can potentially be applied to produce results with the same accuracy and sensitivity as reference methods in a short period of time (Nicolaou and Goodacre, 2008). Measurements were made using a Fourier Transform Infrared Spectrometer (FTIR) (DYNASCAN Border Spectrum, Perkin-Elmer Ltd, England), equipped with a deuterated triglycerin sulfate (DTGS) detector stabilized at an optimized temperature in the far and mid infrared. Infrared spectra were recorded at 64 scans in the range of 8 300 to 50 cm-1 with a resolution of 4 cm-1. KBr separator was used to record milk spectra. The milk sample was ground with KBr powder to be pressed into a tablet. Then, the IR spectrum was collected.
Microbiological analyses
The microbiological analyses were carried out at the Microbiology Laboratory of the University of Khenchela (Algeria). From milk previously homogenized, serial decimal dilutions were prepared in peptone saline diluent using standard methods. (ISO 6887 - 5: 2010). All raw milk samples were analyzed for the presence of total aerobic mesophilic bacteria (TAMB), fecal coliforms (CF), fecal enterococci (FE), yeasts and viable molds and for the detection of Escherichia coli and positive and negative coagulase Staphylococcus using the standard methods described below.
Total aerobic mesophilic bacteria enumeration
Total aerobic mesophilic bacteria (TAMB) were measured according to the standard method (ISO 4833-1: 2013). The Petri dishes were inoculated separately with 1 ml of each dilution to which Plate Count Agar (PCA) was added (Pasteur Institute, Algeria). After 72 h of incubation, all colonies were counted and the results were expressed in units forming colony per ml of milk (CFU/ml).
Fecal coliforms and E. coli counts
Fecal coliforms (FC) and E. coli were measured using the standard method (ISO 4831:2006 and ISO 7251:2005).  Fecal coliforms were counted using the most probable number (MPN) technique in brilliant green bile (2%) broth (Pasteur Institute of Algeria). After the incubation period of 24 to 48 h at 44°C, the pattern of positive results was compared with a table of most probable numbers. The counts were expressed in units forming colony per ml of milk (CFU/ml). For the isolation and identification of E. coli, positive tubes showing turbidity and gas production were cultured on selective medium Hektoen agar (Pasteur Institute, Algeria) and incubated at 37°C for 24 h. Large yellow salmon colonies on Hektoen agar were suspected as E. coli strains and further confirmation was made by following standard microbiological techniques which include colony morphology studies, Gram staining. Biochemical analysis of E. coli isolates was performed using API 20E strips (BioMérieux).
Fecal Enterococci enumeration
Intestinal enterococci were counted using the most probable number method in Rothe broth (Pasteur Institute, Algeria). After incubation from 48 h at 37°C,  the  contents  of  the  positive  tubes, showing turbidity, were inoculated on BEA (Bile Esculine Azide) medium at 37°C for 24 and 48 h, for confirmation. Enterococcal colonies were small, translucent and surrounded by a black halo (positive esculin) (Maury, 1987).
Staphylococcus detection
Staphylococcus detection was performed according to the standard method (ISO 6888-1:2003) on Baird Parker agar supplemented by egg yolk and potassium tellurite (Pasteur Institute, Algeria) by a spread plate technique; after enrichment on Giolitti Cantoni Base broth (Pasteur Institute, Algeria) (ISO, 2003). The agar plate was aerobically incubated for 24 - 48 h at 37°C. The positive result of the test is the appearance of colonies surrounded by a light halo with a black or grey center. Suspected colonies were sub-cultivated on the same selective medium plates and incubated at 37 °C for 24 h to obtain a pure culture. Pure cultures were further examined for morphological staining and cultural characteristics as well as biochemical characteristics (fermentation of mannitol, catalase and coagulase). For the identification of Staphylococcus species, API 20 Staph strips (BioMérieux) were used (Zangerl and Asperger, 2003).
Viable yeasts and molds enumeration
ISO 21527-1:2008 specifies a horizontal method for the enumeration of viable yeasts and molds in products intended for human consumption or animal feeding with a water activity greater than 0.95 (eggs, meat, dairy products (except milk powder), fruit, vegetables, fresh pasta, etc.), using the colony counting technique at 22 -25°C (ISO, 2008).
The spread-plate technique is strongly preferred to the pour-plate technique for enumeration of yeasts and molds in foods using dilution plating. Spread plating avoids any risk of thermal inactivation of fungal propagules which may be associated with the pour-plate technique and facilitates maximum exposure of cells to atmospheric oxygen (Beuchat, 2003). A sample of 0.1 ml of appropriately diluted sample is deposited in duplicate on the surface of the oxytetracycline glucose yeast extract agar (OGYE) (Pasteur Institute, Algeria). Then it was uniformly spread on the surface using a curved sterile glass rod. The rods must not exceed 2 mm in diameter in order to minimize the adhesion of the sample at the end of the spreading procedure. The agar plates were aerobically incubated for 5 days at 22°C (Beuchat, 2003). All colonies were counted and the results were expressed in units forming colony per ml of milk (CFU/ml).
Antimicrobial susceptibility/resistance test
An antimicrobial susceptibility/resistance test by disc diffusion on Mueller-Hinton agar (Pasteur Institute, Algeria) (Bauer et al., 1966) was performed for all E. coli and Staphylococcus isolates according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2017). The antimicrobial agents and disc charges used in this study on E. coli isolates were ampicillin (AMP 10 μg), amoxicillin + clavulanic acid (AMC 30 μg), ceftazidime (CAZ 30 μg), Imipenem (IMP 10 μg), Ofloxacin (OFX 05 μg), Nitrofurantoin (F 300 μg), Gentamicin (CN 10 μg), Amikacin (AK 30 μg), Colistine (CT 50 μg) and Fosfomycin (FF 200 μg) (Thermo Scientific oxoid, France).
The antimicrobial agents and disc charges used in this study on Staphylococcus isolates were Penicillin (P 10 μg), Oxacillin (OX 1 μg), Amikacin (AK 30 μg), Gentamicin (CN 10 μg) and Kanamycin (K 30 μg), Erytromycin (E 15 μg), Clindamycin (DA 2 μg), Pristinamycin (PT 15 μg), Ofloxacin (OFX 5 μg), Levofloxacin (LEV 5μg), Vancomycin (VD 5μg), Rifampicin (RD 5μg) and Cotrimoxazol (SXT 1.25 μg) (Thermo Scientific oxoid, France). The area diameter for each antimicrobial agent was then transformed into sensitive, intermediate and resistant categories according to the performance standards interpretation table for antimicrobial susceptibility testing (MHPHR, 2014). For quality control, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 were used as reference strains.


Fourier transforms infrared (FTIR) spectroscopy
Figure 2a and b. show the Fourier transform infrared absorption spectra of  raw  milk  samples  in  the  spectral range of 400 - 4 000 cm-1. There are numerous peaks that correspond to the different molecular bonds of milk components interacting with infrared radiation. Three main components, fat, protein and lactose, all have strong and characteristic peaks.
Determination of the fat content of milk samples from Fourier Transform Infrared (FTIR) spectra is mainly based on 2 specific regions. The spectral region between 1 725 and 1 850 cm-1 showed bands of low absorption due to the carbonyl group (C=O) of milk lipids (commonly called fat A). Another spectral region of medium intensity between 2 800 and 2 924 cm-1 absorbed infrared light due to the alkyl chain of fatty acids (commonly called fat B)   (Lefier   et   al.,  1996;  Eskildsen  et  al.,  2016).  The infrared peaks at 2 854 and 1 744 cm-1 observed in Fig. 2.a. could be related to the milk fat contents B and A respectively.
Milk protein is expected to have absorption bands around 1 650, 1 550 and 1 250 cm-1 due to amide I, amide II and amide III groups, respectively (Dagnachew et al., 2013). It also has an absorbance peak in the region between 1 060 and 1 100 cm-1, associated with a phosphate group bound to casein protein (Etzion et al., 2004). Figure 2b shows two spectral bands of medium intensity, the peak of 1 242 cm-1 in the wavelength range of 1 225-1 280 cm-1 corresponds to the N-H bending and C-N stretching vibrations of amide III (Lei et al., 2010). The other peak of 1 546 cm-1 in the spectral range of 1 525-1 580 cm-1 was obtained by bending the n-plane N-H with the C-N stretching vibrations of amide II (Moros et al., 2006). 
Lactose is expected to have an absorption peak in the infrared region between 1 030 and 1 150 cm-1 due to the presence of various C-O stretching vibrations in carbohydrates (Grappin et al., 2006; Zhou et al., 2006). Figure 2a shows a high intensity peak of 1 076 cm-1 in the spectral range between 1 000 and 1 150 cm-1 and this peak could be related to the lactose content of the milk. A typical water transmittance spectrum between 3 650 and 3 000 cm-1 was represented in the hydroxyl group (O-H) (Coitinho et al., 2017) (Figure 2a).
Microbiological analyses
The average contamination of milk samples with total flora is 3.7.105 CFU/ml. fecal coliforms were present at an average value of 1.5,103 CFU/ml.  All of the samples (100%) were positive for the count of fecal enterococci with an average value of 2.5.10 CFU / ml. For fungal flora, it was present with an average value of 1.36,103 CFU/ml.
According to the microbiological criteria of the inter-ministerial decree of 04-10-2016 of the OJ No.: 39/17 of the Algerian Republic (Table 1) (JORA, 2017), the overall quality of the study sample is compromised. Fecal enterococci were the first causes of non-compliance: A hundred percent of the samples was unsatisfactory, the total aerobic mesophilic flora and fecal coliforms do not exceed the acceptability limit according to the Algerian standard.
E. coli isolation and identification
For E. coli testing, 25 isolates were isolated, purified and identified. Antibiotic resistance was tested for each of the bacteria identified against 10 different antibiotics. The values of inhibition diameters were compared with the values in the reading table (MHPHR, 2014). Resistance rates for each antibiotic were calculated; the results obtained are grouped in Table 2.
Seventy two percent of E. coli isolates were resistant to Ampicillin (AMP). Forty per cent (40%) of isolates were to Amoxicillin/Clavulanic acid (AMC). The resistance frequencies for Ceftazidime (CAZ) (third generation cephalosporin) and Imipenem (IMP) were 28 and 00% respectively. The resistance rate obtained for antibiotics belonging to the aminoglycosides class is variable, with a resistance rate of 08% for Gentamicin and 32% for Amikacin. This variability would be due to the low consumption of Gentamicin giving the existence of less toxic and more effective molecules. Eight percent of E. coli isolates was resistant to Colistin, 12% to fosfomycin, ofloxacin and Nitrofurantoin.
Staphylococcus isolation and identification
The second cause of non-compliance of the milk sample is the presence of Staphylococcus. All samples were positive for Staphylococcus; colonies that developed on Baird Parker agar after incubation were stained with Gram stain and staphylocoagulase tested to distinguish strains with pathogenic potential (S. aureus) from non-pathogenic  strains. Twenty five strains were isolated and purified; their identification by the API 20 Staph system revealed the predominance of coagulase-negative Staphylococcus (64%) compared to the coagulase-positive S. aureus species (36%). The main species of coagulase negative Staphylococcus isolated and their respective frequencies were: S. hominis (36%), S. xylosus (08%), S. warneri (08%), S. epidermidis (04%), S. chromogenes (04%) and S. lugdunensis (04%).
Antibiotic resistance was tested for each of the bacteria identified against 13 antibiotics. Resistance rates for each antibiotic were calculated and the results obtained are presented in Table 3. It can be observed that resistance rates vary significantly from one antibiotic to another. The Staphylococcus species studied showed a 100% resistance rate to Penicillin, and Oxacillin.  The resistance rate obtained for antibiotics belonging to the aminoglycosides class varies, a zero resistance rate for Gentamicin, 04% for Amikacin and 08% for Kanamycin. For macrolides; the Staphylococcus species studied showed 100% resistance to Erytromycin, 48% to Clindamycin and 64% to Pristinamycin. Eighty per cent (80%) of the isolates were resistant to Rifampicin and 12% to Cotrimoxazole. A resistance rate of 2% for Ofloxacin and a zero resistance rate for Levofloxacin and vancomycin. Multiple antibiotic resistance phenotypes were generated from 25 S. aureus isolates showing resistance to three or more antibiotics. Data indicating the predominant multiple antibiotic resistance phenotypes are shown in Table 4.


Fourier transforms infrared (FTIR) spectroscopy
The possibility of FTIR analysis for milk and dairy products has been mentioned by Lanher (1991), Van de Voort (1992) and Lefier et  al.  (1996).  Milk FTIR  spectra could possibly give more useful information on how the quality of milk is influenced by environmental factors. This could be used to define new traits and also used as a herd management monitoring tool to detect aberrations due to feeding and other environmental changes (Dagnachew et al., 2013). The results obtained showed that the weight composition of water, carbohydrates, lipids and proteins in the milk samples were always balanced in descending order: The vast majority of water, carbohydrates mainly represented by lactose, lipids and finally proteins.  The use of treated wastewater in the study site obviously did not influence the biochemical and nutritional composition of the milk samples.
Bacteriological qualities of raw milk
The water used on the study farm is the wastewater treated and discharged by the treatment plant; this is mixture of urban, industrial, agricultural and hospital wastewater from the city of Khenchela. On the farm, this water is used for three main activities: pasture cultivation, dairy barn farming and cleaning, and for dairy cow consumption. The main objective of this study is to assess the risks associated with the reuse of treated wastewater in agriculture. The dairy industry is particularly concerned about the potential effect on dairy cattle and milk quality following pasture irrigation with waste water. Therefore, monitoring bacterial pathogens, their survival and transfer is of the utmost importance to ensure that milk quality is not compromised.
Contamination of raw cow's milk with microorganisms is influenced by the health status and hygiene of dairy cows (Chambers, 2002; Cempírková, 2007). Due to the high nutritional value, water content and almost neutral pH of milk, many pathogenic and spoilage microorganisms can develop (Ray, 2004). The value of the total mesophilic aerobic flora of raw milk indicates a  very  poor  quality  of raw milk compared to the required standards of 105 CFU/ml (JORA, 2017). In addition, the overall bacterial load was very high; 90% of the samples had a value greater than 105 CFU/ml of flora. This total flora load and the large number of samples exceeding the recommended limits can be attributed mainly to infected udders, unsanitary milking equipment or procedures and/or poor microbiological quality of water used for cleaning utensils and animals, as well as milk storage conditions (Chye et al., 2004; Ghazi and Niar, 2011; Singh and Gupta, 2015; Wanjala et al., 2018). The result of the fecal coliforms showed significant contamination and indicated very poor quality of raw milk compared to the required standards of 103 CFU/ml (JORA, 2017). In general, coliforms indicate fecal contamination and their number is proportional to the degree of pollution produced by the stool (Aggad et al., 2009). However, the presence of coliforms indicates poor hygienic and sanitary conditions during milking and subsequent handling or water supply (Yucel and Ulusoy, 2006).
Some studies have shown that cattle excreta is not a significant source of coliform contamination of raw milk, but that water used for sanitation and milking environments is considered as one of the critical sources (Kagkli et al., 2007; Martin et al., 2016). Therefore, the use of poor quality and unsanitary water during sanitation procedures can indirectly contaminate milk (Robinson, 2005).
E. coli was isolated in 100% of the samples; the presence of this bacterium in milk indicates possible contamination by contaminated manure, soil and water (Chye et al., 2004). The development of antibiotic resistance in bacteria such as E. coli is a serious public health problem. The results show that only one antibiotic, Imipenem, showed 100% efficacy against E. coli strains; of the 25 isolates tested, 72% showed resistance to at least one of the 11 antibiotics.
The highest resistance of E. coli isolates in this study was observed in antibiotics β-lactam. β-lactam antibiotics have low toxicity, a factor that has led to overuse of these drugs in medical therapy (Moyane et al., 2013). Few studies have noted resistance of enterobacteriaceae to the antibiotic β-lactam in milk samples (Ntuli et al., 2016); a study by Geser et al. (2012) reported resistance to antibiotics CTX-M β-lactam in E. coli from milk samples.
The development of bacterial resistance to antimicrobial agents is a serious threat to human health (Zastempowska et al., 2016). Although antibiotic-resistant bacteria and genes encoding antibiotic resistance have been commonly detected in wastewater and treatment system by-products, the role of wastewater treatment processes in the dissemination of antimicrobial resistance is not clear (Mohammadali and Davies, 2017). In recent years, a number of studies have focused on variables that influence the profiles of antibiotic-resistant bacteria and antibiotic resistance genes during treatment (Xia et al., 2012; Yuan et al., 2014).
Hospital wastewater is likely to contribute significantly to the spread of multidrug-resistant pathogenic bacteria in wastewater treatment plants (Lien et al., 2016). Due to the presence of constant sub-inhibitor levels of broad spectrum antimicrobials, hospital wastewater creates an ideal situation for the exchange of antibiotic resistance genes and their combinations between clinical pathogens and environmental bacteria (Basode et al., 2018; Amador et al., 2015). Staphylococcus was detected in all samples. The high number of isolated coagulase-negative Staphylococcus is believed to be due to poor milking hygiene conditions and poor quality washing water (Kouamé et al., 2010; Hamiroune et al., 2016).
S. aureus of environmental origin can easily colonize cow udders (Piessens et al., 2011). In addition, unhygienic cow milking methods, particularly manual milking and the use of contaminated utensils, could lead to contamination of milk with S. aureus from foreign sources (Hamiroune et al., 2016). The presence of S. aureus tends to reduce the quality of milk and milk products traditionally prepared by their metabolic activities and could precipitate food poisoning due to the development of toxins that could cause disease when consumed by humans (Omoshaba et al., 2018). The study of the susceptibility/resistance of S. aureus to antibiotics revealed high resistance frequencies, particularly for penicillin and oxacillin. The mechanism of penicillin resistance is based on the bacterium's synthesis of an enzyme called β-lactamase or penicillinase (Guérin-Faublée and Brun, 1999). This inducible plasmid enzyme hydrolyzes the β-lactam cycle of penicillins A and G and renders them inactive (Kotra and Mobashery, 1998).
Staphylococcus in hospitals, and more recently in communities (present outside the hospital environment) have developed cross-resistance between penicillins M (methicillin, oxacillin) and other β-lactamins through the production of a protein, PLP2a, which binds penicillin (PLP) and has a low affinity for these compounds (Chambers, 2001). The gene encoding PLP2a, mecA, is carried by  a  chromosomal  element  that  also  contains other genes for resistance to heavy metals and other antibiotics, which explains the multi-resistance profile of MRSA (methicillin-resistant S. aureus) (Dumitrescu et al., 2010).
Methicillin-resistant Staphylococcus aureus (MRSA) is an important opportunistic pathogen in humans and cattle (Omoshaba et al., 2018). In this study, methicillin-resistant Staphylococcus aureus could be transferred from livestock to humans through milk and dairy products. Multiple antibiotic-resistant strains of Staphylococcus, defined as isolates resistant to three or more antibiotics, were obtained in a large proportion of the milk samples analyzed. The development of multiple antibiotic resistances in most of these isolates can be attributed to the acquisition of plasmid-mediated resistance (factor R) (Yamamoto et al., 2013; Akindolire et al., 2015). Usually, S. aureus is known to contain a number of multiple antibiotic-resistant plasmids that may explain the observed phenotypes (Yamamoto et al., 2013).
These results reveal that multiple antibiotic-resistant Staphylococcus isolates were isolated from milk samples. It is therefore suggested that these multiple antibiotic resistant isolates can have serious health implications for people who consume such dairy products. The high number of yeasts and molds in this study may be due to poor equipment hygiene during milk handling and processing, and indicating unsanitary conditions of handling and environmental contamination (Bonfoh et al., 2003; Prejit and Latha, 2007). Many foodborne molds, and possibly even yeasts, can also be dangerous to human or animal health because of their ability to produce toxic metabolites called mycotoxins. Human exposure to mycotoxins can result either from the consumption of contaminated food of plant origin or from the ingestion of mycotoxins transported from animal feed into animal tissues, meat, eggs or milk (Zastempowska et al., 2016). Some foodborne molds and yeasts can also cause allergic reactions or infections.


This study aims to assess the impact of the use of chlorine-free treated wastewater in farming and dairy cattle breeding.  Milk samples were collected from a farm in Northeastern Algeria. The treated wastewater from the treatment plant is used on this farm for different activities. The results of this study indicate that the overall microbiological quality of milk samples is well below current Algerian standards; they are heavily contaminated with fecal contamination germs and pathogenic bacteria with worrying antibiotic multi-resistance profiles. The source of contamination in milk samples can be water used for three main activities: pasture farming, dairy barn operations and cleaning, and for    consumption   by   dairy   cows.   The   presence   of multidrug-resistant bacteria in milk can pose a serious threat to public health and has a negative effect on the treatment of infections in humans. Newborns and children appear to be more exposed to milk contaminants than adults because they consume larger amounts of milk and are more sensitive. Urgent and effective measures must be taken to ensure proper wastewater management by the services concerned and farmers. Therefore, it is recommended that training and advice be given to farm owners and workers responsible for milking, emphasizing the need for hygiene practices on farms.


The authors have not declared any conflict of interests.


The authors thank Professor Boumaza A. Charif of the Department of Mechanics, Khenchela University, Algeria, for his valuable assistance in conducting biochemical analysis of samples using the Fourier Transform Infrared Spectroscopy (FTIR) technique at the Laboratory of Structures, Properties and Interatomic Interactions (LASPI2A), University of Khenchela, Algeria.



Aggad H, Mahouz F, Ammar YA, Kihal M (2009). Assessment of milk hygienic quality in Western Algeria. Revue de Médecine Vétérinaire 160:590-595.


Akindolire M, Babalola O, Ateba C (2015). Detection of antibiotic resistant Staphylococcus aureus from milk: A public health implication. International Journal of Environmental Research and Public Health 12(9):10254-10275.


Amador PP, Fernandes RM, Prudêncio MC, Barreto MP, Duarte IM (2015). Antibiotic resistance in wastewater: Occurrence and fate of Enterobacteriaceae producers of Class A and Class C β-lactamases. Journal of Environmental Science and Health, Part A 50(1):26-39.


Barancheshme F, Munir M (2018). Strategies to Combat Antibiotic Resistance in the Wastewater Treatment Plants. Frontiers in Microbiology 8:2603.


Basode VK, Abdulhaq A, Alamoudi M, Tohari HM, Quhal WA, Madkhali AM, Hershan AA (2018). Prevalence of a carbapenem-resistance gene (KPC), vancomycin-resistance genes (van A/B) and a methicillin-resistance gene (mecA) in hospital and municipal sewage in a southwestern province of Saudi Arabia. BioMed Central Research Notes 11(1):30.


Bauer AW, Kirby WMM, Sherris JC, Turck M (1966). Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology 45(4_ts):493-496.


Becerra-Castro C, Lopes AR, Vaz-Moreira I, Silva EF, Manaia CM, Nunes OC (2015). Wastewater reuse in irrigation: A microbiological perspective on implications in soil fertility and human and environmental health. Environment International 75:117-135.


Beuchat LR (2003). Media for detecting and enumerating yeasts and moulds. In Corry JEL, Curtis GDW, Baird RM. (Eds). Handbook of culture media for food microbiology (pp. 369-385). Elsevier, Amsterdam. 


Bonfoh B, Wasem A, Traore AN, Fane A, Spillmann H, Simbé CF, Zinsstag J (2003). Microbiological quality of cows' milk taken at different intervals from the udder to the selling point in Bamako (Mali). Food Control 14(7):495-500.


Cass S, Lowe H (2014). Monitoring Pathogens on Pasture Derived from Treated Wastewater and Biosolids Application. Lowe Environmental Impact. NZ Land Treatment Collective Conference papers. 


Cempírková R (2007). Contamination of cow's raw milk by psychrotropic and mesophilic micro-flora in relation to selected factors. Czech Journal of Animal Science 52(11):387-393.


Chambers HF (2001). The changing epidemiology of Staphylococcus aureus? Emerging Infectious Diseases 7(2):178-82.


Chambers JV (2002). The microbiology of raw milk. In Robinson RK. (Ed.), Dairy microbiology handbook (3rd ed, pp. 39- 90). New York: John Wiley and Sons.


Chye FY, Abdullah A, Ayob MK (2004). Bacteriological quality and safety of raw milk in Malaysia. Food Microbiology 21(5):535-541.


Clinical and Laboratory Standards Institute (CLSI) (2017). Performance Standards for Antimicrobial Susceptibility Testing. 27th ed. CLSI document M100.Wayne, PA: Clinical and Laboratory Standards Institute.


Coitinho TB, Cassoli LD, Cerqueira PHR, Da Silva HK, Coitinho JB, Machado PF (2017). Adulteration identification in raw milk using Fourier transform infrared spectroscopy. Journal of Food Science and Technology 54(8):2394-2402.


Dagnachew BS, Kohler A, Ådnøy T (2013). Genetic and environmental information in goat milk Fourier transform infrared spectra. Journal of Dairy Science 96:3973-3985.


Drechsel P, Scott CA, Raschid-Sally L, Redwood M, Bahri A (2010). Wastewater irrigation and health: assessing and mitigating risk in low-income countries. IWMI. Earthscan, London, UK and Sterling, USA.


Dumitrescu O, Dauwalder O, Boisset S, Reverdy ME, Tristan A, Vandenesch F (2010). Staphylococcus aureus resistance to antibiotics: key points in 2010. Medicine Sciences 26(11):943-949.


Eskildsen CE, Skov T, Hansen MS, Larsen LB, Poulsen NA (2016). Quantification of bovine milk protein composition and coagulation properties using infrared spectroscopy and chemometrics: A result of collinearity among reference variables. Journal of Dairy Science 99(10):8178-8186.


Etzion Y, Linker R, Cogan U, Shmulevich I (2004). Determination of protein concentration in raw milk by mid-infrared Fourier transform infrared/attenuated total reflectance spectroscopy. Journal of Dairy Science 87(9):2779-2788.


Gatto D'Andrea ML, Salas Barboza AGJ, Garcés V, Rodriguez-Alvarez MS, Iribarnegaray MA, Liberal VI, Fasciolo EG, van Lier J B, Seghezzo L (2015). The Use of (Treated) Domestic Wastewater for Irrigation: Current Situation and Future Challenges. International journal of water and wastewater treatment 1(2). 


Geser N, Stephan R, Hächler H (2012). Occurrence and characteristics of extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae in food producing animals, minced meat and raw milk. BioMed Central Veterinary Research 8(1):21.


Ghazi K, Niar A (2011). Hygienic quality of raw cow's milk in the different farms of Tiaret area (Algeria). Tropicultura 29(4):193-196.


Grappin R, Lefier D, Mazerolles G (2006). Analysis of milk and dairy products. In Bertrand D, Dufour E (Eds.), Infrared Spectroscopy and its analytical applications (pp. 583-623). Lavoisier: TEC et Doc.


Guérin-Faublée V, Brun Y (1999). Antimicrobial resistance of animal Staphylococci. Revue de Médecine Vétérinaire, France.


Hamiroune M, Berber A, Boubekeur S (2016). Evaluation of the bacteriological quality of raw cow's milk at various stages of the milk production chain on farms in Algeria. International Office of Epizootics 35(3):925-946.


Hultman J, Tamminen M, Pärnänen K, Cairns J, Karkman A, Virta M (2018). Host range of antibiotic resistance genes in wastewater treatment plant influent and effluent. Federation of European Microbiological Societies (FEMS). Microbiology Ecology 94(4).


International Organization for Standardization (ISO) (2003). Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of coagulase-positive Staphylococci (Staphylococcus aureus and other species) - Part 1: Technique using Baird-Parker agar medium (ISO 6888-1). International Organization for Standardization, Geneva, Switzerland.


International Organization for Standardization (ISO) (2005). Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of presumptive Escherichia coli - Most probable number technique. (EN ISO 7251). International Organization for Standardization, Geneva, Switzerland.


International Organization for Standardization (ISO) (2006). Microbiology of Food and Animal Feeding Stuffs - Horizontal Method for the Detection and Enumeration of Coliforms - Most Probable Number Technique (EN ISO 4831). International Organization for Standardization, Geneva, Switzerland.


International Organization for Standardization (ISO) (2008). Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of yeasts and moulds - Part 1: Colony count technique in products with water activity greater than 0.95. (EN ISO 21527-1). International Organization for Standardization, Geneva, Switzerland.


International Organization for Standardization (ISO) (2010). Microbiology of food and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination - Part 5: Specific rules for the preparation of milk and milk products (EN ISO 6887-5). International Organization for Standardization, Geneva, Switzerland.


International Organization for Standardization (ISO) (2013). Microbiology of the food chain - Horizontal method for the enumeration of microorganisms - Part 1: Colony count at 30 degrees C by the pour plate technique. (EN ISO 4833-1). International Organization for Standardization, Geneva, Switzerland.


JORA (Official Journal of the Algerian Republic) (2017). Inter-ministerial decree of 04-10-2016 of the OJ No.: 39/17 of the Algerian Republic.


Kagkli DM, Vancanneyt M, Vandamme P, Hill C, Cogan TM (2007). Contamination of milk by enterococci and coliforms from bovine feces. Journal of Applied Microbiology 103(5):1393-1405.


Kotra LP, Mobashery S (1998). β-Lactam antibiotics, β-lactamases and bacterial resistance. Bulletin de l'institut Pasteur 96:139-150.


Kouamé-Sina SM, Bassa A, Dadié A, Makita K, Grace D, Djè M, Bonfoh B (2010). Analysis of microbial risks of local raw milk in Abidjan (Côte d'Ivoire). African Journal of Health and Animals Productions 8(S):35-42.


Lanher BS (1991). Fourier transform infrared spectrometry and multidimensional analysis of spectral data: Application to quantification and process control in the field of dairy products. Ph.D. Theses, University of Bourgogne, Dijon, France.


Lefier D, Grappin R, Pochet S (1996). Determination of fat, protein, and lactose in raw milk by Fourier transform infrared spectroscopy and by analysis with a conventional filter-based milk analyzer. Journal of the Association of Official Analytical Chemists 79(3):711-717.


Lei Y, Zhou Q, Ya T, Chen JB, Sun SQ, Noda I (2010). The study of Cistanche deserticola using Fourier transform infrared spectroscopy combined with two-dimensional correlation infrared spectroscopy. Journal of Molecular Structure 974(1-3):156-160.


Li B, Webster TJ (2018). Bacteria antibiotic resistance : New challenges and opportunities for implant-associated orthopedic infections. Journal of Orthopedic Research 36(1):22-32.


Lien LT, Hoa NQ, Chuc NT, Thoa NT, Phuc HD, Diwan V, Dat NT, Tamhankar AJ, Lundborg CS (2016). Antibiotics in Wastewater of a Rural and an Urban Hospital before and after Wastewater Treatment, and the Relationship with Antibiotic Use-A One Year Study from Vietnam. International Journal of Environmental Research and Public Health 13(6):588.


Martin NH, Trmčić A, Hsieh TH, Boor KJ, Wiedmann M (2016). The evolving role of coliforms as indicators of unhygienic processing conditions in dairy foods. Frontiers in Microbiology 7:1549.


Maury M (1987). Milieux et réactifs de laboratoire pasteur. Microbiologie et immunologie. Diagnostic Pasteur: Paris, 727p.


McDermott P, Zhao S, Tate H (2018). Antimicrobial Resistance in Nontyphoidal Salmonella. Microbiology Spectrum 6(4).


Ministry of Health, Population and Hospital Reform (MHPHR) (2014). Standardization of the national antibiogram (human and veterinary medicine) - 7éd. Algerian Network for for Surveillance of Antimicrobial Resistance - OMS, 15-159. WHO Collaborating Centre for Surveillance of Antimicrobial Resistance. 



Mohammadali M, Davies J (2017). Antimicrobial resistance genes and wastewater treatment. In: Keen PL, Fugère R. editors. Antimicrobial Resistance in Wastewater Treatment Processes. h


Moros J, Iñón FA, Khanmohammadi M, Garrigues S, De la Guardia M (2006). Evaluation of the application of attenuated total reflectance-Fourier transform infrared spectrometry (ATR-FTIR) and chemometrics to the determination of nutritional parameters of yogurt samples. Analytical and Bioanalytical Chemistry 385(4):708-715.


Moyane JN, Jideani AIO, Aiyegoro OA (2013). Antibiotics usage in food-producing animals in South Africa and impact on human: Antibiotic resistance. African Journal of Microbiology Research 7(24):2990-2997.


Nicolaou N, Goodacre R (2008). Rapid and quantitative detection of the microbial spoilage in milk using Fourier transform infrared spectroscopy and chemometrics. Analyst 133:1424-1431.


Nicolaou N, Xu Y, Goodacre R (2010). Fourier transform infrared spectroscopy and multivariate analysis for the detection and quantification of different milk species. Journal of Dairy Science 93:5651-5660.


Ntuli V, Njage PMK, Buys EM (2016). Characterization of Escherichia coli and other Enterobacteriaceae in producer-distributor bulk milk. Journal of Dairy Science 99(12):9534-9549.


Omoshaba EO, Ojo OE, Sofela O, Onifade OI (2018). Prevalence and antibiotic resistance patterns of methicillin-resistant Staphylococcus aureus in raw milk and soft cheese (wara) sold in Abeokuta, Nigeria. Sokoto Journal of Veterinary Sciences 16(1):1-8.


Piessens V, Van Coillie E, Verbist B, Supré K, Braem G, Van Nuffel A, De Vuyst L, Heyndrickx M, De Vliegher S (2011). Distribution of coagulase-negative Staphylococcus species from milk and environment of dairy cows differs between herds. Journal of dairy Science 94(6):2933-2944.


Prejit NE, Latha C (2007). Microbial Quality Assurance of Milk during Production, Processing, and Marketing. American Journal of Food Technology 2(3):136-144.


Ray B (2004). Fundamental Food Microbiology. CRC Press, Boca Raton, FL.


Robinson RK (2005). Dairy microbiology handbook: The microbiology of milk and milk products. John Wiley and Sons, New York.


Singh V, Gupta J (2015). Analysis of knowledge and adoption level of the dairy farmers regarding clean milk production (CMP) practices. Asian Journal of Dairy and Food Research 34(3):180-186.


Van de Voort FR (1992). Fourier transform infrared spectroscopy applied to food analysis. Food Research International 25(5):397-403.


Vásquez JL, Ramírez NF, Akineden Ö, Fernández SJA (2017). Presence of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae in bulk-tank milk of bovine dairy farms in Antioquia, Colombia. Revista Colombiana de Ciencias Pecuarias 30:85-100.


Wanjala W, Nduko J, Mwende M (2018). Coliforms Contamination and Hygienic Status of Milk Chain in Emerging Economies. Journal of Food Quality and Hazards Control 5:3-10.


Xia S, Jia R, Feng F, Xie K, Li H, Jing D, Xu X (2012). Effect of solids retention time on antibiotics removal performance and microbial communities in an A/O‐MBR process. Bioresource Technology 106:36-43.


Yamamoto T, Hung WC, Takano T, Nishiyama A (2013). Genetic nature and virulence of community-associated methicillin-resistant Staphylococcus aureus. BioMedicine 3(1):2-18.


Yuan QB, Guo MT, Yang J (2014). Monitoring and assessing the impact of wastewater treatment on release of both antibiotic‐resistant bacteria and their typical genes in a Chinese municipal wastewater treatment plant. Environmental Science: Processes and Impacts 16(8):1930-1937.


Yucel N, Ulusoy H (2006). A Turkey survey of hygiene indicator bacteria and Yersinia enterocolitica in raw milk and cheese samples. Food Control 17(5):383-388.


Zangerl P, Asperger H (2003). Chapter 6: Media used in the detection and enumeration of Staphylococcus aureus. In: Janet EL, Corry GDWC, Rosamund MB. (Eds.), Progress in Industrial Microbiology. Amsterdam, pp. 91-110.


Zastempowska E, Grajewski J, Twarużek M (2016). Food-borne pathogens and contaminants in raw milk-A review. Annals of Animal Science 16(3):623-639.


Zhou Q, Sun SQ, Yu L, Xu CH, Noda I, Zhang XR (2006). Sequential changes of main components in different kinds of milk powders using two-dimensional infrared correlation analysis. Journal of Molecular Structure 799(1-3):77-84.