Efecto experimental de las radiaciones ionizantes en el pulmón:

Original Article

Characterization of Staphylococcus
species isolated from raw milk
and milk products (lben and jben) in North Morocco

Abdrezzak Bendahou,1,2 Mariam Lebbadi,3 Latifa Ennanei,2
Fatima Z. Essadqui,1 Mohammed Abid.2
1Département de Sécurité Alimentaire & Environnementale, Institut Pasteur, Tanger, Maroc
2Département de Recherche scientifique, Laboratoire de biologie moléculaire, Institut Pasteur, Tanger, Maroc 3Département des Sciences de la vie, Faculté des sciences & technique, Tanger, Maroc

Background: To investigate the incidence and antibiotic resistance of staphylococcal strains isolated from milk and milk products
and to trace the ecological origin of the Staphylococcus aureus isolated.
Methodology: Eighty-one samples of raw milk, lben (whey) and jben (cheese) were analyzed for the presence of staphylococcal
strains. Isolates were identified by Gram stains, tests for coagulase, the API staph system and the WalkAway® 40/96, which
also determines the antimicrobial susceptibility profiles. The S. aureus strains were biotyped, and variable regions of the
coagulase gene were amplified using the polymerase chain reaction.
Results: The identification results showed a predominance of coagulase-negative staphylococci (54 %). Coagulase-positive
staphylococci that were identified were divided into 3 groups comprising S. aureus (40%), Staphylococcus intermedius (2 %)
and Staphylococcus hyicus (4%). Among the S. aureus that was isolated, biotype C was the predominant biotype. Among 40
coagulase gene PCR-amplification products, 37 produced a single band, while 3 isolates produced two bands.
The antimicrobial susceptibility-profile of the staphylococcal strains revealed a high incidence of S. aureus to penicillin G. In
addition, Staphylococcus lentus presented considerable resistance to the oxacillin, erythromycin and lincomycin.
Conclusions: The presence of staphylococci in raw milk, lben and jben in areas of northern Morocco poses a health hazard, so it
is necessary for the public health inspectors to properly examine the conditions during production, storage and
commercialization of all products made with unpasteurized milk.
Key Words: milk products, Staphylococcus, coagulase-gene typing, biotyping, antimicrobial susceptibility.
J Infect Developing Countries 2008; 2(3):218-225.
Received 13 March 2008 - Accepted 29 April 2008
Copyright 2008 Bendahou et al. This is an open access article distributed under the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
implicated in SFP, and often contaminated raw Staphylococcal food poisoning (SFP) is one of milk is involved [2]. These products are highly the most prevalent causes of gastroenteritis susceptible to a variety of microorganisms worldwide [1]. Symptoms of SFP have a rapid because of their high nutritive value and complex onset (2 to 6 hours) of abdominal cramps, nausea, chemical composition. The biological changes and vomiting, sometimes followed by diarrhoea produced by these organisms can be either [2,3]. Patients become symptomatic after ingestion desirable or undesirable. They may have a useful of thermostable staphylococcal enterotoxins (SE) function in the preparation of fermented milk of an approximate dose of 0.1 to 1.0 mg/kg of body products such as lben (whey) and jben (cheese) or weight [4]. Since SE are more stable than S. they may have undesirable effects and produce aureus bacteria, it is possible to test a food product changes in the odour, colour, taste, texture or and obtain negative S. aureus culture results and appearance of the food. Furthermore, most of positive SE tests. The hazard to public health is these bacteria produce toxins and cause food particularly linked to the ability of 50% of these poisoning frequently. The presence of the strains to produce thermostable enterotoxins pathogen depends on ingestion of contaminated associated with food poisoning [5]. Milk and milk feed followed by amplification in bovine hosts and products are common vehicles for staphylococcal faecal dissemination in the farm environment. The food poisoning [6-11]. They have frequently been final outcome of this cycle is a self-maintained Bendahou et al. Staphylococcus in artisanal dairy products J Infect Developing Countries 2008; 2(3): 218-225. reservoir of a pathogen that can reach the human trace the ecological origin of the S. aureus strains population by direct contact, ingestion of raw using the simplified scheme of Devriese [15] and contaminated food (raw milk, cheese or whey also to use coA gene polymorphism to identify S. made with raw milk), or contamination during the processing of food. Isolation of strains with similar biotypes from dairy farms and human cases and Materials & Methods
outbreaks substantiate this hypothesis [12]. A total of 81 samples of milk and milk products consisting of raw milk (27), lben (27) and jben (27) (whey) and jben (Moroccan traditional fresh were aseptically collected on a random basis from cheese) are widely manufactured and consumed different localities (weekly rural markets) in North by the peoples of North Morocco. However, these Morocco [Tanger, Tetouan and Larache] between products have not been subjected to hygiene or May 2005 and May 2006. Each locality was visited sanitary control, because they are made at home. monthly (except in bad weather, when farmers The incidence of staphylococcal food poisoning cannot offer their products). Three samples due to the consumption of dairy products is not consisting of 1 raw milk, 1 jben and 1 lben were uncommon in our country. The contamination of collected during each visit so that at least nine these products can be attributed to the occurrence samples were collected monthly. All the samples were placed in sterile plastic bags and immediately organisms can gain access to milk (raw material) taken in a container containing ice cubes to the either by direct excretion from udders with clinical laboratory for bacteriological analysis. and sub-clinical staphylococcal mastitis or by contamination. The contaminants reach the Isolation and identification of Staphylococcus products either during cooling or handling after cooking [13]. Several easy steps can be taken to Twenty-five grams of each cheese sample and lower the risk and to render milk and milk products 25 ml of raw milk and lben were stirred separately safe for consumption. Proper sanitary measures into 225 ml of sterile buffered peptone water. are needed to improve the hygienic conditions Baird-Parker plates were then spread with 0.1ml of the dilution of each sample. Additional plates were manufacturing of cheese and whey in order to prepared with successive 1/10 dilutions. The guarantee the quality of these popular products in plates were incubated for 48 hours at 37° C. The North Morocco. These measures must include a identification of the Staphylococcus genus was program of sanitary education for the milking done by microscopic observation, Gram-staining and catalase determination. All staphylococcal strains were checked for purity and tested for their between origin, biotype and antibiotic resistance of ability to coagulate citrated rabbit plasma. Further staphylococcal strains isolated from milk and milk identification, biochemical system “API Staph products have not yet been conducted, except system” (both from Bio Merieux, Marcy-l’Etoile, works which have dealt with 56 strains isolated from thirty samples of soft fresh traditional WalkAway® 40/96 DADE BEHRING: designed for Moroccan cheeses made from fresh milk and use for identification to the species level and/or collected at three milk farms in the city of Rabat. antimicrobial agent susceptibility of facultative and The results show that 16 (29%) of the strains are some fastidious aerobic gram positive cocci) were enterotoxigenic and 40 (71%), 12 (22%) and 4 used to determine the species more precisely. (7%) belonged to ovine, human and unspecified The objective of the present study was to Each of the 40 S. aureus strains isolated from investigate the incidence and antibiotic resistance fresh milk, cheese [jben] and whey [lben] was of staphylococci isolated from raw milk and biotyped following the simplified system proposed products made with raw milk, such lben and jben, by Devriese [15]. Characteristics examined were collected from various locations of the northern rural areas of Morocco. The study further aimed to coagulation of bovine plasma and the type of Bendahou et al. Staphylococcus in artisanal dairy products J Infect Developing Countries 2008; 2(3): 218-225. growth on crystal violet. The strains were classified of French Company of Microbiology (CA-FCM). All in various biotypes: human (A), bovine (C), ovine the identified Staphylococcus strains were tested (D) and poultry (B). The strains that could not be classified in one of the biotypes was regarded as vancomycin (Van), oxacillin (Ox), penicillin (P), Augmentin (Aug), tetracyclline (Te), fosfomycine lincomycin (Lin), kanamycin (K), tobramycin (To), DNA of all the bacteria was extracted using the InstaGenee Matrix (Bio-Rad, Marnes-la-Coquette, France). The kit was used according to the manufacturer’s instructions. From each sample, 5 pefloxacin (Pef). µl of total cellular DNA were then evaluated by PCR with appropriate primers and cycling conditions. The PCR primers used for the Identification of Staphylococcus Species identification of the coa genes were those reported The 100 staphylococci isolated from the 81 by Hookey et al. [16]. Sequences of the primers samples of milk and dairy products examined (27 lben, 27 milk and 27 jben) were isolated and Coa1: 5’-ATA GAG ATG CTG GTA CAG G-3’, observed on Baird Parker, then tested for the Coa2: 5’-GCT TCC GAT TGT TCG ATG C-3’. production of coagulase on rabbit plasma and For PCR reaction, the conditions described by characteristics. They could be divided into 4 Hookey et al. [16] were used. Amplification was groups: the first comprised the species S. aureus conducted in a thermal cycler (iCycler; Bio-Rad with a total of 40 isolates (40%); the second and Laboratories) as follows: An initial denaturation at third were represented respectively by the species 94° C for 45 seconds. The cycling proceeded for S. intermedius, with 2 isolates (2%) and S. hycius 30 cycles of 94° C for 20 seconds, 57° C for 15 hycius with 4 isolates (4%); the last contained 54 seconds, and 70° C for 15 seconds with a final (54 %) isolates that were found to be coagulase step at 72° C for 2 minutes. The tubes were cooled Table 1. Distribution of staphylococci isolated from milk,
(wt/vol) agarose gel in the presence of ethidium bromide then photographed and analysed under UV light in the gel-doc system (BioRad, MuK nchen, Germany). The 100-bp DNA ladder (EZ Load 100 bp, Bio-Rad Laboratories) was used as a The antimicrobial susceptibility tests were performed by dilution in liquid medium and application to substrates dehydrated in a Mueller-Hinton magnesium or other factors critical for the bacterial suspension and incubation at 35° C for 16 hours, Minimal Inhibitory Concentration (MIC) was determined by the lowest antibiotic concentration presenting growth inhibition. For these panels, dilutions of antibiotics used correspond to the concentrations of the Committee of Antibiorgamme NI: Species not identified (regarded as rare biotype). Bendahou et al. Staphylococcus in artisanal dairy products J Infect Developing Countries 2008; 2(3): 218-225. Biotypes of Staphylococcus aureus were resistant to erythromycin (10%); lincomycin The data shows that of the four coagulase (10%) and kanamycin (10%). Coagulase-negative types, 18 (45%) have been reported to be of staphylococci are more susceptible to penicillin. bovine origin (C) and more dominant than the The overall penicillin resistance rate for CNS was other biotypes. The distribution of the remaining biotypes for the isolates of the S. aureus were respectively 12 (30%), 6 (15%), and 4 (10%) for Figure 1. Electrophoretic profile, in 2% agarose gel, of
polymerase chain reaction (PCR) products of S. aureus unspecified (IND). Isolates that could not be coagulase gene isolated from milk and milk products (whey and jben): determined as biotypes A, B, C bovine or C ovine (A) Isolates with only 1 amplicon. Lane 1: molecular weight marker 100 bp according to Devriese’s scheme were classified as amplicon; Lane 2: 400 bp amplicon. Lanes 3, 4: 560 bp amplicon. Lanes 5, 6: an unspecified biotype. It can be noted that this 720 bp amplicon; lane 7: negative controls, S. epidermidis; lanes 8: S. intermedius. biotype was often very similar to the C bovine (B) Isolates with 1 amplicon or with 2 amplicons. Lane 1: molecular weight marker 100 bp amplicon; Lane 2: 700 bp amplicon. Lane 3: 560-800 bp amplicon; Lane 4: 480-700 bp amplicon; Lane 5: 900 bp; Lane 6: 700 bp amplicon; Lane 7: 700 bp amplicon; Lane 8 : 480-700 bp amplicon. According to culture, chemical properties, and the positive tube coagulase test, 40, 4 and 2 isolates used in the present study could be respectively identified as S. aureus, S. intermedius and S. hyicus. The coagulase gene typing was effective in subdividing strains of S. aureus from milk products [L, J and M] as all yielding a PCR amplification product. In contrast, no amplification product could be obtained from the strains of the other coagulase positive staphylococcal species investigated. The 40 isolates of S. aureus could be differentiated from each other on the basis of two characteristics of their PCR products, i.e., the presence of one or two PCR products and their size(s). A single PCR product with sizes of approximately 400 pb, 560 pb and 720 pb from 37 isolates was found, while two products from 3 isolates were amplified. The sizes of the PCR products ranged approximately from 400 to approximately 900 bp (Figure 1A and 1B). Two isolates of coagulase-negative and positive staphylococci (S. epidermidis which served as negative controls and S. intermedius) produced no coagulase amplification (Figure 1A). Antimicrobial Susceptibility Antibiotic-resistance patterns of the CNS and CPS strains isolated from milk and milk product sources in North Morocco are shown in Table 2. The antimicrobial susceptibility profile revealed a high resistance of S. aureus to penicillin (50%). A low prevalence of resistance was detected for According to our results, it was also shown that tetracyclline (25%), oxacillin (15%). Few strains Bendahou et al. Staphylococcus in artisanal dairy products J Infect Developing Countries 2008; 2(3): 218-225. high number of CNS isolated may be due to the bad conditions of hygiene during milking and lack of hygienic measures in the manufacturing, Table 2. Frequencies of antimicrobial resistance in
preparation, handling and storage of whey and jben. Also, the method of their sale is entirely based on tradition. Because CNS are a part of the The most resistant
normal teat skin flora and mucosa of humans and Antibiotic
animals, some species are also found free-living in aureus
the environment [17], and therefore are a common cause of contamination of milk and milk product. In addition, unpasteurized milk may contain CNS if the cow suffers from mastitis, an inflammation of In the past, CNS were often regarded as skin flora opportunists but emerging data now indicates that they are associated with several sub-clinical It appears from the biotyping results with regard to the 40 S. aureus strains that a high proportion of the strains belonged to the C bovine ecovar. Approximately 45% of the S. aureus isolates belong to this biotype indicating the preponderance of the contaminations coming from raw milk used. Most literature indicates that S. aureus appears in milk from cows afflicted with mastitis [23]. The data from literature also suggest that the persistent colonization of the teat skin occurs and may be an important predisposing factor for S. aureus contaminations. [24]. About 30% of our strains possessed the characters of the B biotype, a fact easily explained by the interchange of staphylococci among different animals due to their frequent contact [25]. Identification of A biotype strains in the other group NID: Staphylococcus species not identified by the MicroScan of isolates suggests contamination of the products with staphylococci of human origin during No resistance for glycopeptides was observed Routine bacteriological tests used in the for S. aureus; however, 10.8 % of CNS showed identification of S. aureus, such as mannitol fermentation, DNAse production, VP, etc., are not For the remainder antibiotics, we found an Nevertheless, coagulase production is one of the most reliable criteria for the identification of S. pefloxacine 21.6% and tobramycin 18.9%. Finally aureus [28]. The PCR products of the gene it should be noted that none of the milk and milk encoding staphylococcal coagulase displayed product isolates had augmentin (amoxicillin- gene polymorphisms and allowed a genotypic characterization of the bacteria. Length and sequence of the polymorphisms of the coagulase Discussion
gene and its use for genotypic characterization of The results showed that coagulase-negative S. aureus had been already shown [16,29-33]. The staphylococcal (CNS) species more frequently coagulase gene has been found polymorphic and occurred in milk and milk products (54%). This Bendahou et al. Staphylococcus in artisanal dairy products J Infect Developing Countries 2008; 2(3): 218-225. genotypically variable among S. aureus strains the S. aureus and CNS strains respectively isolated in this study. The polymorphism obtained isolated from goat mastitis were resistant to was clearly revealed due to multi-allelic forms at penicillin G [22]. Messadi et al. presented very the 3- end of the gene (tandem repeats) which similar data with 64% against 18.6% [39]. No differ in their sequences and restriction sites. Phenotypic variations were demonstrated clearly in clavulanate) was observed both from strains of S. the production of staphylocoagulases among milk, aureus and CNS in this study. Thus, the results whey and jben isolates which may be due to polymorphism of the gene. In order to assess the resistance in S. aureus and CNS isolates could be feasibility of using coagulase gene typing as an due to production of β-lactamases. El-Ghodban et epidemiological marker, a large number of isolates al. found that 75% of Libyan S. aureus strains from different geographic regions and different milk originating from food were resistant to penicillin products were analyzed. The ease of analysing and were positive for β-lactamase [40]. Ann Hébert coagulase gene polymorphism within a large et al. showed that all seven isolates of CNS strains number of strains and the multiple distinct from hospital were β-lactamase positive and polymorphic patterns generated support the use of resistant to penicillin but were susceptible to the this technique in epidemiological investigations of other antibiotics tested [41]. The second-highest resistance was observed to tetracycline (25%) and The primer pair amplified more than one PCR oxacillin (15%) for S. aureus strains, and to product in 3 isolates, which suggests the presence tetracycline, oxacillin, erythromycin, lincomycin, of different allelic forms of the coA gene. With the kanamycin, tobramycin and pefloxacin for CNS, PCR method, an amplification product was not especially S. lentus. Little to no resistance was observed for the DNA of other coagulase-positive seen with the other antimicrobial agents tested (0 species of Staphylococcus (S. hyicus hyicus and to10%). The findings suggest the requirement of S. intermedius). These results are in accordance proper use of β-lactam antibiotics for mastitis with Aarestrup et al. [34], who studied the therapy. To prevent the unnecessary use of β- amplification of sequences of the coA gene in 187 lactams and to achieve effective therapy, isolation strains of S. aureus, 10 strains of S. intermedius, of the microorganisms and determination of 3 strains of S. hyicus, 1 strain of S. delphini and 1 antimicrobial susceptibility is essential before the strain of S. schleiferi subspecies coagulans and verified the presence of bands only in S. aureus In conclusion, our results showed high levels of [35]. These authors suggested that coagulase CNS contamination in the samples of raw milk, gene typing might also be useful as an additional lben and jben. In the classification scheme of identification criterion to differentiate among Devriese (1984), about 45 % of all the CPS strains coagulase positive staphylococci [36, 34]. The tested were found to belong to C (bovine) biotype. extensive polymorphism observed suggests that A high genotypic uniformity of different-sized the coagulase gene may be an important virulence determinant for this organism’s characterization demonstrated. The PCR method based on [30]. coagulase gene typing is able on one hand to In our study, the highest resistance was identify and discriminate between coagulase- registered for penicillin G by both S. aureus (50%) positive Staphylococcus species and on the other and CNS (37, 5%) strains obtained from milk, hand to classify all S. aureus strains. whey and jben. This is not surprising because We conclude also that sanitary measures are ampicillin is one of the most commonly used needed to improve the hygienic conditions during antibiotics for treatment of infections in humans milking and manufacturing of jben and lben, in and animals [37]. Different rates of penicillin order to guarantee the quality of these highly resistance have been reported for S. aureus and CNS obtained from different sources. Acco et al. Finally, it should be noted that for all the S. showed that 70% of strains of S. aureus isolated aureus strains isolated from all samples, none from food handlers were resistant to penicillin [38]. Benhassen et al. reported that 64% and 22.6% of (amoxicillin-clavulanate) or vancomycin. This is Bendahou et al. Staphylococcus in artisanal dairy products J Infect Developing Countries 2008; 2(3): 218-225. important, since although MRSA strains may pose 14. Hamama A. (1989) Qualité bactériologique des fromages a therapeutic problem for staphylococcal infection, they may be controlled by the use of these 15. Devriese LA (1984) A simplified system for biotyping Staphylococcus aureus strains isolated from different animal species. J Appl Bacterial 56: 215-220. Acknowledgements
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