Identification and antimicrobial resistance of fecal enterococci isolated in coastal mediterranean environments of Morocco

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Identification and Antimicrobial Resistance of Fecal Enterococci Isolated In Coastal

Mediterranean Environments of Morocco

Article in European Journal of Scientific Research · February 2012

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European Journal of Scientific Research

ISSN 1450-216X Vol.70 No.2 (2012), pp. 266-275

http://www.europeanjournalofscientificresearch.com

Identification and Antimicrobial Resistance of Fecal Enterococci Isolated In Coastal Mediterranean

Environments of Morocco

Mohamed Bennani

Laboratory of Microbiology and Hygiene of Food and Environment Pasteur Institue, Casablanca, Morocco

E-mail: mohamed.bennani@pasteur.ma Tel: + 212 6 67 94 28 92; Fax: + 212 5 22 26 09 57

Hamid Amarouch

Laboratory of Microbiology, Biology Department, Faculty of Science Hassan II University, Casablanca

Nadia Oubrim

Nozha Cohen

Abstract

The aim of this 2 years field study is to enumerate, to identify and to assess the percentage of antibiotic resistance of enterococcus in the samples (Seawater, Shellfish and Sediments) at three marine sites in the Mediterranean coast of Morocco. The highest densities of entrococcus were recorded at the site 2 and in samples of sediments. The three sites showed abundance of distribution of enterococcus faecalis, enterococcus faecium and enterococcus casseliflavus respectively. Site 2 which has a higher degree of enterococcus presented higher percentages of resistant strains 64,7% and resistance to a larger number of antimicrobials compared with the less polluted sites 1 and 3 with 45,3% and 35,3%

respectively. In seawater samples, the highest frequencies of resistance were obtained against rifanpicine (33%), whereas in Shellfish and sediment, the highest frequencies were detected against erythromycin with (28% and 24% respectively). This study shows that the sites receiving sewage and urban runoff with the high density of entrococcus were the major source of resistance.

Keywords: Enterococcus. Water quality. Coastal environments of Morocco.

Abbreviations: MPN: Most Probable number; FIB: Fecal Indicator Bacteria. AFNOR:

French Association for Standardization; WHO: World Health Organization; VER:

Vancomycin-Resistant Enterococcus NF: Norme Française; EN: Norme Européenne; ISO:

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International Organization for Standardization; API20strept: Analytical Profile Index for Streptococci.

I. Introduction

The potential of waterborne disease transmission at recreational beaches is related to the abundance of fecal indicator bacteria (FIB). When the indicator levels are high, fecal contamination may be present which leads to an increased risk of encountering disease-causing pathogens (Bonilla et al. 2007).

Accordingly, there is increasing evidence that antimicrobial use both in humans as therapeutic measures as well as in animals for treatment and control of infections and for growth promotion (Bower & Daeschel 1999) can select resistant genes and lead to the emergence of antibiotic-resistant strains (Klein et al. 1998, Harwood et al. 2001). Bacteria involved in this context include foodborne pathogens (Bower & Daeschel 1999), opportunistic pathogens and commensal bacteria (Aarestrup 1999).

One of the most important genera with regard to antimicrobial resistance is the Enterococcus genus (Huycke et al. 1998), which showed intrinsic resistance to many antimicrobials. These microorganisms may require resistance to genes present in plasmids and through conjugation and may disseminate them to intra- and inter-specific bacteria (Kühn et al. 2005). Many recent studies have been carried out in freshwater, estuaries, water distribution systems and sewages to determine the distribution of bacteria resistant to antibiotics (Ash et al. 2002, Mudryk, 2005, Manero et al. 2006);

however, very few studies exist on the presence of resistant bacteria in marine waters (Arvanitidou et al. 2001, Tejedor et al. 2001), with even fewer studies on shellfish and sediment, because in unusual circumstances, the marine environments are unlikely to be sources of human infection (Wilson and McAfee 2002). Instead, they represent a sink for these everlasting organisms once excreted.

The present study examined the density, composition and the resistance of enterococcus genera at three sites in Tamouda bay (Mediterranean coastal environments of Morocco) in samples (seawater, shellfish and sediments). The purpose was to assess the presence in the coastal environments of Morocco of resistant enterococci that would indicate the prevalence of antibiotic use and abuse in animal and human medecine and the frequency of contamination by pathogens.

II. Material and Methods

II.1. Description of the Study Area

This study was conducted in Tamouda bay; located in the mediterranean coast of Morocco, between Sebta at the north (35°54’N, 5°17’10”W) and Cap Negron at the south (35°40’N, 5°16’40”W). The climate is typically mediterranean. The average annual temperature is about 18°C, while the annual rainfall average ranges between 800 and 1000 mm.

Three sites were selected in Tamouda bay, Site1: Marina kabila is located on the release of sewage; Site2: is located near of Smir's river and Site3: Marina beach which is an area of swimming, surfing and leisure. Every site is different in geography, population and ecology. The locations of the sites (Figure 1) were determined by the global positioning system.

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268 Mohamed Bennani, Hamid Amarouch, Nadia Oubrim and Nozha Cohen Figure 1: Map showing the geographical location of three sampling sites

II.2. Samples Collection and Preparation

From January 2007 to December 2008, samples were collected bimonthly in three sampling sites. For each exploration, three ecological types of samples were collected, seawater, shellfish and sediments.

Using boat, seawater samples (1L) were collected at a depth of 1m from the surface in sterilized plastic bottles. Shellfishes samples were purchased from local fisherman including Mussels (Mytilus edulis and Mytilus galloprovincialis) and cockles (Cerastoderma edule), while the sediments were collected from the surface of the coast using sterile plastics pots. After collection, the samples were transported immediately to the laboratory in insulated coolers with frozen gel-packs to maintain the temperature around 4°C. A total of 619 samples, composed of seawater (n = 219), seafood (n = 82) and sediment (n

= 115) were collected.

II.3. Bacterial isolation of Enterococcus

The detection and enumeration of fecal enterococci in samples was done by the standardized five-tube MPN method according to standard NF-EN ISO 9308-3, 1999. The density is reported as log₁₀(MNP g-1). A portion of (25g) from shellfish or sediments was placed into a separate sterile stomacher bag with 225 ml of buffered peptone water and then pummeled with a MIX I mixer (AES Laboratory, Combourg, France) For seawater samples, 1L were filtred using 0.45µm membrane filters (Anon 1998, 2000). Azide dextrose broth (Merck KGaA, Germany) was used as presumptive test followed by the confirmation test in Ethyl Violet bouillons Azide (Merck KGaA, Germany). 100 µl of each sample was streaked on m-Enterococcus (Biorad) and incubated at 37 °C for 24 h. For each sample, 5–6 morphologically different colonies were isolated stored at -80°C in a medium containing 15% glycerol.

II.4. Phenotypic Characterization of Isolates

Strains isolates were subjected to preliminary tests to determine their membership in the genus of Enterococcus, including Gram staining, catalase activity, aesculin hydrolysis and growth in the presence of 40% (v/v) bile, in brain heart infusion broth (Merck KGaA, Germany) with 6.5% NaCl (37

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°C), at pH 9.6 (37 °C), and growth at 45°C. Identification to the species level was performed with standard biochemical tests that were prepared in-house. Biochemical tests included carbohydrate fermentation with 1% L-arabinose, D-sorbitol, lactose, D-raffinose, ribose, sorbose, sucrose and methyl-a-D-glucopyranoside, arginine hydrolysis, pyruvate utilization and pigment production. The results of the above tests were analysed using published standard biochemical identification charts (Facklam & Collins, 1989; Teixeira et al. 2007). Motility was detected as described by (Van Horn et al.

2002) using isolates that were inoculated in trypticase soy broth and incubated at 30°C for 2 h. The direct wet mount method was then used to detect motility by dark-field microscopy. The strains isolated were identified by ApiStrep (Bio-merieux).

II.5. Antimicrobial Susceptibility Testing

The microbial sensitivity tests were carried out by the agar disc diffusion method from Kirby–Bauer, using Mueller–Hinton Agar and following the National Committee for Clinical Laboratory Standards.

We tested 212 colonies for the following antimicrobials: Ampicillin ≥ 8, Erythromycin ≥ 4, high level Gentamycin ≥ 500, Rifampin ≥ 2, high level Streptomycin ≥ 1000, Tetracycline ≥ 8 and Vancomycin ≥ 16. Strains of E. faecalis ATCC 29212 and E. faecalis ATCC 51299 were used as control for the results following the criteria established by the (NCCLS 2004).

III. Resultats

III.1. Abundance of Entrococcus spp

This monitoring showed that the abundance of Enterococcus ranged from undetectable to 6.61 log (MPN/g) with an average abundance of 4.57 log(MPN/g).

The results showed very similar patterns of abundance at the three sites, with mean density of 4.6 log (MPN/g), 4.4 log (MPN/g) and 4.3 log (MPN/g) in sites 2, 1 and 3 respectively, but the prevalence at site 2 was consistently higher. Consequently, the prevalence highest among all sites is up to 72% of the collected samples. The dynamic population during the 2 years showed that the minimum abundance was observed during the hot season and the maximum in cold season. Nevertheless, the abundance in seawater was consistently lower (Figure 2).

Figure 2: Average density of Enterococcus in differents sites and samples

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270 Mohamed Bennani, Hamid Amarouch, Nadia Oubrim and Nozha Cohen

On the other hand, a linear positive correlation was recorded between all ecological types of studied samples, a highly significant correlation of enterococcus spp. abundance in seawater with shellfish was found (rs = 0.633; P= 0.0001) as well as with seawater, sediment (rs = 0.584; P= 0.003).

But the highest significant correlation was between shellfish and sediment enterococcus spp.

abundance (rs = 0.871; P<0.0001).

III. 2. Enterococcus Species Distribution

Of the total analyzed samples (n= 619), 370 Enterococcus strains were isolated, which represent a prevalence of 59.7%. The distribution of strains according to the sampling sites and the type of samples analyzed is presented in Table 1. The identification results of enterococcus showed that with samples (seawater, shellfish and sediment respectively), most of the isolated strains were E.faecium with (38.8%, 40% and 35.2%) followed by E.faecalis with (42%, 30,8% and 37,1%), E.casseliflavus was isolated with (7%, 17.3% and 21,7%) and E.durans with (1,2% and 21,7%) respectively.

Table 1: Number of Enterococcus isoleted from all sites

Sources Total E.faecium E.faecalis E.casselifl

avus E.durans E.gallinar um

Enterococ cus spp Site1

Seawater 47 19 22 0 3 0 3

Shellfish 50 27 19 0 4 0 0

Sediment 50 22 19 2 5 1 1

Site2

Seawater 48 3 16 22 1 1 5

Shellfish 43 6 16 19 0 1 1

Sediment 44 7 14 20 0 1 2

Site3 Seawater 44 25 5 0 5 0 9

Sediment 44 24 6 0 0 6 8

Total 370 133 117 63 18 10 29

The frequency of contamination of Enterococcus species by sites are highlighted in (figure 3).

The dominant species in the site 1 were E.faecalis, E.faecium and E.durans with (47,9%,41,6% and 8,3% respectively), followed by E.gallinarum and E.casseliflavus (1%). At site 2, E.casseliflavus and E.faecium with (47% and 36,4% respectively) were predominant species, whilst E.faecalis E.gallinarum and E.durans (12,9%, 2,3% and1,2% respectively) occurred at lower frequencies. At site 3, the dominant species were E.faecalis 70,5% and E.faecium, E.gallinarum, E.durans represented by (14.7%, 7.3% and 7% respectively).

Figure 3: Distribution of Enterococcus in differents sites.

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III.3. Antimicrobial Susceptibility Testing

Of the total Enteroccocus strains identified, 54.4% demonstrated resistance to at least one antibiotic.

The number of resistant strains to various antibiotics is represented in the Table 2. On the whole, the highest strength vis a vis the antibiotic groups was for (Erythromycin and Rifampin, Tetracycline, High level gentamycin, High level streptomycin and Ampicillin and Vancomycin) with (27.2 % for each, 18.5%, 10,8%, 7,6% and 4,3% for each respectively). The average percentage of resistance strains in (sea water, shellfish, sediment) samples are respectively (25%, 49.4%, 67.5%). In the seawater samples the highest frequencies of resistance were found in erythromycin, rifampicin and tetracycline with (44%, 32%; 24% respectively). The strains identified in sediments have shown resistance vis a vis all antitiotics tested. Similar results was found in shellfish samples except ampicillin resistance, the multiresistance strains in shellfish and sediment samples are respectively (2.4% and 4.5 %). The percentage of resistance by sites is recorded as follows (45.3%, 64.7% and 35.5%) in the sites (1, 2 and 3 respectively). In site 1: five antibiotics have been involved in the resistance of the strains identified, with high rates of resistance that were recorded respectively Erythromycin, Tetracycline, Rifampicin;

strains dual and multi resistance were found in this site with an average rate of 6.2%. In site 2, The highest frequency of resistance was recorded, with rifampicin and erythromycin, of low resistance to vancomycin and ampicillin of order 1.13%, showed 10 % vis-à-vis the high level of gentamycin and 7.9% according the high level of streptomycin, multiresistance was recorded with 13.6% in this site. In Site 3, we have recorded the lowest rate of antibiotic resistance.

Table 2: Percentage of Antibiotic-resistance of Enterococci in differents sites and samples

Sources (n=90) Ampicillin Erythrom ycin

High level gentamycin

Rifampi n

High level streptomycin

Tetracycli ne

Vancom ycin Site1

Seawater 0 4.4 0 4.4 0 4.4 0

Shellfish 0 4.4 0 4.4 0 4.4 4.4

Sediment 4.4 13.4 0 6.6 0 8.9 0

Site2

Seawater 0 2.2 4.4 4.4 0 2.2 0

Shellfish 0 11.1 6.6 8.9 4.45 6.6 0

Sediment 4,4 15.5 11.1 20 11.1 8.9 4.4

Site3 Seawater 0 0 0 0 0 0 0

Sediment 0 4.4 0 6.6 0 2.2 0

IV. Discussion

The present study was to evaluate the resistance to antimicrobials from enterococcus spp, which were isolated from samples (seawater, shellfish and sediment) from three sites in mediterranean coast of Morocco, the purpose was known in samples of shellfish and sediments of strains resistance that could contribute to recreational water pollution and water to aquaculture in this coastal zone. In the Tamouda bay, the impact of organic load discharged in seawater by sewage particularly in site1, and the proximity to site2 at the rivier Smir, explains the higher abundance of Enterococcus spp at sites (1 and 2) throughout the study period. This may be explained by the important flow of visitors to this site during summer period. During the two last years, the maximum abundance was observed during winter and the minimum in summer. The positive correlations observed between bacterial density in seawater, shellfish and in sediment show the existence of a continuous flow of microorganisms between all collected samples (Fig. 2). Similar results were obtained by (Alm et al. 2003, Sato et al 2005) which observed a high correlation between bacterial density in water and in sediment. Alm et al. 2003 were explained the resuspension of sediments with elevated numbers of bacteria and caused by recreational activities. In addition, these authors found that rises or reductions in the density of indicators in the sediment were almost always associated positively with their variations in water. While the elevated densities of Enterococcus in sediment indicate that these latter sediments function as tanks for these

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organisms and shelter against sunlight (Davies-Colley et al. 1999) and protection against protozoan predation are found in these environments.

Several studies have shown that Identification of the enterococcus species that are present in the environment may help determine the main sources of faecal contamination (Devriese et al. 1993, Hagedorn et al. 1999, Wiggins et al. 1999 and Wheeler et al. 2002). Throughout this study, E.faecalis and E.faecuim were isolated with (43% and 31%respectively) dominated over all isolated bacteria (Fig.

3), the work of (Oliveira et al. 2008), made on the distribution of species enterococccus isolated from seawater Gonzaguinha in Brazil, showed that E.faecium with a percentage of 42, 8%, and E.faecalis with a rate of 28.6% correlates with our results. On the other hand, E.casseliflavus ranks third, with a percentage of 16%, these results are similar to those found by (Oliveira et al. 2008), in samples of sea water in Brazil which is 11.6%. The two strains of E.guallinarum and E.durans, with a respective percentage is 3% and 5%, these results are consistent with the work of (Moore et al. 2008), which received a lower percentage to 3% for both species in seawater, at a site similar to this bay, the other percentage of enterococcus spp in a closed bay was similar to this study with 8% (Ferguson et al.

2005). The highest frequencies of E.faecalis and E.faecium in site1 and 3 indicate that faecal contamination of samples on these sites is primarily of human origin because as these bacteria are more specific to humans (Oliviera et al. 2008). These species have been identified as predominant in seawater and shellfish with densities of enterococcus (Ferguson et al. 2005). In contrast, higher frequencies of E. casseliflavus and E gallinarum in seawater in site 2 in the waters receiving the urban runoff (Tab. 1) , which correlates with several studies in Scotland (Bayne et al. 1983) and in Italy (Pinto et al. 1999) and (Moore et al. 2008) that E. casseliflavus was the most frequently isolated species in urban runoff making up 36–65%. Recently, Enterococcus-resistant infections have become a great therapeutic challenge due to an increase in their incidence and to the increase in the occurrence of strains resistant to antimicrobials (Arvanitidou et al. 2001). The most important resistance phenotypes are those related to aminoglycosids (streptomycin and gentamicin), betalactamics (amoxicillin and ampicillin) and glycopeptides (teicoplanin and vancomycin) (D’Azevedo et al. 2004, Courvalin 2004).

In this study, 54.4% of strains enterococcus resistance was detected. To sun up, the results showed a broad resistance to erythromycin, rifampicin and tetracycline, which correlates with the work of (Moore et al 2008, Oliveira et al. 2008), in contrast, resistance to betalactamics and glycopeptides has not been as high in this study as for the work of (Junco et al. 2001, Oliviera et al. 2008). Only four strains were resistant to ampicillin and vancomycin (tab. 2). On shellfish, the presence of two strains resistant to vancomycin VRE are in harmony with the various studies on VRE in the marine environment (Moore et al. 2008). We remark that the samples of sediments have a high resistance to major antibiotics and are characterized by a presence of resistance to (Tetracycline, High level gentamicin, High level streptomycin, Vancomycin and Ampicillin). On strains isolated from the site1 in seawater near of sewages, the results showed a broad resistance to Erythromycin, Rifampicin, Tetracycline and to Vancomycin, these results are similar to the work of (Moore et al. 2008). On strains isolated from the site 2 near of urban, the results showed a different profil, 10 % vis-à-vis High level gentamycin and 7.9% vis-à-vis the High level of streptomycin, a multiresistance was recorded with 13.6% in this site (Moore et al. 2008). It is likely that there is a risk to public especially at the site 1. Although the resistance to Vancomycin can be considered low in the present study (2.3%), it is an important issue as this antibiotic is used in the treatment of infections caused by enterococcus with multiple resistances to various antibiotics (Huycke et al. 1998). As a consequence, the occurrence of resistant strains to such antimicrobial and the existence of strains resistant to Ampicillin in the marine environment are an alarming factor due to the possibility that such resistance may be transferred (Blom et al. 2000). Despite the occurrence of multiple resistance to antibiotics in the strains isolated from either site 1 and site 2, this was not highly compared with other studies (Arvanitidou et al. 2001). The detection of triple resistance in strains isolated from waters at site 2 is an indication that the intermittent contamination at this site by domestic and sewage, the insertion of strains enterococcus resistant to various types of antibiotics with multiple resistance in marine recreational waters may

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cause risk to the swimmers, the epidemiological research in Israel and New Zealand demonstrated strong relationships between enterococcus densities and the incidence of illness among swimmers in marine waters receiving raw sewage and treated sewage discharges (Russell et al. 2007, Oubrim et al.

2008).

V. Conclusion

Considering the results of this study, it is reasonable to conclude that sea water and sediment receiving sewage and urban runoff with the high density of entrococcus contribute to the dissemination of resistance to antimicrobials and increasing the Potential of waterborne disease transmission at Recreational Beaches.

References

[1] Aarestrup, F.M., Association between the consumption of antimicrobial agents in animal husbandry and the occurrence of resistant bacteria among food animals. International Journal of Antimicrobial Agents. 1999, 12: 279– 285.

[2] Alm, E.W., Burke, J., Spain, A., Fecal indicator bacteria are abundant in wet sand at freshwater beaches. Water Res. 2003, 37 (16): 3978–3982.

[3] Anon, ISO 9308–3 Water Quality – Detection and Enumeration of Escherichia coli and Coliform Bacteria in Surface and Waste Water – Part 3: Miniaturized Method (Most Probable Number) by Inoculation in Liquid Medium. 1998, ISO, 20 pp.

[4] Anon, NF V 08–600 Microbiologie des aliments – Dénombrement des Escherichia coli présumés dans les coquillages vivants – Technique du nombre le plus propable (Microbiologie of Foods and Foodstuffs Products – Enumeration of Presumptive Escherichia coli in Living Shellfish – MPN Technique). 2000, AFNOR 16 pp.

[5] Arvanitidou, M., Katsouyannopoulos, V and Tsakris, A., Antibiotic resistance patterns of enterococci isolated from coastal bathing waters. JMed Microbiol. 2001, 50: 1001–1005.

[6] Ash, R.J., Mauck, B., Morgan, M., Antibiotic resistance of Gram negative bacteria in rivers, Unites States. Emerg. Infect. Dis. 2002, 8: 713–716.

[7] Blom, M., Sorensen, T.L., Poulsen, R.L., Frimodt-Moller, N., Monnet, D.L., Aarestrup, F.M., Espersen, F., Ingestion of vancomycin-resistant Enterococcus faecium strains of food animal origin by human healthy volunteers: a randomized double-bind study. 2000, In: 40th ICAAC Abstracts, Ontario, Canada. p. 432.

[8] Bonilla T.D., Nowosielski K., Cuvelier M., Hartz A., Green M., Esiobu N., McCorquodale., DS, Nwadiuto Esiobu Donald S., McCorquodale Jay M., & Fleisher Rogerson A., Prevalence and distribution of fecal indicator organisms in South Florida beach sand and preliminary assessment of health effects associated with beach sand exposure. Marine Pollution Bulletin, 2007, 54: 1472-1482.

[9] Bower, C.K., Daeschel, M.A., Resistance responses of microorganisms in food environments.

International Journal of Food Microbiology, 1999, 50: 33– 44.

[10] Devriese, L.A., Pot, B. and Collins, M.D., Phenotypic identification of the genus Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups. J Appl Bacteriol. 1993, 75: 399–408.,

[11] Facklam, R.R., Collins, M.D.,. Identification of Enterococcus species isolated from human infections by a conventional test scheme. J. Clin. Microbiol. 1989, 27 (4): 731–734.

[12] Ferguson, D.M., Moore, D.F., Getrich, M.A., Zhowandai, M.H., Enumeration and speciation of enterococci found in marine and intertidal sediments and coastal water in Southern California.

J. Appl. Microbiol. 2005, 99: 598–608.

(10)

[13] Hagedorn, C., Robinson, S.L., Filtz, J.R., Gurbbs, S.M., Angier, T.A., Reneau Jr., R.B., Determing sources of fecal pollution in a rural Virginia watershed with antibiotic resistance patterns in fecal streptococci. Appl. Environ. Microbiol. 1999, 65 (12): 5522–5531.

[14] Harwood, V.J., Brownell, M., Perusek, W and Whitlock, J.E., Vancomycin-resistant Enterococcus spp. isolated from wastewater and chicken feces in the United States. Appl Environ Microbiol. 2001, 67: 4930–4933.

[15] Huycke, M.M., Sahm, D.F, Gilmore, M.S., Multiple-drug resistant enterococci: the nature of the problem and an agenda for the future. Emerg Infect Dis. 1998. 4 (2): 239–249.

[16] Junco, M.T.T., Martîn, M.G., Toledo, M.L.P., Gómez, P.L., Barrasa, J.L.M., Identification and antibiotic resistance of faecal enterococci isolated from water samples. Int J Hyg Environ Health. 2001, 203: 363–368.

[17] Kauffman, F Serological diagnostics of Salmonella species Copenhagen, 1972 : Munksgaard.

[18] Klein, G., Pack, A., Reuter, G., Antibiotic resistance patterns of enterococci and occurrence of vancomycin-resistant enterococci in raw minced beef and pork in Germany. Appl Environ Microbiol. 1998, 64: 1825– 1830.

[19] Kühn, I., Iversen, A., Finn, M., Greko, C., Burman, L.G., Blanch, A.R., Vilanova, X., Manero, A., Taylor, H., Caplin, J., Domînguez, Z.L, Herrero, I.A, Moreno, M.A, Möllby, R., Occurrence and relatedness of vancomycin-resistant enterococci in animals, humans, and the environment in different European regions. 2005, Appl Environ Microbiol 71 (9): 5383–5390.

[20] Manero, A., Vilanova, X., Cerdá-Cuéllar, M., Blanch, A.R., Vancomycin and erythromycin- resistant enterococci in a pig farm and its environment. Environ Microbiol. 2006, 8: 667–674.

[21] Moore, D.F., Guzman, J.A., McGee, C., Species distribution and antimicrobial resistance of enterococci isolated from surface and ocean water. Journal of Applied Microbiology. 2008, 105 (4): 1017-1025.

[22] Mudryk, Z.J, Occurrence and distribution antibiotic resistance of heterothophic bacteria isolated from a marine beach. Mar Pollut Bull. 2005, 50: 80–86.

[23] National Committee for Clinical Laboratory Standards, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Approved Standard M7-A5. 2000, National Committee for Clinical Laboratory Standards, Wayne, PA, US.

[24] Oliveira, A-J., and Pinhata, M.W., Antimicrobial resistance and species composition of Enterococcus spp. isolated from waters and sands of marine recreational beaches in Southeastern Brazil, Water Research. 2008, 42: 2242-2250.

[25] Oubrim, N. Ennaji, M.M. Hajjami, K. Bennani, M. Hassar, M. Cohen, N. Microbiological impact of treatment lagoons on the economics of water for reuse in agriculture a case study in morocco (settat and soualem regions). Cell. Mol. Biol. 2011, 57 Suppl: OL1567-OL1574.

[26] Pinto, B., Pierotti, R., Canale, G., Reali, D., Characterization of faecal streptococci’ as indicators of faecal pollution and distribution in the environment. Lett Appl Microbiol. 1999, 29: 258–263.

[27] Popoff, M.Y, Le Minor, L, Taxonomie du genre Salmonella. Changements de la nomenclature des sérovars. In: M.Y. POPOFF and L. LE MINOR: Formules antigéniques des sérovars de Salmonella, 7th revision. WHO Collaborating Centre for Reference and Research on Salmonella. Institut Pasteur, Paris, France, 1997, p. 4.

[28] Rhodes, M.W and Kator, H., Enumeration of Enterococcus sp. using a modified mE method. J Appl Microbiol. 1997, 83: 120–126.

[29] Russell, D. Arnone and Joyce, Perdek, Walling, Waterborne pathogens in urban watershed.

Journal of Water and Health. 2007, 5(1): 149-162.

[30] Sato, M.I.Z, Bari, M.D, Lamparelli, C.C, Truzzi, A.C, Coelho, M.C.L.S, Hachich, E.M, Sanitary quality of sands from marine recreational beaches of São Paulo, Brazil. Braz J Microbiol. 2005, 36 (4): 321–326.

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[31] Sinton, L.W, Davies-Colley, R.J, Bell, R.G, Inactivation of enterococci and fecal coliforms from sewage and meat-works effluents in seawater chambers. Appl Environ Microbiol. 1994, 60 (6): 2040–2048.

[32] Teixeira, L.M, Carvalho, M.G.S and Facklam, R.R, Enterococcus. In Manual of Clinical Microbiology, 9th edn, ed. Murray PR, Baron EJ, Jorgensen JH, Landry ML and Pfaller MA p:

430. Washington, 2007 DC: ASM Press.

[33] Tejedor J.M.T, Gonzalez M.M., Pita T.M., Lupiola G.P., Martîn B.J.L, Identification and antibiotic resistance of faecal enterococci isolated from water samples. Int J Hyg Environ Health. 2001, 203: 363–368.

[34] Van Horn, K, Toth, C, Kariyama, R, Mitsuhata, R, Komon, H , Evaluation of 15 motility media and a direct microscopic method for detection of motility in enterococci. J Clin. Microbiol, 2002. 40: 2476–2479.

[35] Wheeler, A.L., Hartel, P.G., Godfrey, D.G., Hill, J.L and Segars, W.I., Potential of Enterococcus faecalis as a human fecal indicator for microbial source tracking. J Environ Qual.

2002, 31: 1286–1293.

[36] Wiggins, B.A., Andrews, R.W., Conway, R.A., Corr, C.L., Dobratz, E.J., Dougherty, D.P, Eppard, J,R., Knupp, S.R., Limjoco, M,C., Mettenburg, J.M., Rinehardt, J.M., Sonsino, J., Torrijos, R.L., Zimmerman, M.E., Use of antibiotic resistance analysis to identify nonpoint sources of fecal pollution. Appl Environ Microbiol. 1999, 65 (8): 3483–3486.

[37] Wilson, I. G and G. G McAfee, Vancomycin-resistant enterococci in shellfish, unchlorinated waters and chicken. Int J Food Microbiol. 2002, 79:143-151.

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