Enterotoxin-Encoding Genes in Staphylococcus spp. from Bulk Goat Milk

Abstract

Although Staphylococcus aureus has been implicated as the main Staphylococcus species causing human food poisoning, recent studies have shown that coagulase-negative Staphylococcus could also harbor enterotoxin-encoding genes. Such organisms are often present in goat milk and are the most important mastitis-causing agents. Therefore, this study aimed to investigate the occurrence of enterotoxin-encoding genes among coagulase-positive (CoPS) and coagulase-negative (CoNS) staphylococci isolated from raw goat milk produced in the semi-arid region of Paraiba, the most important region for goat milk production in Brazil. Enterotoxin-encoding genes were screened in 74 staphylococci isolates (30 CoPS and 44 CoNS) by polymerase chain reaction targeting the genes sea, seb, sec, sed, see, seg, seh, and sei. Enterotoxin-encoding genes were found in nine (12.2%) isolates, and four different genes (sea, sec, seg, and sei) were identified amongst the isolates. The most frequent genes were seg and sei, which were often found simultaneously in 44.5% of the isolates. The gene sec was the most frequent among the classical genes, and sea was found only in one isolate. All CoPS isolates (n=7) harboring enterotoxigenic genes were identified as S. aureus. The two coagulase-negative isolates were S. haemolyticus and S. hominis subsp. hominis and they harbored sei and sec genes, respectively. A higher frequency of enterotoxin-encoding genes was observed amongst CoPS (23.3%) than CoNS (4.5%) isolates (p<0.05), reinforcing the importance of S. aureus as a potential foodborne agent. However, the potential risk posed by CoNS in goat milk should not be ignored because it has a higher occurrence in goat milk and enterotoxin-encoding genes were detected in some isolates.

Introduction

S taphylococcal food poisoning is one of the most common foodborne illnesses worldwide (Ostyn et al., 2010; SVS, 2009) and is caused by the ingestion of food containing staphylococcal enterotoxin (SE) produced mainly by Staphylococcus aureus (Loncarevic et al., 2005). Twenty-three types of SEs (SEA–SEE and SEG–SEV) have been described (Larkin et al., 2009), and the majority of food poisoning outbreaks have been attributed to the classical and most studied enterotoxins (SEA–SEE). Nonetheless, the increasing detection of novel SEs (SEG–SEU) and their putative involvement in staphylococcal food poisoning (Jarraud et al., 1999) suggests the need to understand their impact on public health.

Besides S. aureus and other coagulase-positive staphylococci (CoPS), it is known that coagulase-negative staphylococci (CoNS) species are also capable of producing enterotoxins (Vernozy-Rozand et al., 1996; Cunha et al., 2006; Vasil, 2007; Veras et al., 2008). Moreover, staphylococcal food poisoning outbreaks attributed to CoNS have been reported (Carmo et al., 2002). Interestingly, CoNS are commonly found in goat milk and are the leading mastitis-causing agents in goats (White and Hinckley, 1999). However, information about the occurrence of enterotoxigenic CoNS in goat milk is still scarce and the public health importance of those bacteria is unknown, although some human foodborne illness outbreaks have been linked to the consumption of goat milk (Buyser et al., 2001).

The understanding of the potential hazards of staphylococcal contamination in goat milk is of major importance to the goat industry worldwide. Therefore, the aim of this study was to investigate the occurrence of enterotoxin-encoding genes in CoPS and CoNS Staphylococcus spp. from goat milk produced in the semi-arid Paraiba State, the main goat milk-producing region in South America.

Materials and Methods

Samplings and Staphylococcus isolation

Bulk goat milk samples were randomly collected from 55 smallholder producers in four different municipalities (A, n=18; B, n=16; C, n=12; D, n=5) located in Cariri, a semi-arid region in Paraiba State, Northeastern Brazil. Samples were processed for staphylococci isolation, according to Bennett and Lancette (2001). Briefly, milk samples (25 mL) were diluted in 225 mL of lactose broth (Acumedia, Lansing, MI) and then serially diluted (1:10) in ¼ strength Ringer's solution. Aliquots (100 μL) were spread plated over the surface of Baird-Parker agar (Acumedia) supplemented with potassium tellurite and egg yolk (Laborclin, Pinhais, Brazil). After incubation (35°C, 24–48 h), the total number of colonies on each plate was counted. At least three isolates per bacterial population were picked per dish, considering similar morphological traits. Further confirmation was performed using morphological (Gram staining) and biochemical tests (coagulase, mannitol and maltose fermentation, and hemolysis in blood agar).

Staphylococcus spp. isolates (n=74; 30 CoPS and 44 CoNS) from municipalities A (n=15), B (n=26), C (n=28), and D (n=05) were further screened for enterotoxin-encoding genes by polymerase chain reaction (PCR) assay as described below. Species identification of Staphylococcus-harboring enterotoxigenic genes was determined using a semi-automated system (Autoscan 4; Siemens Healthcare, Malvern, PA) by means of a biochemical panel (Combo PC33; Siemens) and software (LabPro Connect; Siemens).

Detection of enterotoxin-encoding genes by PCR

Genomic DNA was extracted using phenol/chloroform (Fritsch et al., 1989) with minor modifications. The quality and concentration of DNA were measured (Biophotometer Plus; Eppendorf, Wesseling-Berzdorf, Germany).

Detection of the enterotoxigenic genes was performed by individual PCR reactions targeting the genes sea, seb, sec, sed, see, seg, seh, and sei (Blaiotta et al., 2004). Reactions were performed in 25-μL reactions containing 0.4 mM of each primer, 200 mM of each dNTP, 2 mM of MgCl₂, 100 ng of genomic DNA, and 1U of Taq DNA polymerase (Invitrogen, São Paulo, Brazil). Primer details are provided in Table 1.

Table 1.

Primer Sequences and the Expected Amplified Fragment Sizes for Each Uniplex Polymerase Chain Reaction Targeting the Genes sea-see and seg-sei in Staphylococcus spp. Isolated from Bulk Goat Milk Samples in the Semi-Arid Region of Cariri, Paraiba, Brazil

Primer	Sequence (5′→3′)	Fragment size (bp)	Reference
sea-1	TTG GAA ACG GTT AAA ACG AA	120	Johnson et al. (1991)
sea-2	GAA CCT TCC CAT CAA AAA CA
seb-1	TCG CAT CAA ACT GAC AAA CG	478	Johnson et al. (1991)
seb-2	GCA GGT ACT CTA TAA GTG CC
sec-1	GAC ATA AAA GCT AGG AAT TT	257	Johnson et al. (1991)
sec-2	AAA TCG GAT TAA CAT TAT CC
sed-1	CTA GTT TGG TAA TAT CTC CT	317	Johnson et al. (1991)
sed-2	TAA TGC TAT ATC TTA TAG GG
see-1	CAA AGA AAT GCT TTA AGC AAT CTT AGG CCAC	482	Jarraud et al. (1999)
see-2	CTT ACC GCC AAA GCTG
seg-1	AAT TAT GTG AAT GCT CAA CCC GATC	642	Jarraud et al. (1999)
seg-2	AAA CTT ATA TGG AAC AAA AGG TAC TAG TTC
seh-1	CAA TCA CAT CAT ATG CGA AAG CAG	376	Jarraud et al. (1999)
seh-2	CAT CTA CCC AAA CAT TAG CACC
sei-1	CTC AAG GTG ATA TTG GTG TAGG	577	Jarraud et al. (1999)
sei-2	AAA AAA CTT ACA GGC AGT CCA TCTC

Amplification was performed in a thermal cycler (TC-5000; Techne, Stone, Staffordshire, UK) according to the following conditions: initial denaturation step (95°C, 3 min), 30 amplification cycles (95°C, 30 sec; 55°C, 75 sec; 72°C, 30 sec), and final extension step (72°C, 5 min). Products were analyzed by electrophoresis in 2% agarose gel (LGC Biotechnology, Rio de Janeiro, Brazil) stained with GelRed (Biotium, Hayward, CA). The presence or absence of bands was analyzed visually under ultraviolet light. Positive controls used in the reactions included S. aureus American Type Culture Collection (ATCC) 13565 (sea), S. aureus ATCC 14458 (seb), S. aureus ATCC 19095 (sec, seh, and sei), S. aureus ATCC 23235 (sed and seg), and S. aureus ATCC 27664 (see) (Fig. 1).

FIG. 1.

Agarose gel containing polymerase chain reaction–amplified products of enterotoxin-encoding genes in Staphylococcus spp. used as positive controls. M, 100-bp DNA ladder; 1, sea (120pb); 2, seb (478pb); 3, sec (257pb); 4, sed (317pb); 5, see (482pb); 6, seg (642pb); 7, seh (376pb); 8, sei (577pb); 9, femA (132pb).

Statistical analysis

Data was analyzed using SPSS 17.0 (SPSS, Chicago, IL) to generate descriptive analyses, chi-square tests, and Fisher's exact test when appropriate. Mean counts of Staphylococcus were transferred into log format for data normalization and compared between municipalities. Possible associations between the occurrence of enterotoxin-encoding genes and the production of coagulase by the isolates were determined.

Results and Discussion

Staphylococci counts ranged from 2×10² to 3×10⁷ CFU/mL, with a mean value of 2.08×10⁶ CFU/mL. Mean staphylococci counts were significantly lower (p<0.05) in municipality A (1.2×10⁵ CFU/mL) compared to B (2.2×10⁶ CFU/mL), C (5.8×10⁶ CFU/mL), and D (2×10⁵ CFU/mL), suggesting the existence of factors associated with the staphylococcal contamination in raw milk produced in the region. Most important, however, considering that staphylococcal food poisoning is associated with staphylococci levels in food, is the fact that staphylococci contamination was high. It is assumed that S. aureus levels of 10⁵ CFU/g of food may produce enough enterotoxin to cause illness (Seo and Bohach, 2007). Counts exceeding 10⁵ CFU/mL were observed in 41.7% (5/11) of the milk samples collected in municipality A, 61.1% (11/18) in B, 55% (11/20) in C, and 20% (1/5) in D (Table 2).

Table 2.

Enterotoxin-Encoding Genes (sea, sec, seg, and sei) in Nine Staphylococci Isolates Originating from Raw Goat Milk Produced in Four Municipalities (A–D) of the Semi-Arid Region of Cariri, Paraiba, Brazil

Enterotoxin-encoding genes	Staphylococcus species	Municipality	Staphylococcus spp. counts in raw milk (CFU/mL)
sec	S. aureus	C	3.3×10⁵
sec	S. hominis	C	2.8×10⁷
seg	S. aureus	C	6.2×10⁴
sei	S. aureus	C	2.5×10⁵
sei	S. haemolyticus	D	6.9×10⁴
sec+seg	S. aureus	C	5.1×10⁴
seg+sei	S. aureus	B	2.7×10⁵
sea+seg+sei	S. aureus	A	1.2×10⁴
sec+seg+sei	S. aureus	C	3.1×10⁵

Out of the 74 isolates, 44 (59.4%) were coagulase-negative staphylococci (CoNS), and all coagulase-positive (CoPS) isolates (n=7) harboring enterotoxigenic genes were identified as S. aureus. The frequency of CoNS in goat milk was similar to that previously reported (Ndegwa et al., 2002). The two CoNS isolates harboring enterotoxigenic genes were S. hominis and S. haemolyticus, which usually are present in low numbers in milk from healthy goats and sheep (Vasil, 2007; Onni et al., 2012). However, the occurrence of S. haemolyticus was reported in milk from goats and cows with mastitis (Virdis et al., 2010; Fessler et al., 2010).

In the present study, enterotoxin-encoding genes were found in nine (12.2%) isolates, from which seven were CoPS and two were CoNS. A higher frequency (p<0.05) of enterotoxin-encoding genes was observed in CoPS (23.3%) compared with CoNS (4.5%), which corroborates a previous report (Valle et al., 1990). Amongst the positive isolates, four (44.5%) were positive for one gene, three (33.4%) harbored two genes, and one (11.2%) was positive for three genes.

Higher frequencies of enterotoxin-encoding genes in CoPS compared to CoNS were previously described in other cases such as food handlers (Bartels et al., 2009) and sheep's milk (Vasil, 2007). However, a previous report in Brazil indicated a higher enterotoxigenic potential for CoNS than CoPS in milk from goat and other species (Lamaita et al., 2005). Additionally, the genes sea and sec have been reported in CoNS staphylococci from different types of foods in Brazil (Cunha et al., 2006).

The frequency of isolates harboring staphylococcal enterotoxigenic genes in the present study was lower than frequencies reported in samples of bovine milk (Rall et al., 2008) and sheep milk (Maslankova et al., 2009). Although the low number of isolates harboring staphylococcal enterotoxigenic genes in the present study was very similar to that reported for bovine milk produced by smallholders in Norway (Jorgensen et al., 2005), frequencies higher than 50% were reported in samples of different animal species and products elsewhere (Cremonesi et al., 2006; Morandi et al., 2007; Mork et al., 2010).

The sei gene was detected in isolates originating from all four municipalities. The gene seg was detected in municipalities A–C. However, sea was identified in municipality A only, while sec was identified only in staphylococci from municipalities C and D. The genes seb, sed, see, and seh were not detected in the present study.

Amongst the classic enterotoxin-encoding genes, sec was detected in 55.6% of the isolates. The gene sea was observed in one isolate and only in combination with other genes. This gene is described as one of the most common gene found in staphylococci from bovine milk (Rall et al., 2008) and from human samples (Rall et al., 2010). Although there are investigations on the occurrence of enterotoxin-encoding genes in staphylococci goat milk and most of them are restricted to the classic genes, the present study corroborates previous findings (Scherrer et al., 2004; Loncarevic et al., 2005) that reported sec as the gene with higher frequency amongst the classic genes in staphylococci from goat milk, reaching up to 71% (Mork et al., 2010) and 86% (Silva et al., 2005) in staphylococci from healthy and mastitic animals, respectively. The gene sec was detected almost exclusively in staphylococci from goat origin (Cremonesi et al., 2006), and it was also reported as the most frequent (24.1%) in S. aureus from sheep milk and dairy products (Maslankova et al., 2009). A comparative study of staphylococci from goats and cows from the same region (Cremonesi et al., 2006) showed that sed (65.5%) and sea (51.7%) were the most common classical enterotoxin-encoding genes in bovine staphylococci, while goat staphylococci harbored mainly sec (97.3%).

The present study corroborates the majority of reports worldwide indicating a higher frequency of novel as compared to classic enterotoxigenic genes in staphylococci (Jorgensen et al., 2005; Loncarenvic et al., 2005; Rall et al., 2008; Zouharova and Rysanek, 2008; Karahan et al., 2009). The results were similar to those reported by Scherrer et al. (2004) in Switzerland, who detected the genes seg (10.6%), sej (8.9%), and sei (8.2%) in staphylococci from goat milk.

The most common combination of enterotoxin-encoding genes was seg and sei, found in 44.5% of isolates. This finding has been previously reported in Brazil (Rall et al., 2008, 2010; Vasconcelos et al., 2011).

There is a lack of information about the occurrence of enterotoxin-encoding genes in staphylococci from goat milk in Brazil. Although the present study showed that the majority of isolates harboring enterotoxigenic genes were identified as S. aureus, the high numbers of CoNS in goat milk and the detection of enterotoxigenic genes in CoNS reinforce the need to establish regulatory standards for total Staphylococcus spp. counts in goat milk, and not only for CoPS as recommended in the current legislation.

The detection of enterotoxin-encoding genes in goat milk highlights the need for improvements in surveillance programs in order to estimate the real impact of goat milk and dairy products on public health. While one could argue that the frequency of enterotoxin-encoding genes was low in the present study, the cultural habit of consumption of raw milk by rural populations raises special concern, since it potentiates the probability of acquiring zoonotic infections. Public health risks associated with the consumption of bovine raw milk is well documented (Oliver et al., 2009) and should be further investigated for goat milk. Studies to raise knowledge about the host specificity of staphylococci harboring enterotoxin-encoding genes are warranted.

Finally, the results of the present investigation indicate the need to improve veterinary assistance and extension programs targeting education in rural communities in order to decrease microbial contamination in raw milk and warrant market competitiveness for smallholders.

Footnotes

Acknowledgments

We thank CNPq for grants (Proc. 557593/2009-3) and a scholarship to D.L. (Proc. 554302/2010-1).

Disclosure Statement

No competing financial interests exist.

References

Bartels

, Andrade

, Neumann

, Silva

. Identificação de portadores de Staphylococcus enterotoxigênicos e avaliação da sensibilidade a antimicrobianos [Identification of carriers of enterotoxigenic Staphylococcus and evaluation of antimicrobial susceptibility] Arq Bras Med Vet Zootec, 2009; 61:1450–1453. (In Portuguese.)

Bennett

, Lancette

. Staphylococcus aureus. In: Bacteriological Analytical Manual (BAM), 8^th ed. Gaithersburg. Food and Drug Administration. 2001. www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm071429.htm. 2012 December 1.

Blaiotta

, Ercolini

, Pennacchia

, Fusco

, Casaburi

, Pepe

, Villani

. PCR detection of staphylococcal enterotoxin genes in Staphylococcus spp. strains isolated from meat and dairy products. Evidence for new variants of seG and seI in S. aureus. J Appl Microbiol, 2004; 97:719–730.

Buyser

, Dufour

, Maire

, Lafarge

. Implication of milk and milk products in food-borne diseases in France and in different industrialized countries. Int J Food Microbiol, 2001; 67:1–17.

Carmo

, Dias

, Linardi

, Sena

, Santos

, Faria

, Pena

, Jett

, Heneine

. Food poisoning due to enterotoxigenic strains of Staphylococcus present in Minas cheese and raw milk in Brazil. Food Microbiol, 2002; 19:9–14.

Cremonesi

, Castiglioni

, Malferrari

, Biunno

, Vimercati

, Moroni

, Morandi

, Luzzana

. Improved method for rapid DNA extraction of mastitis pathogens directly from milk. J Dairy Sci, 2006; 89:163–169.

Cunha

MLRS

, Peresi

, Calsolari

RAO

, Araújo

Jr . Detection of enterotoxins genes in coagulase-negative staphylococci isolated from foods. Braz J Microbiol, 2006; 37:64–69.

Fessler

, Billerbeck

, Kadlec

, Schwarz

. Identification and characterization of methicillin-resistant coagulase-negative staphylococci from bovine mastitis. J Antimicrob Chemother, 2010; 65:1576–1582.

Fritsch

, Maniatis

, Sambrook

. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.

10.

Jarraud

, Cozon

, Vandenesch

, Bes

, Etienne

, Lina

. Involvement of enterotoxins G and I in staphylococcal toxic shock syndrome and staphylococcal scarlet fever. J Clin Microbiol, 1999; 37:2446–2449.

11.

Jorgensen

, Mork

, Hogasen

, Rovik

. Enterotoxigenic Staphylococcus aureus in bulk milk in Norway. J Appl Microbiol, 2005; 99:158–167.

12.

Karahan

, Açik

, Çetinkaya

. Investigation of toxin genes by polymerase chain reaction in Staphylococcus aureus strains isolated from bovine mastitis in Turkey. Foodborne Pathog Dis, 2009; 6:1029–1035.

13.

Lamaita

, Cerqueira

MMOP

, Carmo

, Santos

, Penna

, Souza

CFAM

. Contagem de Staphylococcus sp e detecção de enterotoxinas estafilocócicas e toxina da síndrome do choque tóxico em amostras de leite cru refrigerado [Counting of Staphylococcus sp. and detection of staphylococcal enterotoxins and toxic shock toxin syndrome from cooled raw milk] Arq Bras Med Vet Zootec, 2005; 57:702–709. (In Portuguese.)

14.

Larkin

, Carman

, Krakauer

, Stiles

. Staphylococcus aureus: The toxic presence of a pathogen extraordinaire. Curr Med Chem, 2009; 16:4003–4019.

15.

Loncarevic

, Jorgensen

, Lovseth

, Mathisen

, Rorvik

. Diversity of Staphylococcus aureus enterotoxin types within single samples of raw milk and raw milk products. J Appl Microbiol, 2005; 98:344–350.

16.

Maslankova

, Pilicincova

, Tkacikova

. Pheno- and genotyping of Staphylococcus aureus isolates of sheep origin. Acta Vet Brno, 2009; 78:345–352.

17.

Morandi

, Brasca

, Lodi

, Cremonesi

, Castiglioni

. Detection of classical enterotoxins and identification of enterotoxin genes in Staphylococcus aureus from milk and dairy products. Vet Microbiol, 2007; 124:66–72.

18.

Mork

, Kvitle

, Mathisen

, Jorgensen

. Bacteriological and molecular investigations of Staphylococcus aureus in dairy goats. Vet Microbiol, 2010; 141:134–141.

19.

Ndegwa

, Mulei

, Munyua

SJM

. Prevalence of various microbial organisms isolated from dairy goat milk samples in central Kenya highlands. The Kenyan Veterinarian, 2002; 25:9–11.

20.

Oliver

, Boor

, Murphy

, Murinda

. Food safety hazards associated with consumption of raw milk. Foodborne Pathog Dis, 2009; 6:793–806.

21.

Onni

, Vidili

, Bandino

, Marogna

, Leori

, Tola

. Identification of coagulase-negative staphylococci isolated from caprine milk samples by PCR-RFLP of groEL gene. Small Rumin Res, 2012; 104:185–190.

22.

Ostyn

, De Buyser

, Guillier

, Groult

, Félix

, Salah

, Delmas

, Hennekinne

. First evidence of a food poisoning outbreak due to staphylococcal enterotoxin type E, France, 2009. Euro Surveill, 2010; 15. 19528 http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19528. 2012 December 1.

23.

Rall

VLM

, Vieira

, Rall

, Vieitis

, Fernandes

Jr , Candeias

JMG

, Cardoso

KFG

, Araújo

Jr . PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk. Vet Microbiol, 2008; 132:408–413.

24.

Rall

VLM

, Sforcin

, Augustini

VCM

, Watanabe

, Fernandes

Jr , Rall

, Silva

, Araújo

Jr . Detection of enterotoxin genes of Staphylococcus sp. isolates from nasal cavities and hands of food handlers. Braz J Microbiol, 2010; 41:59–65.

25.

Scherrer

, Corti

, Muehlherr

, Zweifel

, Stephan

. Phenotypic and genotypic characteristics of Staphylococcus aureus isolates from raw bulk-tank milk samples of goats and sheep. Vet Microbiol, 2004; 101:101–107.

26.

Seo

, Bohach

. Staphylococcus aureus. In: Food Microbiology: Fundamentals and Frontiers. Doyle

, Beuchat

. Washington, DC: ASM Press, 2007; 493–519.

27.

Silva

, Carmo

, Silva

. Detection of the enterotoxins A, B, and C genes in Staphylococcus aureus from goat and bovine mastitis in Brazilian dairy herds. Vet Microbiol, 2005; 106:103–107.

28.

[SVS] Secretaria de Vigilância em Saúde, Ministério da Saúde, Brasil. 2009. Análise epidemiológica dos surtos de doenças transmitidas por alimentos no Brasil, 1999–2009 [Epidemiological analysis of disease outbreaks transmitted by food in Brazil, 1999–2009] http://portal.saude.gov.br/portal/arquivos/pdf/analise_ep_surtos_dta_brasil_2009.pdf. 2012 December 1. (In Portuguese.)

29.

Valle

, Goméz-Lucia

, Piriz

, Goyache

, Orden

, Vacillo

. Enterotoxin producing by staphylococci isolated from healthy goats. Appl Environ Microbiol, 1990; 56:1323–1326.

30.

Vasconcelos

, Pereira

, Araújo

Jr , Cunha

MLRS

. Molecular detection of enterotoxins E, G, H and I in Staphylococcus aureus and coagulase-negative staphylococci isolated from clinical samples of newborns in Brazil. J Applied Microbiol, 2011; 111:749–762.

31.

Vasil

. Aetiology of mastites and enterotoxin production by Staphylococcus sp. isolated from milk of two sheep herds. Slovak J Anim Sci, 2007; 40:189–195.

32.

Veras

, Carmo

, Tong

, Shupp

, Cummings

, Santos

, Cerqueira

MMOP

, Cantini

, Nicoli

, Jett

. A study of the enterotoxigenicity of coagulase-negative and coagulase-positive staphylococcal isolates from food poisoning outbreaks in Minas Gerais, Brazil. Int J Infect Dis, 2008; 12:410–415.

33.

Vernozy-Rozand

, Mazuy

, Prevost

, Lapeyre

, Bes

, Brun

, Fleurette

. Enterotoxin production by coagulase-negative staphylococcal isolated from goats and cheese. Int J Food Microbiol, 1996; 30:271–280.

34.

Virdis

, Scarano

, Cossu

, Spanu

, Santis

EPL

. Antibiotic resistance in Staphylococcus aureus and coagulase negative staphylococci isolated from goats with subclinical mastitis. Vet Med Int, 2010; 2010:517060.

35.

White

, Hinckley

. Prevalence of mastitis pathogens in goat milk. Small Rumin Res, 1999; 33:117–121.

36.

Zouharova

, Rysane

. Multiplex PCR and RPLA identification of Staphylococcus aureus enterotoxigenic strains from bulk tank milk. Zoonoses Public Health, 2008; 55:313–319.