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Assessing the microbiological quality of raw goats' and ewes' tank milk samples in Switzerland
Journal Pre-proof Assessing the microbiological quality of raw goats’ and ewes’ tank milk samples in Switzerland Brian Friker, Marina Morach, Sabrina Püntener, Nicole Cernela, Jule Horlbog, Roger Stephan PII:
S0958-6946(19)30246-8
DOI:
https://doi.org/10.1016/j.idairyj.2019.104609
Reference:
INDA 104609
To appear in:
International Dairy Journal
Received Date: 11 September 2019 Revised Date:
19 November 2019
Accepted Date: 20 November 2019
Please cite this article as: Friker, B., Morach, M., Püntener, S., Cernela, N., Horlbog, J., Stephan, R., Assessing the microbiological quality of raw goats’ and ewes’ tank milk samples in Switzerland, International Dairy Journal, https://doi.org/10.1016/j.idairyj.2019.104609. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Ltd. All rights reserved.
1
Assessing the microbiological quality of raw goats’ and ewes’ tank milk
2
samples in Switzerland
3 4 5 6 7
Brian Frikera, Marina Morachb, Sabrina Püntenerb, Nicole Cernelab, Jule Horlbogb,
8
Roger Stephanb*
9 10 11 12 13
a
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Schwarzenburgstrasse 161, 3097 Liebefeld, Switzerland
15
b
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Winterthurerstrasse 272, 8057 Zürich, Switzerland
Veterinary Public Health Institute, Vetsuisse Faculty, University of Bern,
Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich,
17 18 19 20 21
* Corresponding author. Tel.: +41 44 635 86 51
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E-mail address: [email protected] (R. Stephan)
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1
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___________________________________________________________________
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ABSTRACT
28 29
In recent years, popularity of raw milk has increased in many industrialised countries.
30
This study (i) enumerated total viable counts (TVC) and Escherichia coli counts, (ii)
31
assessed prevalence of Staphylococcus (S.) aureus, Salmonella spp. and STEC, (iii)
32
screened for methicillin resistant S. aureus (MRSA) and extended-spectrum β-
33
lactamases (ESBL)-producing Enterobacteriaceae in sheep and goat tank milk
34
samples collected throughout Switzerland and (iv) provided further strain
35
characteristics on isolated pathogens and MRSA. One hundred and twenty-three
36
tank milk samples from 116 farms were analysed. The median TVC was 3.8 log cfu
37
mL-1. E. coli was detected in 16 (13.0%) and S. aureus in 18 (14.6%) samples.
38
Polymerase chain reaction for stx genes was positive in 14 (11.4%) samples. MRSA
39
were isolated from 4 (3.3%) samples. Salmonella spp. and ESBL-producing
40
Enterobacteriaceae were not isolated.
41
___________________________________________________________________
42
2
43
1.
Introduction
44 45
In recent years, food consumption habits have dramatically changed under the
46
influence of lifestyle changes and new technologies. The importance of ewes’ and
47
goats’ milk has increased considerably. Even though ewes’ and goats’ milk only
48
accounts for 0.7% of the total milk production in Switzerland, the amount brought to
49
market has doubled during the last 10 years (Anonymous, 2008, 2018a).
50
In the context of the trend toward “consuming natural” and “purchasing
51
locally”, the popularity of raw milk has increased in many industrialised countries; the
52
consumers tend to prefer raw milk due to better taste and believe in better nutritional
53
values. Moreover, a variety of health benefits associated with raw milk consumption
54
(as improved immunity, less lactose intolerance, less diabetes, and many others) are
55
propagated, but convincing scientific evidence is hardly available (Katafiasz &
56
Bartett, 2012; LeJeune & Rajala‐Schultz, 2009). These trends are rising despite the
57
fact that selling raw milk is prohibited or strictly regulated in many of these countries
58
(Table 1). Particularly noteworthy are the "cow share programmes" in the USA; this
59
means that a number of people share the ownership of a number of animals.
60
Therefore, people can drink raw milk "from their own animals" without having to buy
61
it. Like this, it is possible to acquire raw milk even in some states that generally
62
prohibit the sale of raw milk.
63
However, there is a well-established association between raw milk
64
consumption and infection with pathogenic bacteria as e.g., Salmonella spp., or
65
Shiga toxin-producing Escherichia coli (STEC). For decades, it has been repeatedly
66
shown that raw milk is a major risk factor for food-borne diseases (EFSA, 2015;
67
Klinger & Rosenthal, 1997; LeJeune & Rajala‐Schultz, 2009).
3
68
According to a review of outbreaks in the United States of America, 65.5% of
69
the dairy-related outbreaks were associated with raw milk. Goat milk was involved in
70
4.6% of the raw milk outbreaks even though goats’ and ewes’ milk only account for
71
0.1% of the total milk production (Milani & Wendorff, 2011; Whitehead & Lake, 2018).
72
Apart from reports of well-known food-borne pathogens like Staphylococcus aureus
73
(Vitale et al., 2015), there are also publications on milk-borne outbreaks of diseases
74
that are more commonly known to be transmitted by some other route, e.g., tick
75
borne encephalitis (Brockmann et al., 2018; Dorko et al., 2018; Markovinović et al.,
76
2016).
77
The last study focusing on human pathogens in goats’ and ewes’ milk in
78
Switzerland was more than 15 years ago (Muehlherr, Zweifel, Corti, Blanco, &
79
Stephan, 2003). Such knowledge is scarce not only in Switzerland but in general,
80
especially when comparing with cows’ milk, on which much more research is
81
conducted.
82
Therefore, this study (i) assessed the microbiological quality of ewes’ and
83
goats’ milk by enumerating total viable counts (TVC) and E. coli counts, (ii) generated
84
up-to-date prevalence data for selected foodborne pathogens (S. aureus, Salmonella
85
spp., STEC), (iii) screened for methicillin resistant S. aureus (MRSA) and extended-
86
spectrum β-lactamases (ESBL)-producing Enterobacteriaceae and (iv) provided
87
further strain characteristics on isolated foodborne pathogens and MRSA.
88 89
2.
Methods
2.1.
Raw milk and sampling
90 91 92
4
93
In this study, 123 raw milk samples (99 from goats and 24 from ewes)
94
collected on farm level from tank milk or from milk churn (depending on how milk was
95
stored on the farm) were analysed. They originated from 116 farms located in 24 out
96
of the 26 cantons of Switzerland. From each farm additional data, e.g., number of
97
animals, farming on a regular or side-line basis, membership of the Sanitary Service
98
for Small Ruminants (SSSR), were collected. The SSSR is a private association that
99
provides consultancy on animal health, feeding and proper housing. Members of the
100
SSSR can voluntarily participate in surveillance (e.g., for gastrointestinal parasites) or
101
eradication programmes (e.g., Maedi-Visna for sheep or pseudotuberculosis in
102
goats). Moreover, the SSSR provides support in case of milk quality or herd health
103
problems.
104
Sampling was performed over 4 months (February to May 2019). Milk storage
105
on farm before sampling depended on the farm's frequency of milk delivery, which
106
ranged from daily delivery to schemes like "delivery on Mondays and Thursdays".
107
Samples were transported chilled to the laboratory and analysed within 24 h.
108 109
2.2.
Total viable counts, E. coli, and coagulase-positive staphylococci
110 111
Samples were quantitatively analysed according to ISO standard methods
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(ISO 4833; ISO 16649; ISO 6888) by the surface spreading technique. The following
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agars and conditions were used: plate count agar (Oxoid, Pratteln, Switzerland; 72 h,
114
30 °C) for total viable counts (TVC), RAPID’E. coli 2 agar (Bio-Rad, Reinach,
115
Switzerland; 24 h, 37 °C), AFNOR validated and corresponds to ISO 16649, for E.
116
coli, and EASY Staph agar (Biokar Diagnostics Beauvais, France; 48 h, 37 °C),
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AFNOR validated and corresponds to ISO 6888, for coagulase-positive staphylococci
118
(CPS). 5
119 120
2.3.
Salmonella spp.
121 122
Ten millilitres of each sample were enriched at a 1:10 ratio in peptone water
123
(Oxoid; 24 h, 37 °C) and then 0.1 mL transferred in Rappaport Vassiliadis (RV) broth
124
(Oxoid; 41.5 h, 37 °C). Thereafter, the enriched RV broth was sub-cultured on the
125
chromogenic RSAL agar (Oxoid; 24 h, 37 °C).
126 127
2.4.
Shiga toxin-producing E. coli
128 129
Ten millilitres of each sample were enriched at a 1:10 ratio in
130
Enterobacteriaceae enrichment broth (Becton Dickinson, Heidelberg, Germany; 24 h,
131
37 °C). One loopful of each of the enrichment cultures was cultured on sheep blood
132
agar (Difco™ Columbia blood agar base EH; Becton Dickinson AG, Allschwil,
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Switzerland) using the streak plate technique. After an incubation of 24 h at 37 °C,
134
the resulting colonies were washed off with 2 mL 0.85% NaCl. These suspensions
135
were screened by the Assurance GDS® assay for Shiga toxin genes (Bio Control
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Systems, Bellevue, WA, USA), a system which is an AOAC validated method
137
(Feldsine et al., 2005). In the event of a stx positive result in the polymerase chain
138
reaction (PCR), one loopful each of the suspension was streaked onto RAPID’E. coli
139
Agar (BioRad, Basel, Switzerland) to obtain single presumptive E. coli colonies. From
140
each plate, 10 individual colonies were picked and confirmed to possess stx (stx1
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and/or stx2) using the Assurance GDS® for Shiga toxin genes (Bio Control Systems).
142
From plates yielding more than one stx positive colony, one isolate was randomly
143
chosen for subsequent characterisation. The determination of stx1 subtypes (stx1a,
144
stx1c, stx1d) and stx2 subtypes (stx2a, stx2b, stx2c, stx2d, stx2e, stx2g) was 6
145
performed by conventional PCR amplification (Scheutz et al., 2012). Screening for
146
eae and the top 5 serogroups (O26, O103, O111, O145 and O157) was performed
147
by real-time PCR according to the guidelines of the European Union Reference
148
laboratory (EURL, 2013).
149 150
2.5.
Extended-spectrum β-lactamases (ESBL)-producing Enterobacteriaceae
151 152
Ten millilitres of each sample were enriched at a 1:10 ratio in
153
Enterobacteriaceae enrichment broth (Becton Dickinson, Heidelberg, Germany; 24 h,
154
37 °C). Thereafter, one loopful was sub-cultured on chromogenic Brilliance ESBL
155
agar (Oxoid; 24 h, 37 °C).
156 157
2.6.
Methicillin-resistant Staphylococcus aureus
158 159
Ten millilitres of each sample were enriched at a 1:10 ratio with 90 mL
160
Mueller-Hinton broth supplemented with 6.5% salt (Oxoid; 24 h, 37 °C). Thereafter, 1
161
mL was transferred to 5 mL tryptic soy broth (Oxoid) supplemented with 75 mg L-1
162
aztreonam and 3.5 mg L-1 cefataxime and incubated at 37 °C for 24 h. One loopful
163
each was streaked onto a MRSA2 agar (Oxoid) and incubated at 37 °C for 24 h. This
164
procedure is in accordance with the guidelines of the European Food Safety
165
Authority (Anonymous, 2012b).
166 167
2.7.
Further characterisation of CPS and presumptive positive MRSA
168 169 170
For confirmation of CPS and presumptive positive MRSA as S. aureus and for further strain characterisation, the StaphType DNA microarray assay was used (Alere 7
171
Technologies, Jena, Germany). This assay covers a variety of target sequences,
172
including species markers, enterotoxin genes, and resistance-associated genes.
173
Resulting DNA microarray profiles were used to assign the S. aureus isolates to
174
clonal complexes (Wattinger, Stephan, Layer, & Johler, 2012).
175 176
2.8.
Antibiotic resistance profiles of E. coli
177 178
From each E. coli positive milk sample, one typical colony from the RAPID’E.
179
coli Agar was subjected to susceptibility testing against 16 antimicrobial agents by
180
the disc diffusion method according to the Clinical and Laboratory Standards Institute
181
protocols and criteria (CLSI, 2017). The panel included amoxicillin-clavulanic acid (30
182
µg), ampicillin (10 µg), azithromycin (15 µg), cefazolin (30 µg), cefepime (30 µg),
183
cefotaxime (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), fosfomycin (200
184
µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), nitrofurantoin (300
185
µg), streptomycin (10 µg), sulfamethoxazole/trimethoprim (23.75/1.25 µg), and
186
tetracycline (30 µg) (Becton Dickinson, Allschwil, Switzerland).
187 188
2.9.
Statistical analysis
189 190
Data handling and minor calculations were done using Excel 2016 (Microsoft,
191
Redmond, WA, USA) and statistical tests were run in R version 3.6.1 (https://cran.r-
192
project.org).
193 194
3.
Results
195
8
196
The median TVC for tank milk from small ruminants was 3.8 log cfu mL-1 with
197
a minimum of 2.0 log cfu mL-1 and a maximum of 7.1 log cfu mL-1. Farms with more
198
animals had significantly higher TVC (p = 0.0031). A tenfold change in farm size (i.e.,
199
number of animals on farm) leads to a 2.5-fold increase in TVC [95% confidence
200
interval (CI) = 1.4- to 4.6-fold increase]. Spearman's correlation coefficient is rs = 0.24
201
(95% CI = 0.07 to 0.40). On the other hand, farms of SSSR members showed
202
significantly lower TVC than non-members (medians of 3.7 and 4.3 log cfu mL-1,
203
respectively; p = 0.0057).
204
E. coli were quantitatively detected in 10 (10.1%) goats’ milk and 6 (25.0%)
205
ewes’ milk samples (in total 16 = 13.0%; 95% CI: 8.2% to 20.1%). In E. coli positive
206
samples counts ranged from 1.0 to 3.9 log cfu mL-1 with a median of 1.5 log cfu mL-1.
207
E. coli was detected more frequently in farms that were not SSSR members (p =
208
0.0143, odds ratio = 5.6, 95% CI: 1.6% to 20.2%). From each of the positive samples
209
one isolate was further tested for its antibiotic resistance profile. Isolates from 6
210
samples (37.5%) were resistant to one or more antibiotic agents: 2 isolates (12.5%)
211
were resistant against only one agent (one against azithromycin and one against
212
streptomycin). The other 4 isolates (25.0%) showed multidrug resistance, all of them
213
being resistant against ampicillin, sulfamethoxazole/trimethoprim and streptomycin
214
as well as 1-2 additional agents that differed between the isolates (2 against
215
gentamicin and tetracycline, 1 against tetracycline only and 1 against kanamycin).
216
The observed prevalence of antimicrobial resistances and intermediate
217
susceptibilities are shown in Table 2.
218
Stx positive PCR results were obtained from 9 (9.1%) of the goats’ milk and 5
219
(20.8%) of the ewes’ milk samples (total 14 = 11.4%; 95% CI: 6.9% to 18.2%). From
220
these 14 PCR positive samples 9 STEC isolates could be recovered (Table 3). All of
221
them harboured stx1 genes (7 of subtype stx1c and 2 of subtype stx1a). Five isolates 9
222
were also positive for stx2. These included 4 isolates of subtype stx1c possessing
223
stx2b as well as 1 isolate of subtype stx1a that harboured stx2a and stx2d. Only 1
224
isolate was positive for eae. None of the STEC isolates belonged to the top 5
225
serogroups (O26, O103, O111, O145, O157). STEC tends to be more frequently
226
detected in farms that were operated on a regular basis (p = 0.0249).
227
CPS were quantitatively detected in 16 (16.2%) samples of goats’ milk and 2
228
(8.3%) samples of ewes’ milk (in total 18 = 14.6%; 95% CI: 9.5% to 21.9%). CPS
229
counts of positive samples ranged from 2.0 to 5.4 log cfu mL-1 with a median of 2.3
230
log cfu mL-1. From each of these samples one CPS isolate was further characterised.
231
These results are summarised in table 4. Enterotoxin genes were detected in 13
232
isolates (72.2%). The most prevalent combination was the presence of sec, sel and
233
tst1 (12 isolates = 66.7%).
234
Presumptive positive MRSA were isolated from 4 goats’ milk samples (4.0%),
235
but from none of the ewes’ milk samples (in total 3.3%; 95% CI: 1.3% to 8.11%).
236
From those 4 positive samples, 1 showed a CPS count of 3.0 log cfu mL-1. The other
237
3 samples showed no growth on the EasyStaph plates. Therefore, their CPS count
238
must have been below the detection limit of 2.0 log cfu mL-1. From each of the 4
239
samples one isolate was further characterised. The results are summarised in Table
240
5.
241 242
Salmonella spp. and ESBL-producing Enterobacteriaceae were not isolated from any of the samples.
243 244
4.
Discussion
4.1.
Hygiene parameters
245 246 247 10
248
Regarding TVC only 3 (2.4%) samples failed to comply with the Swiss and EU
249
legal limits for raw milk from other species than cows intended for the manufacture of
250
products without any heat treatment (Anonymous, 2004, 2017). Therefore,
251
compliance with legal limits has increased during the last years (Muehlherr et al.,
252
2003) and, compared with the last such study in Switzerland, TVC has decreased
253
(Muehlherr et al., 2003). The effect of delivery frequency on TVC could not be
254
reproduced in our study. However, it must be considered that the sampling period of
255
the present study was limited to springtime (February to May) and potential cooling
256
problems might be masked by the fact that outdoor temperatures were quite low
257
during that period. On the other hand, the effect of farm size on TVC could be
258
reaffirmed in a regression model (p = 0.0034). The fact that members of the SSSR
259
had lower TVC results indicates that additional efforts taken by motivated farmers or
260
private organisations might help improving the milk hygiene status on farms.
261
However, these effects are only small. Farm size, especially, might only be a
262
confounder of various other management factors that could possibly influence milk
263
hygiene (e.g., less time to observe animal health or more transport contacts to other
264
farms). On the other hand, animal health consultancy or veterinary support in case of
265
herd level problems seem to be plausible factors as to how SSSR membership could
266
lead to lower TVC.
267 268
4.2.
Foodborne pathogens
269 270 271 272 273
CPS were quantitatively detected less often compared with other studies (see Table 6). The characteristics of the CPS isolates revealed similar clonal complexes and a similar spectrum of virulence factors as observed in other studies (Linage, 11
274
Rodríguez-Calleja, Otero, García-López, & Santos, 2012; Merz, Stephan, & Johler,
275
2016; Murphy, O’Mahony, Buckley, O’Brien, & Fanning, 2010). Remarkably, all
276
enterotoxin gene harbouring CPS isolates of this study encoded multiple
277
staphylococcal enterotoxin genes as well as the tst1 gene. The high prevalence of
278
tst1 in CPS strains from small ruminant milk was already described by Merz et al.
279
(2016).
280
The STEC prevalence of our study in goats’ and ewes’ milk was comparable
281
to what can be found in literature (Table 7). However, there are only very few studies
282
available. Several stx variants were detected among the isolates, including stx2a and
283
stx2d that are associated with severe disease in STEC infected humans, and stx2b
284
that is linked to mild clinical symptoms or asymptomatic faecal carriage (Fierz,
285
Cernela, Hauser, Nüesch-Inderbinen, & Stephan, 2017; Fuller, Pellino, Flagler,
286
Strasser, & Weiss, 2011). Other stx variants included stx1a and stx1c that are stx
287
variants associated with a milder course of disease but are frequently found among
288
STEC from diseased humans and small ruminants (Fierz et al., 2017; Zweifel,
289
Blanco, Blanco, Blanco, & Stephan, 2004). The intimin gene eae, one of the most
290
prominent virulence factors contributing to pathogenesis, was detected in one isolate.
291
Salmonella spp. has not yet been isolated from goats’ and ewes’ BTM in Switzerland.
292
This is in line with a report of goats’ milk from Spain (Cortés et al., 2006), but
293
Salmonella spp. have already been reported in ewes’ milk in Italy (Amagliani et al.,
294
2016).
295 296
4.3.
Antibiotic resistant bacteria
297 298
In our study, MRSA was detected more frequently than in international
299
comparisons (Ou et al., 2018). However, the number of studies focusing on MRSA in 12
300
milk is limited. Higher MRSA prevalence has only been reported by Tegegne et al.
301
(2018). The clonal complexes of the MRSA isolates revealed that they mainly belong
302
to the livestock associated MRSA.
303
To the authors' knowledge, our study is the first of broad screening for ESBL-
304
producing Enterobacteriaceae in goats’ and ewes’ milk. Even though no sample was
305
tested positive, it is still reasonable to keep track of this situation as sheep can be
306
carriers of ESBL-producing Enterobacteriaceae (Geser, Stephan, & Hächler, 2012;
307
Teale et al., 2011) and it has been shown in cattle that bulk tank milk can be
308
contaminated (Kaesbohrer et al., 2019; Odenthal, Akineden, & Usleber, 2016).
309
Comparative studies on resistance profiles of E. coli isolated from goats’ or ewes’
310
milk are very rare. Malissiova et al. (2017) found 18.2% of the isolates to be resistant
311
against ampicillin.
312 313
5.
Conclusions
314 315
This study generated further baseline data on the microbiological quality of
316
goats’ and ewes’ tank milk samples. The results showed that the overall
317
microbiological quality of goats’ and ewes’ milk in Switzerland is favourable
318
compared with studies from other countries. As additional efforts (e.g., within the
319
framework of the SSSR) seem to help improving milk hygiene status, such efforts or
320
programmes should be further encouraged. However, the exact factors of SSSR
321
membership leading to better milk hygiene status would need further investigation.
322
With regard to foodborne pathogens, S. aureus harbouring staphylococcal
323
enterotoxin genes (including sea, sec, and sed) and tst1, and STEC harbouring stx
324
gene patterns of pathogenic strains, were detected. This shows that the occurrence
325
of foodborne pathogens can never be ruled out. From the authors' point of view, no 13
326
supposed, but poorly evidenced, benefit of drinking raw milk outweighs taking a risk
327
of food-borne infection. Even if it conflicts with people’s beliefs, raw milk should
328
therefore always be properly heated before consumption.
329 330
Acknowledgements
331 332
The authors very much appreciate the support by the Sanitary Service for
333
Small Ruminants and the Swiss Goat Breeding Federation, who provided the contact
334
details to get in touch with the farmers. This study was supported by the Swiss Army
335
Veterinary Service.
336 337
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Dorko, E., Hockicko, J., Rimárová, K., Bušová, A., Popaďák, P., Popaďáková, J., et
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EURL. (2013). Identification and characterization of Verocytotoxin-producing
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406
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Fierz, L., Cernela, N., Hauser, E., Nüesch-Inderbinen, M., & Stephan, R. (2017).
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study. Journal of AOAC International, 88, 1334-48.
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Fuller, C. A., Pellino, C. A., Flagler, M. J., Strasser, J. E., & Weiss, A. A. (2011).
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Geser, N., Stephan, R., & Hächler, H. (2012). Occurrence and characteristics of
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extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae in food
420
producing animals, minced meat and raw milk. BMC Veterinary Research, 8,
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Giacinti, G., Carfora, V., Caprioli, A., Sagrafoli, D., Marri, N., Giangolini, G., et al.
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(2017). Prevalence and characterization of methicillin-resistant
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Staphylococcus aureus carrying mecA or mecC and methicillin-susceptible
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Staphylococcus aureus in dairy sheep farms in central Italy. Journal of Dairy
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Science, 100, 7857–7863.
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Jaakkonen, A., Castro, H., Hallanvuo, S., Ranta, J., Rossi, M., Isidro, J., et al. (2019).
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Longitudinal study of Shiga toxin-producing Escherichia coli and
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Campylobacter jejuni on Finnish dairy farms and in raw milk. Applied and
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Environmental Microbiology, 85, Article e02910-18.
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Kaesbohrer, A., Bakran-Lebl, K., Irrgang, A., Fischer, J., Kämpf, P., Schiffmann, A.,
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et al. (2019). Diversity in prevalence and characteristics of ESBL/pAmpC
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producing E. coli in food in Germany. Veterinary Microbiology, 233, 52–60.
434 435
Katafiasz, A. R., & Bartett, P. (2012). Motivation for unpasteurized milk consumption in Michigan, 2011. Food Protection Trends, 32, 124–128.
436
Klinger, I., & Rosenthal, I. (1997). Public health and the safety of milk and milk
437
products from sheep and goats. Revue Scientifique et Technique
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(International Office of Epizootics), 16, 482–488.
439 440 441
LeJeune, J. T., & Rajala‐Schultz, P. J. (2009). Unpasteurized milk: A continued public health threat. Clinical Infectious Diseases, 48, 93–100. Linage, B., Rodríguez-Calleja, J. M., Otero, A., García-López, M. L., & Santos, J. A.
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(2012). Characterization of coagulase-positive staphylococci isolated from
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tank and silo ewe milk. Journal of Dairy Science, 95, 1639–1644.
444
Malissiova, E., Papadopoulos, T., Kyriazi, A., Mparda, M., Sakorafa, C., Katsioulis,
445
A., et al. (2017). Differences in sheep and goats milk microbiological profile
446
between conventional and organic farming systems in Greece. Journal of
447
Dairy Research, 84, 206–213.
448
Markovinović, L., Kosanović Ličina, M. L., Tešić, V., Vojvodić, D., Vladušić Lucić, I.,
449
Kniewald, T., et al. (2016). An outbreak of tick-borne encephalitis associated
18
450
with raw goat milk and cheese consumption, Croatia, 2015. Infection, 44, 661–
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665.
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Mehmeti, I., Bytyqi, H., Muji, S., Nes, I. F., & Diep, D. B. (2017). The prevalence of
453
Listeria monocytogenes and Staphylococcus aureus and their virulence genes
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in bulk tank milk in Kosovo. Journal of Infection in Developing Countries, 11,
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Article 247.
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Merz, A., Stephan, R., & Johler, S. (2016). Staphylococcus aureus isolates from Goat
457
and sheep milk seem to be closely related and differ from isolates detected
458
from bovine milk. Frontiers in Microbiology, 7, Article 319.
459 460 461
Milani, F. X., & Wendorff, W. L. (2011). Goat and sheep milk products in the United States (USA). Small Ruminant Research, 101, 134–139. Muehlherr, J. E., Zweifel, C., Corti, S., Blanco, J. E., & Stephan, R. (2003).
462
Microbiological quality of raw goats’ and ewes’ bulk-tank milk in Switzerland.
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Journal of Dairy Science, 86, 3849–3856.
464
Murphy, B. P., O’Mahony, E., Buckley, J. F., O’Brien, S., & Fanning, S. (2010).
465
Characterization of Staphylococcus aureus Isolated from dairy animals in
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ireland. Zoonoses and Public Health, 57, 249–257.
467
Odenthal, S., Akineden, Ö., & Usleber, E. (2016). Extended-spectrum β-lactamase
468
producing Enterobacteriaceae in bulk tank milk from German dairy farms.
469
International Journal of Food Microbiology, 238, 72–78.
470
Ou, Q., Zhou, J., Lin, D., Bai, C., Zhang, T., Lin, J., et al. (2018). A large meta-
471
analysis of the global prevalence rates of S. aureus and MRSA contamination
472
of milk. Critical Reviews in Food Science and Nutrition, 58, 2213–2228.
473 474
Scheutz, F., Teel, L. D., Beutin, L., Pierard, D., Buvens, G., Karch, H., et al. (2012). Multicenter evaluation of a sequence-based protocol for subtyping Shiga
19
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toxins and standardizing stx nomenclature. Journal of Clinical Microbiology,
476
50, 2951–2963.
477
Sonnier, J. L., Karns, J. S., Lombard, J. E., Kopral, C. A., Haley, B. J., Kim, S.-W., et
478
al. (2018). Prevalence of Salmonella enterica, Listeria monocytogenes, and
479
pathogenic Escherichia coli in bulk tank milk and milk filters from US dairy
480
operations in the National Animal Health Monitoring System Dairy 2014 study.
481
Journal of Dairy Science, 101, 1943–1956.
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Teale, C. J., Barker, L., Foster, A. P., Liebana, E., Batchelor, M., Livermore, D. M., et
483
al. (2011). Extended-spectrum beta-lactamase detected in E. coli recovered
484
from calves in Wales. Veterinary Record, 156, 186–187.
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Tegegne, H. A., Florianová, M., Gelbíčová, T., Karpíšková, R., & Koláčková, I.
486
(2018). Detection and molecular characterization of methicillin-resistant
487
Staphylococcus aureus Isolated from bulk tank milk of cows, sheep, and
488
goats. Foodborne Pathogens and Disease, 16, 68–73.
489
Vitale, M., Scatassa, M. L., Cardamone, C., Oliveri, G., Piraino, C., Alduina, R., et al.
490
(2015). Staphylococcal food poisoning case and molecular analysis of toxin
491
genes in Staphylococcus aureus Strains isolated from food in Sicily, Italy.
492
Foodborne Pathogens and Disease, 12, 21–23.
493
Wattinger, L., Stephan, R., Layer, F., & Johler, S. (2012). Comparison of
494
Staphylococcus aureus isolates associated with food intoxication with isolates
495
from human nasal carriers and human infections. European Journal of Clinical
496
Microbiology & Infectious Diseases, 31, 455–464.
497
Whitehead, J., & Lake, B. (2018). Recent trends in unpasteurized fluid milk
498
outbreaks, legalization, and consumption in the United States. PLoS Currents,
499
2018, Article 10.
20
500
Zweifel, C., Blanco, J., Blanco, M., Blanco, J., & Stephan, R. (2004). Serotypes and
501
virulence genes of ovine non-O157 Shiga toxin-producing Escherichia coli in
502
Switzerland. International Journal of Food Microbiology, 95, 19–27.
21
Table 1 Overview of legal status of raw milk sales in different countries.
Country / Union
Description of legal status
Reference
Switzerland
Raw milk sales permitted with additional labelling.
(Anonymous, 2019b)
European Union
Member states may maintain or establish national rules.
(Anonymous, 2004)
Germany
Raw milk sales are prohibited.
(Anonymous, 2018b)
Austria
Raw milk sales are permitted from producer to end consumer.
(Anonymous, 2006)
France
Raw milk sales permitted only after being granted official authorisation.
(Anonymous, 2012a)
USA
Legislation differs between states. Raw milk sale possible in 30 states.
(Whitehead & Lake, 2018)
Canada
Raw milk sales are prohibited.
(Anonymous, 2019a)
1
Table 2 Observed prevalence of antimicrobial resistances and intermediate susceptibilities of isolated E. coli strains. a Characteristic
Number of strains
% of isolates
% of total samples
Antimicrobial resistance streptomycin ampicillin sulfamethoxazole/trimethoprim tetracycline gentamicin kanamycin azithromycin
5 4 4 3 2 1 1
31.3 25.0 25.0 18.8 12.5 6.3 6.3
4.1 3.3 3.3 2.4 1.6 0.8 0.8
Intermediate susceptibility cefazolin amoxicillin-clavulanic
7 2
43.8 12.5
5.7 0.8
2
Table 3 Characteristics of the STEC isolates from goat and ewes’ tank milk samples. a Species
Number of strains
Virulence genes
Goat
1
stx1a, eae
Goat
2
stx1c
Goat
3
stx1c, stx2b
Ewe
1
stx1a, stx2a, stx2d
Ewe
1
stx1c
Ewe
1
stx1c, stx2b
a
Abbreviations are: stx1a, stx1c, Shigatoxin 1 gene subtypes; stx2a, stx2b, stx2d:
Shigatoxin 2 gene subtypes; eae, E. coli attaching and effacing gene.
3
Table 4 Characterisation data of methicillin susceptible S. aureus (MSSA) isolated from goat and ewes’ tank milk samples. a Species
Number of strains Clonal complex
Toxin genes
Goat
1
CC30
sea, egc, tst1
Goat
4
CC130
sec, sel, tst1
Goat
3
CC130
-
Goat
6
CC133
sec, sel, tst1
Goat
1
CC398
-
Goat
1
CC522
-
Ewe
2
CC133
sec, sel, tst1
a
Abbreviations are: sea, sec, sel, staphylococcal enterotoxin genes A, C and L,
respectively; egc, cluster comprising staphylococcal enterotoxin genes I, M, N, O, U; tst1, toxic shock toxin 1 gene. Capsule type 8 was determined in all cases.
4
Table 5 Characterisation data of methicillin resistant S. aureus (MRSA) isolated from different goat tank milk samples. a Number of strains
Clonal complex
mec gene
Toxin genes
3
CC398
mecA
-
1
CC8
mecA
sec, sel, egc
a
Abbreviations are: sec, sel, staphylococcal enterotoxin genes C and L, respectively;
egc, cluster comprising staphylococcal enterotoxin genes I, M, N, O, U. Capsule type 5 was determined in both cases.
5
Table 6 Comparison of S. aureus prevalence with other studies of goats’, ewes’ and cows’ milk. a Reference
Country
Species
Prevalence
This study
CH
GE
14.6%
Muehlherr et al. (2003)
CH
GE
31.9%
Ou et al. (2018)
n/a
G
25.8%
Malissiova et al. (2017)
GR
GE
60.0%
Giacinti et al. (2017)
IT
E
53.5%
Cortimiglia et al. (2016)
IT
C
47.2%
Mehmeti et al. (2017)
XK
C
39.8%
a
Abbreviations are: GE, goats’ and ewes’ milk; G, goats’ milk only; E, ewes’ milk
only; C, cows’ milk; CH, Switzerland; GR, Greece; CZ, Czech Republic; IT, Italy; XK, Kosovo; n/a indicates study was a meta-analysis of studies from different countries.
6
Table 7 Comparison of STEC prevalence with other studies of goats’, ewes’ and cows’ milk. a Reference
Country
Species
Prevalence
This study
CH
GE
11.4%
Muehlherr et al. (2003)
CH
GE
15.2%
Sonnier et al. (2018)
USA
C
11.2%
Jaakkonen et al. (2019)
FI
C
6.6%
a
Abbreviations are: CH, Switzerland; USA, United States of America; FI, Finland;
GE, goats' and ewes' milk; C, cows' milk.
7
S0958-6946(19)30246-8
DOI:
https://doi.org/10.1016/j.idairyj.2019.104609
Reference:
INDA 104609
To appear in:
International Dairy Journal
Received Date: 11 September 2019 Revised Date:
19 November 2019
Accepted Date: 20 November 2019
Please cite this article as: Friker, B., Morach, M., Püntener, S., Cernela, N., Horlbog, J., Stephan, R., Assessing the microbiological quality of raw goats’ and ewes’ tank milk samples in Switzerland, International Dairy Journal, https://doi.org/10.1016/j.idairyj.2019.104609. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Ltd. All rights reserved.
1
Assessing the microbiological quality of raw goats’ and ewes’ tank milk
2
samples in Switzerland
3 4 5 6 7
Brian Frikera, Marina Morachb, Sabrina Püntenerb, Nicole Cernelab, Jule Horlbogb,
8
Roger Stephanb*
9 10 11 12 13
a
14
Schwarzenburgstrasse 161, 3097 Liebefeld, Switzerland
15
b
16
Winterthurerstrasse 272, 8057 Zürich, Switzerland
Veterinary Public Health Institute, Vetsuisse Faculty, University of Bern,
Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich,
17 18 19 20 21
* Corresponding author. Tel.: +41 44 635 86 51
22
E-mail address: [email protected] (R. Stephan)
23 24 25
1
26
___________________________________________________________________
27
ABSTRACT
28 29
In recent years, popularity of raw milk has increased in many industrialised countries.
30
This study (i) enumerated total viable counts (TVC) and Escherichia coli counts, (ii)
31
assessed prevalence of Staphylococcus (S.) aureus, Salmonella spp. and STEC, (iii)
32
screened for methicillin resistant S. aureus (MRSA) and extended-spectrum β-
33
lactamases (ESBL)-producing Enterobacteriaceae in sheep and goat tank milk
34
samples collected throughout Switzerland and (iv) provided further strain
35
characteristics on isolated pathogens and MRSA. One hundred and twenty-three
36
tank milk samples from 116 farms were analysed. The median TVC was 3.8 log cfu
37
mL-1. E. coli was detected in 16 (13.0%) and S. aureus in 18 (14.6%) samples.
38
Polymerase chain reaction for stx genes was positive in 14 (11.4%) samples. MRSA
39
were isolated from 4 (3.3%) samples. Salmonella spp. and ESBL-producing
40
Enterobacteriaceae were not isolated.
41
___________________________________________________________________
42
2
43
1.
Introduction
44 45
In recent years, food consumption habits have dramatically changed under the
46
influence of lifestyle changes and new technologies. The importance of ewes’ and
47
goats’ milk has increased considerably. Even though ewes’ and goats’ milk only
48
accounts for 0.7% of the total milk production in Switzerland, the amount brought to
49
market has doubled during the last 10 years (Anonymous, 2008, 2018a).
50
In the context of the trend toward “consuming natural” and “purchasing
51
locally”, the popularity of raw milk has increased in many industrialised countries; the
52
consumers tend to prefer raw milk due to better taste and believe in better nutritional
53
values. Moreover, a variety of health benefits associated with raw milk consumption
54
(as improved immunity, less lactose intolerance, less diabetes, and many others) are
55
propagated, but convincing scientific evidence is hardly available (Katafiasz &
56
Bartett, 2012; LeJeune & Rajala‐Schultz, 2009). These trends are rising despite the
57
fact that selling raw milk is prohibited or strictly regulated in many of these countries
58
(Table 1). Particularly noteworthy are the "cow share programmes" in the USA; this
59
means that a number of people share the ownership of a number of animals.
60
Therefore, people can drink raw milk "from their own animals" without having to buy
61
it. Like this, it is possible to acquire raw milk even in some states that generally
62
prohibit the sale of raw milk.
63
However, there is a well-established association between raw milk
64
consumption and infection with pathogenic bacteria as e.g., Salmonella spp., or
65
Shiga toxin-producing Escherichia coli (STEC). For decades, it has been repeatedly
66
shown that raw milk is a major risk factor for food-borne diseases (EFSA, 2015;
67
Klinger & Rosenthal, 1997; LeJeune & Rajala‐Schultz, 2009).
3
68
According to a review of outbreaks in the United States of America, 65.5% of
69
the dairy-related outbreaks were associated with raw milk. Goat milk was involved in
70
4.6% of the raw milk outbreaks even though goats’ and ewes’ milk only account for
71
0.1% of the total milk production (Milani & Wendorff, 2011; Whitehead & Lake, 2018).
72
Apart from reports of well-known food-borne pathogens like Staphylococcus aureus
73
(Vitale et al., 2015), there are also publications on milk-borne outbreaks of diseases
74
that are more commonly known to be transmitted by some other route, e.g., tick
75
borne encephalitis (Brockmann et al., 2018; Dorko et al., 2018; Markovinović et al.,
76
2016).
77
The last study focusing on human pathogens in goats’ and ewes’ milk in
78
Switzerland was more than 15 years ago (Muehlherr, Zweifel, Corti, Blanco, &
79
Stephan, 2003). Such knowledge is scarce not only in Switzerland but in general,
80
especially when comparing with cows’ milk, on which much more research is
81
conducted.
82
Therefore, this study (i) assessed the microbiological quality of ewes’ and
83
goats’ milk by enumerating total viable counts (TVC) and E. coli counts, (ii) generated
84
up-to-date prevalence data for selected foodborne pathogens (S. aureus, Salmonella
85
spp., STEC), (iii) screened for methicillin resistant S. aureus (MRSA) and extended-
86
spectrum β-lactamases (ESBL)-producing Enterobacteriaceae and (iv) provided
87
further strain characteristics on isolated foodborne pathogens and MRSA.
88 89
2.
Methods
2.1.
Raw milk and sampling
90 91 92
4
93
In this study, 123 raw milk samples (99 from goats and 24 from ewes)
94
collected on farm level from tank milk or from milk churn (depending on how milk was
95
stored on the farm) were analysed. They originated from 116 farms located in 24 out
96
of the 26 cantons of Switzerland. From each farm additional data, e.g., number of
97
animals, farming on a regular or side-line basis, membership of the Sanitary Service
98
for Small Ruminants (SSSR), were collected. The SSSR is a private association that
99
provides consultancy on animal health, feeding and proper housing. Members of the
100
SSSR can voluntarily participate in surveillance (e.g., for gastrointestinal parasites) or
101
eradication programmes (e.g., Maedi-Visna for sheep or pseudotuberculosis in
102
goats). Moreover, the SSSR provides support in case of milk quality or herd health
103
problems.
104
Sampling was performed over 4 months (February to May 2019). Milk storage
105
on farm before sampling depended on the farm's frequency of milk delivery, which
106
ranged from daily delivery to schemes like "delivery on Mondays and Thursdays".
107
Samples were transported chilled to the laboratory and analysed within 24 h.
108 109
2.2.
Total viable counts, E. coli, and coagulase-positive staphylococci
110 111
Samples were quantitatively analysed according to ISO standard methods
112
(ISO 4833; ISO 16649; ISO 6888) by the surface spreading technique. The following
113
agars and conditions were used: plate count agar (Oxoid, Pratteln, Switzerland; 72 h,
114
30 °C) for total viable counts (TVC), RAPID’E. coli 2 agar (Bio-Rad, Reinach,
115
Switzerland; 24 h, 37 °C), AFNOR validated and corresponds to ISO 16649, for E.
116
coli, and EASY Staph agar (Biokar Diagnostics Beauvais, France; 48 h, 37 °C),
117
AFNOR validated and corresponds to ISO 6888, for coagulase-positive staphylococci
118
(CPS). 5
119 120
2.3.
Salmonella spp.
121 122
Ten millilitres of each sample were enriched at a 1:10 ratio in peptone water
123
(Oxoid; 24 h, 37 °C) and then 0.1 mL transferred in Rappaport Vassiliadis (RV) broth
124
(Oxoid; 41.5 h, 37 °C). Thereafter, the enriched RV broth was sub-cultured on the
125
chromogenic RSAL agar (Oxoid; 24 h, 37 °C).
126 127
2.4.
Shiga toxin-producing E. coli
128 129
Ten millilitres of each sample were enriched at a 1:10 ratio in
130
Enterobacteriaceae enrichment broth (Becton Dickinson, Heidelberg, Germany; 24 h,
131
37 °C). One loopful of each of the enrichment cultures was cultured on sheep blood
132
agar (Difco™ Columbia blood agar base EH; Becton Dickinson AG, Allschwil,
133
Switzerland) using the streak plate technique. After an incubation of 24 h at 37 °C,
134
the resulting colonies were washed off with 2 mL 0.85% NaCl. These suspensions
135
were screened by the Assurance GDS® assay for Shiga toxin genes (Bio Control
136
Systems, Bellevue, WA, USA), a system which is an AOAC validated method
137
(Feldsine et al., 2005). In the event of a stx positive result in the polymerase chain
138
reaction (PCR), one loopful each of the suspension was streaked onto RAPID’E. coli
139
Agar (BioRad, Basel, Switzerland) to obtain single presumptive E. coli colonies. From
140
each plate, 10 individual colonies were picked and confirmed to possess stx (stx1
141
and/or stx2) using the Assurance GDS® for Shiga toxin genes (Bio Control Systems).
142
From plates yielding more than one stx positive colony, one isolate was randomly
143
chosen for subsequent characterisation. The determination of stx1 subtypes (stx1a,
144
stx1c, stx1d) and stx2 subtypes (stx2a, stx2b, stx2c, stx2d, stx2e, stx2g) was 6
145
performed by conventional PCR amplification (Scheutz et al., 2012). Screening for
146
eae and the top 5 serogroups (O26, O103, O111, O145 and O157) was performed
147
by real-time PCR according to the guidelines of the European Union Reference
148
laboratory (EURL, 2013).
149 150
2.5.
Extended-spectrum β-lactamases (ESBL)-producing Enterobacteriaceae
151 152
Ten millilitres of each sample were enriched at a 1:10 ratio in
153
Enterobacteriaceae enrichment broth (Becton Dickinson, Heidelberg, Germany; 24 h,
154
37 °C). Thereafter, one loopful was sub-cultured on chromogenic Brilliance ESBL
155
agar (Oxoid; 24 h, 37 °C).
156 157
2.6.
Methicillin-resistant Staphylococcus aureus
158 159
Ten millilitres of each sample were enriched at a 1:10 ratio with 90 mL
160
Mueller-Hinton broth supplemented with 6.5% salt (Oxoid; 24 h, 37 °C). Thereafter, 1
161
mL was transferred to 5 mL tryptic soy broth (Oxoid) supplemented with 75 mg L-1
162
aztreonam and 3.5 mg L-1 cefataxime and incubated at 37 °C for 24 h. One loopful
163
each was streaked onto a MRSA2 agar (Oxoid) and incubated at 37 °C for 24 h. This
164
procedure is in accordance with the guidelines of the European Food Safety
165
Authority (Anonymous, 2012b).
166 167
2.7.
Further characterisation of CPS and presumptive positive MRSA
168 169 170
For confirmation of CPS and presumptive positive MRSA as S. aureus and for further strain characterisation, the StaphType DNA microarray assay was used (Alere 7
171
Technologies, Jena, Germany). This assay covers a variety of target sequences,
172
including species markers, enterotoxin genes, and resistance-associated genes.
173
Resulting DNA microarray profiles were used to assign the S. aureus isolates to
174
clonal complexes (Wattinger, Stephan, Layer, & Johler, 2012).
175 176
2.8.
Antibiotic resistance profiles of E. coli
177 178
From each E. coli positive milk sample, one typical colony from the RAPID’E.
179
coli Agar was subjected to susceptibility testing against 16 antimicrobial agents by
180
the disc diffusion method according to the Clinical and Laboratory Standards Institute
181
protocols and criteria (CLSI, 2017). The panel included amoxicillin-clavulanic acid (30
182
µg), ampicillin (10 µg), azithromycin (15 µg), cefazolin (30 µg), cefepime (30 µg),
183
cefotaxime (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), fosfomycin (200
184
µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), nitrofurantoin (300
185
µg), streptomycin (10 µg), sulfamethoxazole/trimethoprim (23.75/1.25 µg), and
186
tetracycline (30 µg) (Becton Dickinson, Allschwil, Switzerland).
187 188
2.9.
Statistical analysis
189 190
Data handling and minor calculations were done using Excel 2016 (Microsoft,
191
Redmond, WA, USA) and statistical tests were run in R version 3.6.1 (https://cran.r-
192
project.org).
193 194
3.
Results
195
8
196
The median TVC for tank milk from small ruminants was 3.8 log cfu mL-1 with
197
a minimum of 2.0 log cfu mL-1 and a maximum of 7.1 log cfu mL-1. Farms with more
198
animals had significantly higher TVC (p = 0.0031). A tenfold change in farm size (i.e.,
199
number of animals on farm) leads to a 2.5-fold increase in TVC [95% confidence
200
interval (CI) = 1.4- to 4.6-fold increase]. Spearman's correlation coefficient is rs = 0.24
201
(95% CI = 0.07 to 0.40). On the other hand, farms of SSSR members showed
202
significantly lower TVC than non-members (medians of 3.7 and 4.3 log cfu mL-1,
203
respectively; p = 0.0057).
204
E. coli were quantitatively detected in 10 (10.1%) goats’ milk and 6 (25.0%)
205
ewes’ milk samples (in total 16 = 13.0%; 95% CI: 8.2% to 20.1%). In E. coli positive
206
samples counts ranged from 1.0 to 3.9 log cfu mL-1 with a median of 1.5 log cfu mL-1.
207
E. coli was detected more frequently in farms that were not SSSR members (p =
208
0.0143, odds ratio = 5.6, 95% CI: 1.6% to 20.2%). From each of the positive samples
209
one isolate was further tested for its antibiotic resistance profile. Isolates from 6
210
samples (37.5%) were resistant to one or more antibiotic agents: 2 isolates (12.5%)
211
were resistant against only one agent (one against azithromycin and one against
212
streptomycin). The other 4 isolates (25.0%) showed multidrug resistance, all of them
213
being resistant against ampicillin, sulfamethoxazole/trimethoprim and streptomycin
214
as well as 1-2 additional agents that differed between the isolates (2 against
215
gentamicin and tetracycline, 1 against tetracycline only and 1 against kanamycin).
216
The observed prevalence of antimicrobial resistances and intermediate
217
susceptibilities are shown in Table 2.
218
Stx positive PCR results were obtained from 9 (9.1%) of the goats’ milk and 5
219
(20.8%) of the ewes’ milk samples (total 14 = 11.4%; 95% CI: 6.9% to 18.2%). From
220
these 14 PCR positive samples 9 STEC isolates could be recovered (Table 3). All of
221
them harboured stx1 genes (7 of subtype stx1c and 2 of subtype stx1a). Five isolates 9
222
were also positive for stx2. These included 4 isolates of subtype stx1c possessing
223
stx2b as well as 1 isolate of subtype stx1a that harboured stx2a and stx2d. Only 1
224
isolate was positive for eae. None of the STEC isolates belonged to the top 5
225
serogroups (O26, O103, O111, O145, O157). STEC tends to be more frequently
226
detected in farms that were operated on a regular basis (p = 0.0249).
227
CPS were quantitatively detected in 16 (16.2%) samples of goats’ milk and 2
228
(8.3%) samples of ewes’ milk (in total 18 = 14.6%; 95% CI: 9.5% to 21.9%). CPS
229
counts of positive samples ranged from 2.0 to 5.4 log cfu mL-1 with a median of 2.3
230
log cfu mL-1. From each of these samples one CPS isolate was further characterised.
231
These results are summarised in table 4. Enterotoxin genes were detected in 13
232
isolates (72.2%). The most prevalent combination was the presence of sec, sel and
233
tst1 (12 isolates = 66.7%).
234
Presumptive positive MRSA were isolated from 4 goats’ milk samples (4.0%),
235
but from none of the ewes’ milk samples (in total 3.3%; 95% CI: 1.3% to 8.11%).
236
From those 4 positive samples, 1 showed a CPS count of 3.0 log cfu mL-1. The other
237
3 samples showed no growth on the EasyStaph plates. Therefore, their CPS count
238
must have been below the detection limit of 2.0 log cfu mL-1. From each of the 4
239
samples one isolate was further characterised. The results are summarised in Table
240
5.
241 242
Salmonella spp. and ESBL-producing Enterobacteriaceae were not isolated from any of the samples.
243 244
4.
Discussion
4.1.
Hygiene parameters
245 246 247 10
248
Regarding TVC only 3 (2.4%) samples failed to comply with the Swiss and EU
249
legal limits for raw milk from other species than cows intended for the manufacture of
250
products without any heat treatment (Anonymous, 2004, 2017). Therefore,
251
compliance with legal limits has increased during the last years (Muehlherr et al.,
252
2003) and, compared with the last such study in Switzerland, TVC has decreased
253
(Muehlherr et al., 2003). The effect of delivery frequency on TVC could not be
254
reproduced in our study. However, it must be considered that the sampling period of
255
the present study was limited to springtime (February to May) and potential cooling
256
problems might be masked by the fact that outdoor temperatures were quite low
257
during that period. On the other hand, the effect of farm size on TVC could be
258
reaffirmed in a regression model (p = 0.0034). The fact that members of the SSSR
259
had lower TVC results indicates that additional efforts taken by motivated farmers or
260
private organisations might help improving the milk hygiene status on farms.
261
However, these effects are only small. Farm size, especially, might only be a
262
confounder of various other management factors that could possibly influence milk
263
hygiene (e.g., less time to observe animal health or more transport contacts to other
264
farms). On the other hand, animal health consultancy or veterinary support in case of
265
herd level problems seem to be plausible factors as to how SSSR membership could
266
lead to lower TVC.
267 268
4.2.
Foodborne pathogens
269 270 271 272 273
CPS were quantitatively detected less often compared with other studies (see Table 6). The characteristics of the CPS isolates revealed similar clonal complexes and a similar spectrum of virulence factors as observed in other studies (Linage, 11
274
Rodríguez-Calleja, Otero, García-López, & Santos, 2012; Merz, Stephan, & Johler,
275
2016; Murphy, O’Mahony, Buckley, O’Brien, & Fanning, 2010). Remarkably, all
276
enterotoxin gene harbouring CPS isolates of this study encoded multiple
277
staphylococcal enterotoxin genes as well as the tst1 gene. The high prevalence of
278
tst1 in CPS strains from small ruminant milk was already described by Merz et al.
279
(2016).
280
The STEC prevalence of our study in goats’ and ewes’ milk was comparable
281
to what can be found in literature (Table 7). However, there are only very few studies
282
available. Several stx variants were detected among the isolates, including stx2a and
283
stx2d that are associated with severe disease in STEC infected humans, and stx2b
284
that is linked to mild clinical symptoms or asymptomatic faecal carriage (Fierz,
285
Cernela, Hauser, Nüesch-Inderbinen, & Stephan, 2017; Fuller, Pellino, Flagler,
286
Strasser, & Weiss, 2011). Other stx variants included stx1a and stx1c that are stx
287
variants associated with a milder course of disease but are frequently found among
288
STEC from diseased humans and small ruminants (Fierz et al., 2017; Zweifel,
289
Blanco, Blanco, Blanco, & Stephan, 2004). The intimin gene eae, one of the most
290
prominent virulence factors contributing to pathogenesis, was detected in one isolate.
291
Salmonella spp. has not yet been isolated from goats’ and ewes’ BTM in Switzerland.
292
This is in line with a report of goats’ milk from Spain (Cortés et al., 2006), but
293
Salmonella spp. have already been reported in ewes’ milk in Italy (Amagliani et al.,
294
2016).
295 296
4.3.
Antibiotic resistant bacteria
297 298
In our study, MRSA was detected more frequently than in international
299
comparisons (Ou et al., 2018). However, the number of studies focusing on MRSA in 12
300
milk is limited. Higher MRSA prevalence has only been reported by Tegegne et al.
301
(2018). The clonal complexes of the MRSA isolates revealed that they mainly belong
302
to the livestock associated MRSA.
303
To the authors' knowledge, our study is the first of broad screening for ESBL-
304
producing Enterobacteriaceae in goats’ and ewes’ milk. Even though no sample was
305
tested positive, it is still reasonable to keep track of this situation as sheep can be
306
carriers of ESBL-producing Enterobacteriaceae (Geser, Stephan, & Hächler, 2012;
307
Teale et al., 2011) and it has been shown in cattle that bulk tank milk can be
308
contaminated (Kaesbohrer et al., 2019; Odenthal, Akineden, & Usleber, 2016).
309
Comparative studies on resistance profiles of E. coli isolated from goats’ or ewes’
310
milk are very rare. Malissiova et al. (2017) found 18.2% of the isolates to be resistant
311
against ampicillin.
312 313
5.
Conclusions
314 315
This study generated further baseline data on the microbiological quality of
316
goats’ and ewes’ tank milk samples. The results showed that the overall
317
microbiological quality of goats’ and ewes’ milk in Switzerland is favourable
318
compared with studies from other countries. As additional efforts (e.g., within the
319
framework of the SSSR) seem to help improving milk hygiene status, such efforts or
320
programmes should be further encouraged. However, the exact factors of SSSR
321
membership leading to better milk hygiene status would need further investigation.
322
With regard to foodborne pathogens, S. aureus harbouring staphylococcal
323
enterotoxin genes (including sea, sec, and sed) and tst1, and STEC harbouring stx
324
gene patterns of pathogenic strains, were detected. This shows that the occurrence
325
of foodborne pathogens can never be ruled out. From the authors' point of view, no 13
326
supposed, but poorly evidenced, benefit of drinking raw milk outweighs taking a risk
327
of food-borne infection. Even if it conflicts with people’s beliefs, raw milk should
328
therefore always be properly heated before consumption.
329 330
Acknowledgements
331 332
The authors very much appreciate the support by the Sanitary Service for
333
Small Ruminants and the Swiss Goat Breeding Federation, who provided the contact
334
details to get in touch with the farmers. This study was supported by the Swiss Army
335
Veterinary Service.
336 337
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21
Table 1 Overview of legal status of raw milk sales in different countries.
Country / Union
Description of legal status
Reference
Switzerland
Raw milk sales permitted with additional labelling.
(Anonymous, 2019b)
European Union
Member states may maintain or establish national rules.
(Anonymous, 2004)
Germany
Raw milk sales are prohibited.
(Anonymous, 2018b)
Austria
Raw milk sales are permitted from producer to end consumer.
(Anonymous, 2006)
France
Raw milk sales permitted only after being granted official authorisation.
(Anonymous, 2012a)
USA
Legislation differs between states. Raw milk sale possible in 30 states.
(Whitehead & Lake, 2018)
Canada
Raw milk sales are prohibited.
(Anonymous, 2019a)
1
Table 2 Observed prevalence of antimicrobial resistances and intermediate susceptibilities of isolated E. coli strains. a Characteristic
Number of strains
% of isolates
% of total samples
Antimicrobial resistance streptomycin ampicillin sulfamethoxazole/trimethoprim tetracycline gentamicin kanamycin azithromycin
5 4 4 3 2 1 1
31.3 25.0 25.0 18.8 12.5 6.3 6.3
4.1 3.3 3.3 2.4 1.6 0.8 0.8
Intermediate susceptibility cefazolin amoxicillin-clavulanic
7 2
43.8 12.5
5.7 0.8
2
Table 3 Characteristics of the STEC isolates from goat and ewes’ tank milk samples. a Species
Number of strains
Virulence genes
Goat
1
stx1a, eae
Goat
2
stx1c
Goat
3
stx1c, stx2b
Ewe
1
stx1a, stx2a, stx2d
Ewe
1
stx1c
Ewe
1
stx1c, stx2b
a
Abbreviations are: stx1a, stx1c, Shigatoxin 1 gene subtypes; stx2a, stx2b, stx2d:
Shigatoxin 2 gene subtypes; eae, E. coli attaching and effacing gene.
3
Table 4 Characterisation data of methicillin susceptible S. aureus (MSSA) isolated from goat and ewes’ tank milk samples. a Species
Number of strains Clonal complex
Toxin genes
Goat
1
CC30
sea, egc, tst1
Goat
4
CC130
sec, sel, tst1
Goat
3
CC130
-
Goat
6
CC133
sec, sel, tst1
Goat
1
CC398
-
Goat
1
CC522
-
Ewe
2
CC133
sec, sel, tst1
a
Abbreviations are: sea, sec, sel, staphylococcal enterotoxin genes A, C and L,
respectively; egc, cluster comprising staphylococcal enterotoxin genes I, M, N, O, U; tst1, toxic shock toxin 1 gene. Capsule type 8 was determined in all cases.
4
Table 5 Characterisation data of methicillin resistant S. aureus (MRSA) isolated from different goat tank milk samples. a Number of strains
Clonal complex
mec gene
Toxin genes
3
CC398
mecA
-
1
CC8
mecA
sec, sel, egc
a
Abbreviations are: sec, sel, staphylococcal enterotoxin genes C and L, respectively;
egc, cluster comprising staphylococcal enterotoxin genes I, M, N, O, U. Capsule type 5 was determined in both cases.
5
Table 6 Comparison of S. aureus prevalence with other studies of goats’, ewes’ and cows’ milk. a Reference
Country
Species
Prevalence
This study
CH
GE
14.6%
Muehlherr et al. (2003)
CH
GE
31.9%
Ou et al. (2018)
n/a
G
25.8%
Malissiova et al. (2017)
GR
GE
60.0%
Giacinti et al. (2017)
IT
E
53.5%
Cortimiglia et al. (2016)
IT
C
47.2%
Mehmeti et al. (2017)
XK
C
39.8%
a
Abbreviations are: GE, goats’ and ewes’ milk; G, goats’ milk only; E, ewes’ milk
only; C, cows’ milk; CH, Switzerland; GR, Greece; CZ, Czech Republic; IT, Italy; XK, Kosovo; n/a indicates study was a meta-analysis of studies from different countries.
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Table 7 Comparison of STEC prevalence with other studies of goats’, ewes’ and cows’ milk. a Reference
Country
Species
Prevalence
This study
CH
GE
11.4%
Muehlherr et al. (2003)
CH
GE
15.2%
Sonnier et al. (2018)
USA
C
11.2%
Jaakkonen et al. (2019)
FI
C
6.6%
a
Abbreviations are: CH, Switzerland; USA, United States of America; FI, Finland;
GE, goats' and ewes' milk; C, cows' milk.
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