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Food Microbiology 26 (2009) 910–914

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Food Microbiology

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Occurrence of foodborne pathogens in Irish farmhouse cheese

Martina O’Brien, Karen Hunt, Sara McSweeney, Kieran Jordan*

Teagasc, Moorepark Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland

a r t i c l e i n f o

Article history:Received 3 February 2009Received in revised form17 June 2009Accepted 20 June 2009Available online 27 June 2009

Keywords:Farmhouse cheeseListeriaS. aureusE. coliFood safety

* Corresponding author. Tel.: þ353 2542451; fax: þE-mail address: kieran.jordan@teagasc.ie (K. Jorda

0740-0020/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.fm.2009.06.009

a b s t r a c t

Food safety is a critical factor in the production of farmhouse cheese. In Ireland the varieties of farmhousecheese produced reflect a much broader range than those produced commercially and some of thesecheese varieties are associated with greater microbiological risk. These include cheese produced fromunpasteurised milk and soft ripened cheese such as mould or smear-ripened cheeses which have high pHand relatively short ripening times. The aim of this study was to determine the microbiological quality offarmhouse cheeses in Ireland. Three hundred and fifty one cheese samples, from 15 cheese producers,were analysed for microbiological quality on a monthly basis throughout the year. The analyses includedenumeration of Escherichia coli, Staphylococcus aureus and Listeria monocytogenes (using the relevantagars) and enrichment for L. monocytogenes. The cheeses selected were produced from ovine, caprineand bovine milk. Both unpasteurised and pasteurised milk cheeses were sampled and these includedhard, semi-hard and soft cheeses, internal/external mould-ripened and smear-ripened cheeses and thecheeses represented different geographic regions. Of the cheeses tested, 94% were free of L. mono-cytogenes, all were within the EU limits for E. coli and only one cheese variety had S. aureus levels abovethe recommended numbers for the first 6 months of the year. Due to a modified production process thenumbers were within the guidelines for the second six months. The results indicate that Irish farmhousecheeses are of a high microbiological quality.

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1. Introduction

The production and consumption of speciality cheeses havea long tradition in Europe and the diversity of cheeses produced iswidely recognised. In Ireland, such speciality cheeses are oftenmade on a small scale at the farm where the milk is produced.Farmhouse cheese production in Ireland, which began on a verysmall scale in the 1970s, has expanded at a rate of between 5 and10% per annum over the last number of years. The value of thissector is about V9 million (An Bord Bia, personnel communication),the National Dairy Council estimates the volume at 1000 tonnes peryear and there are approximately 55 farmhouse cheesemakersoperating in this country (Teagasc Farmhouse Cheese Fact Sheet No.8, 2006). Of the approximately 55 producers, 6% exceed 100 tonnesper year, while a further 8% produce between 50 and 100 tonnes;45% of the producers have an output of 10 tonnes or less. Goat’smilk is used in the production of cheese by 30% of the cheese-makers. It is estimated that 30% of producers are responsible for70% of production (An Bord Bia, personnel communication). Cais,which is the representative body for farmhouse cheesemakers, has

353 2542340.n).

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members involved in the small scale production of hand-madecheese, with milk coming from one or a limited number of sources.The varieties manufactured include those made from pasteurisedand unpasteurised milk, hard, semi-hard, soft, semi-soft and bluecheeses from cow’s, sheep and goat’s milk.

Listeria monocytogenes is the cause of listeriosis, a potentiallylethal disease with a mortality rate of about 30%. Particularly at riskare the young, the elderly, the immunocompromised and pregnantwomen (FAO, 2004). There was a significant increase in thenumber of cases of listeriosis in Ireland in 2007. The crude inci-dence rate increased from 0.17 per 100,000 population in 2006 to0.50 per 100,000 population in 2007 (Garvey et al., 2008) andIreland is listed as one of the six European Union countries witha statistically significant increase in human listeriosis casesbetween the years 1999 and 2006 (Denny and McLauchlin, 2008).In 2006, there were 1583 reported cases in the EU (a crude inci-dence rate of 0.3 per 100,000 population;), while an incidence of0.27 per 100,000 population was reported for the U.S. by theCentres for Disease Control and Prevention (MMWR, 2007).Although the numbers of reported cases are relatively low, theseverity of the disease is high. In England and Wales it wasreported in 2000 that while listeriosis accounted for less than 0.1%of foodborne illness, it accounted for 17% of foodborne illnessrelated deaths (Adak et al., 2002). Set against this background,

M. O’Brien et al. / Food Microbiology 26 (2009) 910–914 911

determination of the occurrence of L. monocytogenes in farmhousecheese is important.

Staphylococcal food poisoning is caused by ingestion of a heatstable toxin produced by coagulase-positive Staphylococcus aureus.It is generally considered that the numbers of S. aureus need to be>105 cfu/g of cheese for production of sufficient toxin to causeillness (Le Loir et al., 2003). In raw milk cheeses, S. aureuscontamination can occur from raw milk produced from cowssuffering from mastitis, and in all cheeses from food handlers whoare carriers of S. aureus, or from poor hygiene practices. It can growduring processing, but once salt is added there is inhibition. Forthese reasons, a regulation of <105 cfu coagulase-positive S. aureus/g of cheese has been set for raw milk cheese. Pasteurisation kills S.aureus, and therefore it is less likely to be an issue with pasteurisedmilk cheese (Lancette and Bennett, 2001).

Escherichia coli is used as an indicator of direct or indirect faecalcontamination of foods, and therefore the possible presence ofenteric pathogens. The greatest application of testing for E. coli is inthe assessment of the overall quality of the food and the hygienicconditions during food processing. In cheese, E. coli is used as anindicator to assess post-pasteurisation contamination and itspresence may indicate inadequate pasteurisation, poor hygieneconditions during processing or post-processing contamination(Kornaki and Johnson, 2001).

Irish farmhouse cheesemakers have secured a number of awardsat international level, raising the profile of Irish farmhouse cheeses.However, microbiological quality of the cheeses, particularly withraw milk and surface/smear-ripened cheeses can occasionally be anissue (Coveney et al., 1994; FSAI, 2005, 2006). Since these surveyswere completed, EU regulations have changed. As a requirement ofEC regulation 2073 of 2005, as amended by EC regulation 1441/2007 (Commission Regulation (EC) No 2073/2005; CommissionRegulation (EC) No 1441/2007) on the microbiological criteria forfoodstuffs, there is an onus on Food Business Operators (FBOs) toensure their food products comply with certain food safety andprocess hygiene criteria. For cheese, the required food safetycriteria include testing for L. monocytogenes. There is also an onuson FBOs who produce foodstuffs capable of supporting the growthof Listeria to sample their processing environment.

The aim of this study was to monitor the microbiological qualityof 2 cheeses (ready for retail) from 15 different farmhouse cheesemanufacturers on a monthly basis throughout the year. The focus ofthe study was on L. monocytogenes, E. coli and S. aureus.

2. Materials and methods

2.1. Sampling plan

From January to December 2007, two cheeses (representingdifferent batches; for this study, a batch is defined as the cheeseproduced in one day) were collected from 15 different cheesemanufacturers every month. In order to represent the wide varietyof different farmhouses cheeses in Ireland, these included 3 caprine,2 ovine and 10 bovine milk cheeses. There were 6 raw milk cheesesand the remainder were pasteurised. There were 2 mould ripened,3 blue mould, 2 smear-ripened, 1 fresh, 1 semi-soft and 6 hardcheeses. In order to represent a wide geographic area, cheeses weresourced from nine different counties in three provinces. Cheesesthat were ready for distribution to the market were selected, asthese represented products that were entering the food chain.

2.2. Sample collection

Samples, individually wrapped to prevent cross-contamination,were collected from the farms, placed in a cool box with ice packs

and transported directly to the laboratory. They were either ana-lysed immediately or stored at less than 4 �C and analysed thefollowing day.

2.3. Microbiological analyses

In general, a composite of rind and core was analysed. However,for cheeses with a higher risk of L. monocytogenes (smear-ripenedcheeses), the rind and core were assayed separately. All sampleswere analysed for L. monocytogenes, S. aureus and E. coli using theappropriate agars (see below). Ten grams were aseptically homo-genised in the appropriate volume of 2% tri-sodium citrate solution(to give a 1:10 dilution) in a Colworth stomacher 400 for 4 min. Thehomogenate was serially diluted in maximum recovery diluent(MRD; Oxoid, UK, CM0733). E. coli was enumerated according to ISO16649-2, using CTBX (Chromocult Trytone Bile X-glucuronide) agar(Merck, Darmstadt, Germany, 1.16122.0500). Typical E. coli coloniesare coloured blue-green. Coagulase-positive S. aureus wasenumerated according to ISO 6888-2, using Baird Parker RPF(Rabbit Plasma Fibrinogen) agar (Oxoid, Basingstoke, UK, CM0961).Typical coagulase-positive S. aureus colonies are black, grey orwhite and are surrounded by an opaque halo of precipitation whichsignifies the coagulase reaction. Presence/absence of L. mono-cytogenes was determined by the FDA method. Samples wereenriched in Listeria enrichment broth (LEB; Oxoid, Basingstoke, UK,CM862). Twenty five grams of sample were added to 225 ml of LEB(with selective agents added), homogenised in a Colworth stom-acher 400 for 4 min and incubated at 37 �C for 48 h. After thisenrichment step, 20 and 100 ml were spread on ALOA (Agosti &Ottaviani Listeria Agar; LabM, Lancs, UK, HAL010) agar plates (toaccount for low or high numbers) which were incubated at 37 �C for24–48 h. For enumeration of L. monocytogenes, 100 ml of homoge-nate (1:10 dilution in 2% tri-sodium citrate, homogenised as above)was spread on an ALOA agar plate which was incubated at 37 �C for48 h. Typical L. monocytogenes colonies (which are green witha surrounding halo) were selected for confirmation (see below). Forcomparative purposes, presence/absence of L. monocytogenes wasalso determined in 30 samples by the ISO 12900-1 method usingFrazer Broth (Merck, Darmstadt, Germany, 1.10398.0500).

2.4. Confirmation by PCR

For confirmation of isolates as L. monocytogenes, a positivecolony from an ALOA plate was added to 50 ml of sterile PCR gradewater, which was heat treated at 95 �C for 15 min to release theDNA. From this, 5 ml was assayed by real-time PCR using a Light-Cycler 2.0 (Rodrıguez-Lazaro et al., 2004). The primers and probeused targeted the haemolysin (hly) gene:

Forward primer 50 CAT GGC ACC ACC AGC ATC T-30

Reverse primer 50 ATC CGC GTG TTT CIT TTC GA-30

Taqman probe 50 FAM-CGC CTG CAA GTC CTA AGA CGCCA-TAMRA-30

Prior to freezing, confirmed positive isolates were purified by re-streaking onto TSA, followed by streaking onto ALOA agar.

3. Results

3.1. Analysis for coagulase-positive S. aureus

For 339 of the cheeses (96%), S. aureus was within the regula-tions, i.e. either absent or present in very low numbers (Regulations<100,000 cfu/g for raw milk cheeses, <1000 cfu/g for heat treatedmilk cheese). There were consistently high counts of S. aureus

Table 1Comparison of FDA and ISO methods for analysis of L. monocytogenes in 30 samples(2 samples by each method from 3 different farms over a 5-month period).

Month Farm 6 Farm 10 Farm 15

FDAc ISOd FDA ISO FDA ISO

Aug. �a þb � þ � � � � þ þ � �Sept. � � � � � � � þ � � � �Oct. � � � � � � � � � þ � þNov. � � � � � � þ þ � � � þDec. � � � � � � � � � � � �

a Sample was negative for L. monocytogenes.b Sample was positive for L. monocytogenes.c Samples were analysed by the FDA method using Listeria Enrichment Broth.d Samples were analysed by the ISO method using Frazer Broth.

M. O’Brien et al. / Food Microbiology 26 (2009) 910–914912

(over 10,000 cfu/g), which exceeded the legal limits, in twelvecheeses from one manufacturer. All these cheeses were negative forenterotoxin (data not shown). These cheeses represented the first6 months of the sampling period. For the second 6 month period,all the cheeses from this manufacturer were all within the legalrequirement.

3.2. Analysis for E. coli

E. coli was absent, or present in very low numbers in all 351cheeses tested (100%). All cheeses were within the limits specifiedby the EU regulations. For cheese made from heat treated milk, thelimit is<1000 cfu/g; there are no stated limits for raw milk cheeses.Seventy nine per cent of the raw milk cheeses had E. coli counts<10 cfu/g. All of the raw milk cheese counts where <10,000 cfu/gand the pasteurised milk cheeses <250 cfu/g.

3.3. Analysis for L. monocytogenes

L. monocytogenes was not detected in 330 of the cheeses tested(94%). L. monocytogenes was detected by direct count in 12 of thecheeses (3%). For the remaining 9 cheeses (2%), L. monocytogeneswas only detected by enrichment. Isolates from all the positivecheeses were confirmed as L. monocytogenes by PCR. All of thesamples that were positive for L. monocytogenes were obtainedduring the months April to November (Fig. 1).

3.4. Comparison of ISO and FDA methods for L. monocytogenesdetection

For 15 cheese samples (in duplicate) analysis for presence/absence of L. monocytogenes was determined by both the FDA andISO 12900-1 methods. The results are shown in Table 1. For 80% ofthe samples a similar result was obtained; 73% of the samples werenegative by both methods and 7% were positive by both methods.Of the 20% of samples that were different, 13% were positive by theISO method only and 7% were positive by the FDA method only.

4. Discussion

Food safety policy in the EU is governed by, among other regu-lations, Commission Regulation (EC) No 2073/200, which lays downthe general principles and requirements of food law and calls for ‘acomprehensive integrated approach’ to ‘ensure a high level of humanhealth and consumer protection’. This legislation places responsibilityfor safety on the food producer (Section 4, Article 17) and alsorequires that food law be based on risk analysis, where appropriate.

With regular monthly sampling, this work aimed to establishthe level of E. coli, S. aureus and L. monocytogenes in a variety of Irishfarmhouse cheeses and thus establish some measure of the riskassociated with these products. In a previous (limited) survey

L. monocytog

0

1

2

3

4

5

6

jan feb mar apr may junMo

No

. o

f ch

eeses p

ositive

Fig. 1. Seasonality of L. mo

(Coveney et al., 1994), coliform, faecal coliform, S. aureus andL. monocytogenes were measured. They were detected at levels of68%, 24%, 48% and 0%, respectively. At that time, the regulationsspecified numbers of coliforms. The current regulations specifynumbers of E. coli. As E. coli is killed by pasteurisation, its presencein pasteurised milk cheese is an indicator of pasteurisation failureor poor hygiene during cheese manufacture and ripening.

The FSAI undertook a survey of raw milk and pasteurised milkcheeses in 2004 and 2005, respectively (FSAI, 2004, 2005). In eachsurvey, samples were taken at both production and retail level. Theorigin of the retail samples was not defined, therefore these samplescannot be compared to the current survey. The origin of the samplestaken at production level were known to be Irish, therefore thesesamples are comparable to the current survey. For Raw milk cheeses(n¼ 28), S. aureus was the only organism of concern, 14.3% beingclassified as unsatisfactory. For pasteurised milk cheeses (n¼ 54),3.7% were classified as unsatisfactory for E. coli.

In the current study, E. coli was absent from 210 pasteurised milkcheeses tested. While the results of both surveys are not directlycomparable, the lack of an issue with E. coli in the current surveyindicates an improvement in hygiene. Although limits for E. coli arenot specified for cheese produced from raw milk, samples wereanalysed in order that a baseline level could be identified over theproduction season.

Coagulase-positive S. aureus can produce an enterotoxin thatcauses illness. High numbers of the organism are necessary toproduce the toxin in sufficient quantities to be a threat to publichealth (Le Loir et al., 2003). S. aureus can cause infections in themammary gland of animals and, therefore, contaminate milk.However, it is killed by pasteurisation, and therefore, it should beabsent in pasteurised milk cheeses; their presence is due to unhy-gienic handling or poor manufacturing practice. In the survey byCoveney et al. (1994), S. aureus was present in 48% of the cheesestested, and in all cheese types, including pasteurised milk cheeses. Inthe FSAI survey, 14.3% of the raw milk cheeses tested were unsatis-factory with respect to S. aureus. In the current survey, coagulase-positive S. aureus were absent or very low in all the pasteurised milk

enes in cheese

jul aug sep oct nov decnth

nocytogenes in cheese.

M. O’Brien et al. / Food Microbiology 26 (2009) 910–914 913

cheeses tested, again indicating an improvement in hygiene stan-dards. In unpasteurised milk cheeses, the number of S. aureus canincrease during manufacture if they are present in the raw milk.However, if the numbers remain below 105 cfu/g, insufficient toxinwould be produced to cause illness (Le Loir et al., 2003). For thisreason, the regulation states that S. aureus numbers must be<105 cfu/g. If the numbers exceed this, the cheese must test negativefor enterotoxin before it can be released to the market. In the currentsurvey, only 12 cheeses exceeded 105 cfu/g and all were negative forenterotoxin. In an effort to address the issue of high S. aureusnumbers, the manufacturer made minor modifications to themanufacturing process half way through the study period. Thesemodifications resulted in a reduction of S. aureus numbers to withinthe regulations for the remainder of the study period.

EU regulation 2073/2005 on the microbiological criteria forfoodstuffs, has placed stringent criteria on ready-to-eat (RTE)foods able to support the growth of Listeria, including many farm-produced speciality foods such as cheese and dairy products(Commission Regulation (EC) No 2073/2005). The legislationrequires an absence of L. monocytogenes in 25 g before the foodhas left the immediate control of the FBO, who has produced it,and <100 cfu/g on products placed on the market during theirshelf-life. Despite this, Listeria spp. have been isolated from varietyof foods including ready-to-eat foods, fish, meat and raw milk,and the general occurrence has been recently reviewed (Lianouand Sofos, 2007). In most cases, contamination is due to post-processing environmental cross-contamination of foods (D’Amicoet al., 2008; Jacquet et al., 1993), as Listeria is killed by pasteur-isation and cooking. Ripened soft cheeses and smear-ripenedcheeses (Rudol and Scherer 2001) are in general considered high-risk products.

Listeria spp. have been isolated from foods in Ireland. Sheridanet al. (1994) examined 549 varied food samples, including pre-packed cooked meats, frozen meats, frozen fish and cooked meatspackaged at retail level. In Northern Ireland, Harvey and Gilmour(1993) examined 513 food samples of which 35% were positive forListeria spp., and 18.3% were positive for L. monocytogenes. Kells andGilmore (2004) studied and examined two milk processing facili-ties in Northern Ireland and detected L. monocytogenes in 22% ofraw milk samples. In a recent survey of bovine, ovine and caprinemilks used for farmhouse cheese manufacture, 5% of samples (bulktank milk 1%, and milk filters 8%) were positive for L. monocytogenes(Brenda Murphy, personal communication). L. monocytogenes wasnot detected in any of the seventeen soft and semi-soft farmhousecheeses analysed as part of Coveney et al. (1994).

In this survey, L. monocytogenes was absent in 330 of the 351cheeses tested. Of the twenty one cheeses testing positive, only 3%had numbers exceeding 100 cfu/g. In the cases where L. mono-cytogenes was detected by enrichment, the ability of the cheeses tosupport the growth of the organism is important. Further work isrequired to determine the ability of the cheeses to support thegrowth of L. monocytogenes. All the cheese samples that testedpositive for L. monocytogenes where collected between April andNovember; the highest number of positives samples were found inJune. The seasonal variation of L. monocytogenes in raw milk hasbeen reported (Ryser, 2007; Abdel-Aziz et al., 2000), with higheroccurrence in Spring and Winter. This variation is generally thoughtto be due to feeding contaminated silage. In the current study, theSummer was very wet (Irish Meteorological Services Online, 2009),resulting in animals being kept indoors and fed silage duringSummer months. In addition to this, the higher humidity andincreased Summer temperatures could result in higher survival ofL. monocytogenes in the environment. These factors could explainthe higher occurrence of L. monocytogenes in the cheeses duringSummer and Autumn.

As Frazer Broth is currently used for detection of L. mono-cytogenes by the Department of Agriculture in Ireland (ISOmethod), it is possible that the occurrence of L. monocytogenes wasunder-estimated by the use of an alternative method, such as LEB(FDA method). Of the 30 samples tested by both methods, 80% gaveexactly the same result (Table 1). For 6 samples (20%; 4 ISO and 2FDA) the results were positive by only one method. Similar resultswere obtained by Moreno-Enriquez et al. (2007) in a comparativestudy of two different isolation methods. The present results indi-cate that the isolation rates obtained by the FDA method would nothave been dramatically different had the ISO method been used.

When L. monocytogenes was detected, the manufacturer wasimmediately alerted and appropriate action taken. This includedfurther testing to identify the possible source/cause and extent ofthe problem and targeted cleaning regimes to reduce thecontamination. The responsibility for informing the authorities waswith the manufacturer.

5. Conclusions

This work has contributed to a knowledge of the microbiologicalquality of Irish farmhouse cheeses over an entire season, and hadindicated an improvement in hygiene since a previous survey in1994. The low occurrence of food pathogens is an indication of thehigh quality of Irish farmhouse cheeses.

Acknowlegdements

This work was supported by the EU 6th Framework Programmeunder the project BIOTRACER, project number 036272 and by theIrish Government under the FIRM Programme, project number06RDTMFRC434. The authors wish to acknowledge the assistanceof CAIS – the Irish Farmhouse Cheesemakers Association – and thecooperation of all 15 cheesemakers who participated in this work.

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