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Food Microbiology

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Use of indicator bacteria for monitoring sanitary quality of raw milk cheeses– A literature review

Monica Metza, John Sheehana,*, Peter C.H. Fengb

a Division of Dairy, Egg and Meat Safety, Office of Food Safety, FDA, College Park, MD, 20740, USAbDivision of Microbiology, Office of Regulatory Science, FDA, College Park, MD, 20740, USA

A R T I C L E I N F O

Keywords:E. coliColiformIndicatorRaw milkCheese

A B S T R A C T

Many countries use Escherichia coli and coliforms as indicators of sanitary quality of foods and have set limits forcheeses, including raw-milk cheeses. This paper reviewed the scientific literature for E. coli and coliform levelsthat are found in different types of raw milk, the fate of indicators during the manufacturing and ripening ofdifferent cheeses and the indicator levels that have been found in the finished cheeses. These studies fromworldwide showed that E. coli and coliforms are found in different types of raw milk but usually at< 100 CFU/ml or not found. Instances where raw milk contained indicator levels> 1000 CFU/ml have mostly been at-tributed to unsanitary conditions/production. During cheese-making, indicators present in raw milk will oftenincrease in numbers, but the levels decline as the acidity from lactose fermentation decreases the pH. Except forfresh cheeses that are not aged, indicator levels are further reduced by 2–3 log10 CFU/g or more, during theripening process. As a result, indicator levels in finished cheeses are often low and within the limits of< 10or<100 CFU/g set by many countries. The cited studies also show that raw milk cheeses that are made withquality raw milk, under hygienic conditions and properly aged, should not contain high levels of indicatorbacteria in the final product.

1. Introduction

Typhoid fever was recognized as a waterborne disease in the mid-1800s and was suspected to be spread via fecal contamination of water.However, Salmonella Typhi, the causative agent of typhoid fever wasnot identified until much later and even then, no methods were avail-able to test for the pathogen. In 1885, Escherichia coli was identified andfound to be a normal inhabitant of the human intestine which lead tothe idea that it may be useful as an indicator of fecal contamination andtherefore, indirect evidence that pathogens may be present. The de-tection of E. coli was initially based on fermentation of glucose but waslater changed to lactose which made it difficult to distinguish E. colifrom other enteric bacteria that also ferment lactose. This group oflactose-fermenting bacteria, known as the coliform, is comprised ofseveral genera, including Enterobacter, Klebsiella, Citrobacter andEscherichia. In the U.S. in 1914, the coliform group was adopted as anindicator of fecal pollution to ensure the sanitary quality of drinkingwater (Tortorello, 2003). However, since coliforms may not alwaysoriginate from fecal sources, the concept of fecal coliforms, which havethe ability to ferment lactose at 45 °C, was introduced to better reflectfecal origin. Fecal coliforms are comprised mostly of E. coli, however,

some Klebsiella spp. also ferment lactose at the elevated temperature, asa result, E. coli are more often used as indicator of fecal origin. Cur-rently in the U.S., fecal coliforms are used mostly as indicators of in-sanitation in shellfish and seldom used for other foods. Coliform, fecalcoliform and E. coli all belong in the family Enterobactericeae, which isalso used by some countries as indicator for insanitation.

The initial concept of testing for indicator bacteria was for evidenceof fecal contamination, which served as an indirect evidence that pa-thogens may be present and therefore, posed safety concerns (Smootand Pierson, 1997). However, numerous studies over the years showeda lack of correlation between the presence of indicators and pathogens(Martin et al., 2016; Miskimin et al., 1976), thereby lessening theusefulness of indicators for assessing product safety. Furthermore, thepresence of certain coliforms in the environment has also diminishedthe association of coliforms as a group as indicators of fecal con-tamination (Boor et al., 2017; Trmčić et al., 2016). E. coli can also befound in the environment, but its origin is most likely intestinal, andtherefore, it is still advocated as an appropriate indicator of fecal con-tamination and a hygiene indicator (Smoot and Pierson, 1997; Trmčićet al., 2016).

The concept of indicators has evolved over the years, from being an

https://doi.org/10.1016/j.fm.2019.103283Received 26 November 2018; Received in revised form 6 June 2019; Accepted 30 July 2019

* Corresponding author. FDA, 5001 Campus Drive, College Park, MD, 20740, USA.E-mail address: John.Sheehan@fda.hhs.gov (J. Sheehan).

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indicator of safety to become mostly an indicator of sanitary quality orunsanitary conditions. As stated by the International Life SciencesInstitute (ILSI) (ILSI, 2011), “Indicator organisms are bacteria that areused to provide evidence of poor hygiene, inadequate processing or post-process contamination of foods… Their absence in food provides a degree ofassurance that the hygiene and food manufacturing processes have beencarried out appropriately, whereas their presence usually indicates that apotential problem or failure in the process has occurred.“. Similar positionshave been stated by the International Commission on MicrobiologicalSpecifications for Foods (ICMSF), which provides science-based gui-dance to government and industry (ICMSF, 1986), by the NationalAcademy of Science (NAS, 1985), by a report of the National AdvisoryCommittee on Microbiological Criteria for Foods (NACMCF, 2018) andby the WHO/FAO JEMRA report on “Shiga toxin-producing Escherichiacoli (STEC) and food: attribution, characterization and monitoring”(JEMRA, 2018). Consistent with these scientific positions and re-commendations, many industrialized and developing countries world-wide have established indicator limits for monitoring the sanitaryquality of cheeses. These hygiene indicators have been used by thedairy industry to monitor lapses in sanitation, and post-processingcontamination for over 100 years (Boor et al., 2017).

In this review we examined the use of coliforms and E. coli as in-dicators of sanitary quality in cheeses. Sanitary quality encompasses theentire process, from the quality of the raw milk used, the cheese-makingprocess and aging, to the levels of indicators found in finished cheeses,therefore, we conducted a comprehensive scientific literature reviewon: 1) the levels of indicators found in raw milk, 2) the fate of indicatorsduring cheese-making, processing and aging and 3) the levels of in-dicators reported in finished cheese products worldwide. For referencepurposes, the review also compiled tables showing the coliform and E.coli limits, in both raw and pasteurized cheeses and dairy products, setby countries worldwide. Some countries also use Enterobacteriaceae asan indicator and some of the cited studies also tested for fecal coliformsand therefore, these were included in the discussion of those cited pa-pers.

2. Process for literature search

The terms we used to do the literature search for raw milk included:coliforms, fecal coliforms, E. coli, milk and raw milk. For the literaturesearch on cheeses, the same terms were used and in addition: cheese,processing, ripening, aging, diversity, microflora and microbiota. Thecitations used in this review are those that provided information on thelevels of coliforms, fecal coliforms and E. coli in raw milk, the impact ofcheese manufacturing and ripening on bacteria and the levels of in-dicators found in finished cheese products. The citations we used in-cluded publications dated from 1968 to 2018 and covered a variety ofcheeses made in many countries worldwide.

To compile the indicator limits in dairy foods and cheeses estab-lished by the different countries (Tables 1 and 2), we consulted thepublications of the USDA Global Agricultural Information Network(GAIN), performed internet searches on indicator limits for cheeses, andcommunicated directly with regulators worldwide to obtain existingmicrobial limits for raw milk, raw-milk cheeses or cheeses for the dif-ferent countries. Some countries have set sampling criteria and limitsfor coliforms in cheese (Table 1), which range from<3 to 5000 colonyforming units (CFU)/g, but most limits are at 100 CFU/g or lower.However, most countries and regional authorities have set limits for E.coli in cheese (Table 2), which, depending on cheese types, rangefrom<3 to 2000 CFU/g, with most limits between 10 and 100 CFU/g(New Zealand Food Safety Authority, 2010; Humphrey and Hart,1986). The limits for E. coli and coliforms we compiled are from 59countries, which encompass 62% of the world's population (CIA WorldFact Book). Similarities in these indicator limits have also set relativeglobal parity and in doing so, facilitated trade and the import/export ofcheeses worldwide.

3. Prevalence and levels of coliforms in raw milk

Coliforms can be found in fecal sources, but some genera are alsopresent in the environment, so are often used as indicators of in-sanitation rather than fecal contamination. Coliforms are not inherentmicroflora of raw milk and can be introduced into milk from the en-vironment, udder and milking equipment during and after milking(Hayes and Boor, 2001). Many studies worldwide have surveyed dif-ferent types of milk for the presence of coliforms (Table 3). Indicatorbacteria are usually enumerated using direct methods, like a plate count(colony forming units [CFU]/g) or indirect method, such as mostprobable number (MPN), which is a statistical estimation of the num-bers of cells present (MPN/g). Pantoja et al. (2009) examined 7275samples of raw bovine milk from 16 farms in Wisconsin over a one yearperiod and found coliform levels that ranged from not found to1520 CFU/ml with a mean count of 1.7 CFU/ml. The observed fre-quency distribution of coliform levels was: 20% at< 10 CFU/ml, 60%had< 70 CFU/ml, 70% had<120 CFU/ml, 80% had<250 CFU/ml,90% had<1140 CFU/ml and 10% of the samples had coliforms at>1140 CFU/ml. D'Amico et al. (2008) tested 67 samples of raw bovinemilk from 5 farms in Vermont and showed that 89% had coliform levelsof< 100 CFU/ml, with 62% showing<10 CFU/ml and< 10% withlevels at 1001–10,000 CFU/ml. Of the other milk types tested, 76% ofthe 49 samples of caprine milk had coliforms at< 100 CFU/ml, 55%had< 10 CFU/ml and no samples exceeded 1000 CFU/ml. Of the 22samples of ovine milk analyzed, 92% had coliforms at< 100 CFU/ml,with 75% at< 10 CFU/ml and<10% had coliforms in the range of1001–10,000 CFU/ml. This Vermont study was followed up two yearslater by the analysis of 85 samples from 21 farms (12 cow, 5 goat and 4sheep) but the results were similar in that 96% of the bovine milksamples had coliforms at< 100 CFU/ml, 69% at< 10 CFU/ml andonly< 10% exceeded 1000 CFU/ml. Analysis of caprine milk samplesshowed 84% contained coliforms at< 100 CFU/ml, 51% at< 10 CFU/ml and none had>1000 CFU/ml. With respect to the ovine milksamples, 93% had< 100 CFU/ml, 60% had<10 CFU/ml and<10%exceeded 1000 CFU/ml (D'Amico and Donnelly, 2010). Other studiesanalyzed bulk tank raw-milk samples taken from different dairy herdsin different States and found similar levels of coliforms (Boor et al.,1988; Jayarao et al., 2004; Jayarao and Wang, 1999) (Table 3) and onestudy also found a significant association between coliform levels andherd sizes with smaller herds often showing lower counts (Jayaraoet al., 2004). Costello et al. (2003) tested 833 samples of bulk tank milkover a period of 11 years in Washington State and found that ap-proximately 88% had coliforms at< 100 CFU/ml with 65% at≤20 CFU/ml, 33.9% at< 10 CFU/ml and only 3.9% had> 1000 CFU/ml. The coliform levels found ranged from 1 to 90,000 CFU/ml and thegeometric mean was 14 CFU/ml. From these studies, it can be surmisedthat between 50 and 96% of the U.S raw-milk samples had coliformsat< 100 CFU/ml and 10–75% of samples were at levels of< 10 CFU/ml.

Analyses of samples from other countries showed that raw caprineand ovine milks can sometimes have very high coliform counts. Forexample, de Garnica et al. (2012) tested 751 bulk tank samples of ovinemilk in Spain, collected over one year and showed mean coliformcounts ranging from 3.42 to 4.57 log10 CFU/ml throughout the fourseasons. Compared to the study by Jayarao et al. (2004), the U.S. bulktank bovine milk samples only had a mean coliform count of 70 CFU/ml, thus the ovine milk had much higher coliform counts. Differences inmilking facilities, production and practices, such as the absence of teatwashing before milking are a few of the factors that maybe contributingto higher coliform counts in ovine milk. Morgan et al. (2003) tested rawcaprine milk sampled from reception tanks at seven small and mediumdairy plants in Greece, Portugal and France and found that samplesfrom Greece and Portugal had the highest coliform levels with means of5–6 log10 CFU/ml. But, those from French dairies only had coliforms at2 log10 CFU/ml. The study noted that France had implemented

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intensive breeding systems, where modern techniques of livestockbreeding were used to produce large herds of improved breeds andtherefore concluded that improved farming and milking systems areneeded in Greece and Portugal to improve the hygiene of the caprinemilk produced. Foschino et al. (2002) found coliform levels in caprinemilk in Italy to be similar to those found by Morgan et al. (2003) inFrance and also noted that coliform levels were significantly higher inmilk from farms which did not have refrigeration.

These studies from the various countries showed that coliforms arefound in different types of raw milk with most samplescontaining<100 CFU/ml, regardless of the lactating species or thecountry. Occasionally, some samples contained coliform levels of up to6 log10 CFU/ml, but this does not appear to be the norm. Also, thecoliform levels varied greatly, perhaps due to seasonal and geographicvariations, differences in milking practices, sampling, testing methods,hygienic practices or other factors that may be particular to the dif-ferent countries.

4. Prevalence and levels of E. coli in raw milk

E. coli is generally regarded as being of fecal origin and has beenused as an indicator of both insanitation and fecal contamination. Manystudies worldwide have surveyed raw milk samples for the presenceand levels of E. coli (Table 4). Ruusunen et al. (2013) tested 183 sam-ples of raw bovine milk collected from bulk tanks on dairy farms inFinland and found E. coli in 45% of the samples and the levels rangedfrom 1 to 885 CFU/ml with the average at 5 CFU/ml. Pyz-Łukasik et al.

(2015) tested 50 samples of raw bovine milk collected from five directpoints of sale in Poland and found that 76% had no E. coli and theremaining 24% of the samples had E. coli at levels ranging from 5 to110 CFU/ml. A survey of 495 raw milk samples taken over a 24-monthperiod in South-East Scotland found that 96.8% had E. coli at< 100CFU/ml (Coia et al., 2001). Desmasures and Guéguen (1997) testedgood-quality milk samples (defined by the authors as milk from farmsthat met a standard of< 50,000 CFU/ml total count and<250,000somatic cells/ml) over a two year-period in France and found that lessthan one third of the samples contained E. coli. Desmasures et al. (1997)tested 69 raw milk samples collected from bulk tanks in the Camembertregion of France and found that 80% of the samples had E. coli at≤10 CFU/ml with only one sample having>30 CFU/ml. A study fromIreland showed that approximately 80% of the 386 raw milk samplescollected over a year from 70 Irish milk farms had E. coli counts of<10 CFU/ml (Rea et al., 1992). Analyses of 143 raw milk samples frombulk tanks in Belgium showed that 90.8% had E. coli at ≤100 CFU/mland 46.8% at≤10 CFU/ml (De Reu et al., 2004). In contrast, Chye et al.(2004) examined raw milk from four different regions of Malaysia andfound that 64.5% of the samples contained E. coli. The mean counts,which varied among the regions, ranged from 1900 - 15,000 CFU/mland the authors concluded that the milk samples that had high E. colicounts were most likely produced under unsanitary conditions.

Some studies also examined the prevalence of E. coli in other milktypes. Foschino et al. (2002) tested 60 samples of raw caprine milk inItaly and showed that 10% of the samples had no E. coli and in the other90% of the samples, the counts ranged from 0.5 to 7.9 CFU/ml with a

Table 1Coliform limits set for cheeses by different countries.

Country Cheese type Sampling plana Comments

n c m M

Brazilc Low moisture 5 2 100 500Medium moisture 5 2 500 1000High moisture 5 2 1000 5000Very high moisture 5 2 500 500046–55% moisture 5 2 1000 5000Dried 5 2 <3 10Grating 5 2 100 1000Pasteurized, processed 5 2 <3 10Very high moisture - condiments, herbs, other ingredients 5 2 50 100Low-medium moisture - condiments, herbs, other ingredients 5 2 100 500

Chinad 5 2 100 1000Mexicoe Fresh 100 fecal coliforms

Aged 50Processed NBTb

Philippinesf products, pasteurized 5 1 11 1000Processed, Spread 5 1 10 100

Peru g Processed 5 1 10 100Non-matured 5 2 500 1000Matured 5 2 200 1000

Russia and EACUb, h AbsenceU.S. DODb/NACMCFb, i Pasteurized <100 routine sampling; applies to DOD only

a n=number of sample units selected from a lot of food to be examined. c=maximum number of allowable defective or marginally acceptable units.m= acceptable level (/g) of microorganism determined by a specific method. M= level (/g) which when exceeded in one or more samples would cause the lot to berejected as this indicates a hazard or imminent spoilage.

b Acronyms: NBT – negative by test; EACU - Eurasian Customs Union; DOD – Department of Defense; NACMCF – National Advisory Committee for MicrobiologicalCriteria for Foods.

c ResolucÃo-RDC Nº 12, de 02 de Janeiro de 2001, Anexo-Regulamento técnico sobre os padrÕes microbiológicos para alimenotsd China National Food Safety Standard – cheese (GB5420-2010). 2010. http://www.nhfpc.gov.cn/zhuz/psp/201005/47387/files/

4035eedabe8a4a3d97fe6963c0c8fb7f.pdf.e Norma Oficial Mexicana NOM-121-SSA1-1994, Bienes y Servicios. Quesos frescos, madurados y procesados. Especificaciones Sanitarias.f Philippines Revised Guidelines for the Assessment of Microbiological Quality of Processed Foods. 2013.g Peru NTS No. 071, MINSA/DIGESA-V.01, Norma sanitaria que establece los criterios microbiologicos de calidad sanitaria e inocuidad para los alimentos y

bebidas de consumo humano.h Technical Regulation of the Russia-Kazakhstan-Belarus Customs Union on Safety of Milk and Dairy Products (TR TS 033/2013).i NACMCF Report - https://www.fsis.usda.gov/wps/wcm/connect/2ea3f473-cd12-4333-a28e-b2385454c967/NACMCF-Report-Process-Control-061015.pdf?

MOD=AJPERES.

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mean of 2.9 CFU/ml.The results of these surveys (Table 4) showed that raw milk in some

countries may occasionally contain E. coli, some with mean counts ashigh as 15,000 CFU/ml (Chye et al., 2004), but most of the studiesshowed that over 90% of the samples have E. coli levels that were<100 CFU/ml, thus indicating that E. coli is not often found at high levelsin various types of raw milk (Table 4).

5. Fate of indicator bacteria during the cheese-making process

It is evident from the above surveys that when raw milk containscoliforms and/or E. coli, it is typically at low levels. However, this doesnot necessarily translate into a presence or corresponding levels ofcoliforms and/or E. coli in the finished product, as the cheese-making

and ripening processes can greatly affect bacterial growth and survival.The cheese manufacturing process, including milk ripening conditions,the starter cultures used, the mode and extent of salting and the con-ditions and duration of aging all contribute toward the ultimate com-position, body and texture of the cheese and the fate of indicator or-ganisms within a finished cheese product. Thus, the variability involvedin the production of hundreds of different raw-milk cheeses and thegreat diversity of intrinsic characteristics of cheeses makes it difficult togeneralize the fate of indicator organisms in the various cheese types.Nevertheless, from the large amount of data collected over the yearsfrom different kinds of raw milk cheeses, a common scenario emerges.During the initial milk ripening process there is an increase in thenumbers of the indicator organisms followed by a decline of the num-bers as manufacturing proceeds and throughout the ripening of the

Table 2Generic Escherichia coli limits set for dairy foods by different countries.

Country Cheese product Sampling plana Comments

n c m M

Argentinac All types 5 0 < 3Australia and New Zealandd All cheese 5 1 10 100 Unified Standards for Australia and New ZealandCanadae Pasteurized 5 2 100 2000

Raw 5 2 500 2000EUb,f Pasteurized 5 2 100 1000 Process hygiene criterionGulf Cooperation Councilg Hard and semi-hard 5 0 0

Processed, in non-metal containers 5 0 0Soft, pasteurized milk 5 1 10 100

Hong Kongh All cheeses excluding those ripened with Hafniaalvei or Proteus vulgaris and raw milk cheeses

>100

Indiai Processed and spreads 0 AbsentAll others 5 0 >10

New Zealandj Raw milk used to make raw milk products < 100 Code of PracticePeruk Non-matured 5 1 3 10Philippinesl Cheese products, pasteurized 5 1 11 110 MPN/gSouth Africam All dairy products 0 AbsenceSouth Korean Processed 5 2 <3 100

Natural or raw 5 1 10 100Taiwano Cheese 100Turkeyp Heat treated milk or cheeses produced from whey 5 2 100 1000 Sample must be collected from the stage where the E. coli count is

estimated to be the highest level throughout the production processUnited States DOD/

NACMCFb,qPasteurized < 10 >100 non-routine sampling; for DOD only

a n=number of sample units selected from a lot of food to be examined. c=maximum allowable number of defective or marginally acceptable units.m= acceptable level (/g) of microorganism determined by a specific method. M= level (/g) which when exceeded in one or more samples would cause the lot to berejected as this indicates a hazard or imminent spoilage.

b Acronyms: EU – European Union; DOD – Department of Defense; NACMCF – National Advisory Committee for Microbiological Criteria for Foods.c Código Alimentario Argentino. Capítulo IIIArtículos: 155 al 183 - De los Productos Alimenticios. - Actualizado al 1/2017.d Australia New Zealand Food Standards Code Schedule 27 Microbiological limits in food. http://www.foodstandards.gov.au/code/Documents/Sched%2027%

20Micro%20limits%20v157.pdf.e Standards and Guidelines for Microbiological Safety of Foods. An Interpretive Summary 2008. Food Directorate, Health Products and Food Branch, Government

of Canada.f Commission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs http://eur-lex.europa.eu/legal-content/EN/TXT/

HTML/?uri=CELEX:32005R2073&from=EN.g Microbiological Criteria for Foodstuffs, Gulf Cooperation Council Standardization Organization, GSO 1016/2015.h Hong Kong Centre for Food Safety. 2014. Microbiological Guidelines for Food. http://www.cfs.gov.hk/english/food_leg/files/food_leg_Microbiological_

Guidelines_for_Food_e.pdf.i India Food Safety and Standards Regulation. 2010. http://indiaenvironmentportal.org.in/files/finalregualtion.pdf.j New Zealand Food Safety Authority. 2010. Code of Practice: Additional Measures for Raw Milk Products http://www.foodsafety.govt.nz/elibrary/industry/raw-

milk-products-cop/code-of-practice-additional-measures-for-raw-milk-products.pdf.k Peru NTS No. 071, MINSA/DIGESA-V.01, Norma sanitaria que establece los criterios microbiologicos de calidad sanitaria e inocuidad para los alimentos y

bebidas de consumo humano.l Philippines Revised Guidelines for the Assessment of Microbiological Quality of Processed Foods. 2013.m South Africa GNR 1551 (1997).n Republic of Korea Processing Standards and Ingredient Specifications for Livestock Products [Ministry of Food and Drug Safety Notice No. 2015–94, 12/16/

2015)].o https://www.fda.gov.tw/EN/lawContent.aspx?cid=16&id=1387.p Regulation on Turkish Food Codex Microbiological Criteria, Official Gazette of Publication 29.12.2011–28157 http://www.tarim.gov.tr/Belgeler/eng/

Legislation/regulation_microbiological_criteria.pdf.q NACMCF Report - https://www.fsis.usda.gov/wps/wcm/connect/2ea3f473-cd12-4333-a28e-b2385454c967/NACMCF-Report-Process-Control-061015.pdf?

MOD=AJPERES.

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product. The increase in cell numbers during the early stages of cheesemanufacture could be at least partially attributed to the entrapment ofthe organisms during the curd-making process, but it is thought to bedue largely to the multiplication of the organisms. The following are afew examples which depicts how the behavior of indicator bacteriavaries in different cheeses and their relationship with the load of theindicator organisms in the raw-milk used to make these cheeses.

Many studies worldwide have examined the effect of making dif-ferent types of cheeses on the fate of indicator bacteria like coliforms,fecal coliforms, E. coli and Enterobacteriaceae that were present in theraw milk. Other studies examined the fate of pathogens like Shiga toxin-producing E. coli (STEC) that were seeded into milk prior to making thecheese. Examples of some of these studies conducted with various re-gional cheeses are presented below.

The fate of fecal coliforms in the making of the Spanish La Serena, acreamy spreadable cheese, was examined by Fernandez del Pozo et al.(1988). The study showed that fecal coliforms present in raw ewes’milkinitially rose during the early stages of manufacturing, but then de-clined to<10 CFU/g after aging for 60 days with a mean reduction of4.71 logs from the highest level found at day 2 to the end of ripening.Alessandria et al. (2010) studied the survival of fecal coliforms andcoliforms in Bolona, a pressed fresh cheese from the Cape Verde Islandof Sant Antao, which is made without starter culture or thermal treat-ment. The study showed that the milk and cheese samples taken fromthe two producers participating in the study had very different counts.The milk used by producer A contained 0.57 log10 CFU/ml of fecalcoliforms and 1.55 log10 CFU/ml of coliforms and the resulting cheesehad fecal coliform and coliform levels of 0.65 and 0.53 log10 CFU/g,respectively. In comparison, milk used by producer B had 1.40

log10 CFU/ml of fecal coliforms and 1.48 log10 CFU/ml of coliforms butthe resulting cheese had fecal coliform and coliform levels of 4.25 and4.29 log10 CFU/g, respectively. Bolona and other local cheeses areusually made in traditional stone huts with straw and matting roofs;water is scarce and so equipment is cleaned with whey. These peculiarproduction practices and conditions and the quality of raw milk usedfor production are thought to have accounted for the discrepancy incounts observed between the producers. Similarly, in the making ofCabrales, a Spanish blue cheese, made with raw bovine milk with addedovine and caprine milk, Nuñez (1978) found that the level of coliforms,both in the milk and in the finished cheeses varied between two smalldairies. In the first dairy, coliform levels of 4 log10 CFU/ml found in theraw milk increased to 4.9 log10 CFU/g in the curd, but gradually de-creased to 1.8 log10 CFU/g by day 15 and were not found by day 30.Conversely, at the second dairy, initial coliform levels of 4.5 log10 CFU/ml in the raw milk, increased to 6.1 log10 CFU/g in the curd, but de-creased more rapidly over the ripening period and were not found byday 15. The more rapid microbial decline in the second cheese wasprobably due to the lower pH and the higher salt content of the cheeseand it was also deemed by a taste panel to be of a “finer quality” thanthe first cheese. Zárate et al. (1997) examined Tenerife, a hard, Spanishraw caprine milk cheese traditionally made without added starter cul-tures and showed that the raw milk used can contain 4 log10 CFU/ml offecal coliforms but after aging the cheese for 60 days, the fecal coliformlevels in the interior and on the surface of the cheese were at< 1 and1.87 log10 CFU/g, respectively. Tornadijo et al. (2001) examined themaking of San Simon, a smoked, hard traditional Spanish cheese madewith raw bovine milk and showed that Enterobacteriaceae and coliformlevels of 2–3 log10 CFU/g present in the raw milk increased to the

Table 3Levels of coliforms reported in raw milk from various countries.

Study Country Milk type No of tested samples Levels (CFU/ml) % <100 CFU/ml % <10 CFU/ml

de Garnica et al. (2012) Spain ovine 196 (Winter) mean 4.57 log NRa NR191 (Spring) mean 3.69 log NR NR190 (Summer) mean 3.42 log NR NR174 (Fall) mean 3.55 log NR NR

D'Amico and Donnelly (2010) USA bovine 45 range < 1–68,336 96 69caprine 25 range < 1–10,668 84 51ovine 15 range 6–146 93 60

Pantoja et al. (2009) USA bovine 7275 range 0–1520 60 10D'Amico et al. (2008) USA bovine 67 mean 343 median 6.5 89 62

caprine 49 mean 212 median 4.5 76 55ovine 22 mean 24.6 median 2.8 92 75

Jayarao et al. (2004) USA bovine 126 range 5–4130 mean 70 50 NRCostello et al. (2003) USA bovine 833 range 1–90,000 mean 500 88 34Morgan et al. (2003) Greece caprine NR mean 5–6 log NR NR

Portugal caprine NR mean 6 log NR NRFrance caprine NR mean 2 log NR NR

Foschino et al. (2002) Italy caprine 60 mean 910 NR NRJayarao and Wang (1999) USA bovine 130 range 0–50,000 73.1 43.9Boor et al. (1988) USA bovine 855 range 14–290 77 30

aNR – not reported.

Table 4Levels of Escherichia coli reported in raw milk from various countries.

Study Country Milk type No of tested samples Level (CFU/ml) % <100 CFU/ml % <10 CFU/ml

Pyz-Łukasik et al., 2015 Poland bovine 50 range 5–110 NRa >76Ruusunen et al. (2013) Finland bovine 183 range 1–885 NR NRChye et al. (2004) Malaysia bovine 930 range 1900–15,000 NR 35.5 (negative)De Reu et al. (2004) Belgium bovine 143 NR 90.8 46.8Foschino et al. (2002) Italy caprine 60 mean 2.9 90 NRCoia et al. (2001) Scotland bovine 495 NR 96.8 NRDesmasures et al. (1997) France bovine 69 NR NR 80Desmasures and Guéguen, 1997 France bovine 60 NR NR 66.6 (negative)Rea et al. (1992) Ireland bovine 386 NR 95 80

aNR – not reported.

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highest level of 5–8 log10 CFU/g in the cheese after one week of ri-pening, but after 6 weeks of aging, a 1 to 5-log reduction for the interiorand a 2 to 4-log reduction on the surface was observed. Gerasi et al.(2003) examined the microbiological characteristics of Manura, a hard,raw ovine milk cheese made in Greece. After aging for 100 days, nocoliforms or Enterobacteriaceae were found either on the surface or inthe interior of the cheese. Log reductions for Enterobacteriaceae for theinterior and surface were 4.65 and 4.88, respectively, while for coli-forms, the interior log reduction was 3.73 and the surface log reductionwas 4.51.

The effects of ripening on bacterial reduction have also been re-ported for coliforms in Canestrato Pugliese, a hard Italian cheese madefrom raw ovine milk (Albenzio et al., 2001; De Pasquale et al., 2014);for fecal coliforms in Spanish Manchego, a hard cheese made from rawovine milk (Cabrezas et al., 2007); for Enterobacteriaceae and coliformsin Anevato, a soft, spreadable Greek cheese usually made from rawcaprine and/or ovine milk without any starter culture (Vassiliadis et al.,2009); for Enterobacteriaceae in Fior di Latte di Agerola, a high–-moisture Mozzarella cheese made from raw bovine milk (Coppola et al.,2006); for coliforms in Majorero, an artisanal hard cheese from theCanary Islands made with raw caprine milk (Fontecha et al., 1990); andfor coliforms in Roncal, a hard, raw ovine milk cheese from France(Ordonez et al., 1980).

The fate of Enterobacteriaceae and coliforms in raw-milk Feta cheesemade either with no added starter culture or with thermized milk withadded starter and aged for 60 days was examined by Vassiliadis et al.(2009). Enterobacteriaceae and coliforms reached their highest levels onday 1 but decreased by 4.76 and 5.24 log10 CFU/g, respectively, in thefinished raw-milk Feta cheese. The decline was even more rapid inthermized-milk Feta, where reductions of 6.19 and 6.27 log10 CFU/g,respectively, were observed from the highest level found on day 1 ofmanufacturing. In a limited study of six samples of Montasio cheese,Maifreni et al. (2013) demonstrated that Enterobacteriaceae counts de-clined by 1.87–2.84 log10 CFU/g in five of the six samples over the 120-day ripening period and E. coli was not found after 60 days of aging.Marino et al. (2003) also showed similar declines in coliform and fecalcoliform levels during ripening of Montasio cheese. Alegría et al. (2009)reported that neither E. coli nor other “undesirable microorganisms”were found in the batches of Casin, a Spanish raw-milk cheese, at theend of the 30-day ripening period. Dolci et al. (2008) studied the mi-crobial dynamics of Castelmagno and observed that no coliforms werefound at the end of the 90-day ripening period. To study the metabo-lomics of the ripening process, Piras et al. (2013) made four batches ofFiore Sardo, a raw ovine milk cheese, but three batches were made withautochthonous adjunct cultures and one batch was made with com-mercial starter to serve as control. At the onset of ripening for all fourbatches, Enterobacteriaceae and E. coli were present at levels of 6–7log10 CFU/g and 4–6 log10 CFU/g, respectively, but neither were foundafter 90 days of aging. The study also noted that higher levels of E. coliwere present in the control cheese, and the high level, which persistedduring most of the maturation, may have contributed to product texturedefect (irregularly shaped eyes). Similarly, Lück and Dunkeld (1981)found that high fecal coliform levels can be associated with texture andflavor defects in cheeses and that the defects were more prominent incheeses with higher levels. Manolopoulou et al. (2003) examined Fetacheeses produced by three different dairies in Greece and showed thatin two dairies, the highest levels of E coli were found at day 4 of agingand at day 16 at the third dairy. The extent of reduction during agingranged from 4.5 to 5 log10 CFU/g but the rate of reduction varied be-tween the dairies. At one dairy, E. coli was not found after aging for 60days, but at the two other dairies, E. coli was still found after 60 days,but not after 120 days.

The fate of coliforms in the making of Roquefort cheese was ex-amined by Devoyod et al. (1968) at two manufacturing plants inFrance. The raw milk used had initial coliform levels of 5 log10 CFU/ml,but after production and ripening for 10 days, the cheese surfaces had

coliform levels of 4 log10 CFU/g, a reduction of only 1 log; but at thecenter of the cheeses, a 4 to 5-log reduction was attained and only 1 to2-log10 CFU/g was found. Several other studies have also reported dif-ferences in indicator counts between the inside and the surfaces ofcheeses (Gerasi et al., 2003; Lioliou et al., 2001; Menéndez et al., 2001;Torres-Llanez et al., 2006). The source of the surface indicator popu-lation may be uncertain as they could be the original coliform popu-lation in the raw milk or perhaps have come from other sources duringprocessing, aging or both. However, since the center of the cheese is notexposed to possible cross contamination during handling and aging,counts from the centers of the cheeses are most likely representative ofthe original indicator population in the raw milk and therefore betterreflect the fate of these bacteria during the making of cheeses.

The impact of cheese ripening on the reduction of coliforms, E. coliand Enterobacteriaceae is dependent on many factors including usage ofstarter cultures, rate of fermentation, finished product composition,conditions used for aging, and even seasonal variations. For example,Psoni et al. (2003) studied Batzos, a semi-hard, low-fat, traditionalGreek cheese made from raw caprine milk and showed that initial levelsof Enterobacteriaceae and coliforms in the milk were similar at each ofthe seasons. However, products made in the spring had a 6.5-log re-duction for both group of microorganisms and none were found in thefinished product. In contrast, Batzos made in winter and summer onlyhad 4.5 to 5 log-reductions and both indicators were enumerated atlevels of 1.5–2 log10 CFU/g in the final product. The authors suggestedthat low storage temperatures, variations in salt concentration andantibacterial activity by lactic acid bacteria were some of the factorsthat affected the seasonal survival of these organisms (Psoni et al.,2003). A study by Nikolaou et al. (2002) also showed seasonal varia-tions in the reduction of indicator levels in Batzos. Comparing springand summer productions and calculating from the levels of indicatorsfound in the curd to the end of ripening, Enterobacteriaceae counts de-clined 1.97 log10 CFU/g and 3.49 log10 CFU/g, for spring and summer,respectively, and the coliform counts also had a sharper decline insummer (3.71 log10 CFU/g) as compared to spring (2.59 log10 CFU/g).Batzos made in the summer had roughly 0.5 to 1 log10 CFU/g higherinitial indicator levels than those made in the spring, but summerBatzos also had faster decline in counts during aging, resulting in lowerindicator levels in the finished product than spring Batzos. Similarseasonal effect of aging on the fate of Enterobacteriaceae and coliformindicators have also been reported for Greek Manouri (Lioliou et al.,2001) and Anevato cheeses (Hatzikamari et al., 1999) and for E. coli inItalian Caprino d’Aspromonte cheese (Caridi et al., 2003).

Aging is usually effective in reducing indicator flora levels incheeses. Tabla et al. (2016) reported a rapid decline in E. coli levelsduring the manufacturing and ripening of two raw ovine milk cheesevarieties, but also attributed the lack of finding E. coli in the finishedproduct to the low levels of E. coli that were present in the raw milkused for cheese manufacture. However, some finished cheeses haveoccasionally been found to contain elevated indicator levels. This canoccur if the raw milk used for production had high indicator flora le-vels, the cheese wasn't aged properly, or the product was processedunder insanitary conditions or a combination of these factors. For ex-ample, Prodromou et al. (2001) tested fresh Greek Orinotyri, a rawovine milk cheese and found initial counts of nearly 8 log10 CFU/g ofEnterobacteriaceae and coliforms. After aging for three months, the le-vels of Enterobacteriaceae and coliforms had declined by 3.01log10 CFU/g and 2.76 log10 CFU/g, respectively; however, counts re-mained at nearly 5 log10 CFU/g in the finished product and thereforethe cheese was deemed to pose public health concerns that affected themarketing of the product. The study further suggested that the higherlevels of indicators in Orinotyri cheese as compared to other fresh rawovine milk cheeses were due to the much higher pH of Orinotyri andthat lactic culture inoculation would have enhanced the acidifyingability and the proteolytic and lipolytic activity, which would haveimproved both the sanitary and technological qualities of the cheese.

M. Metz, et al. Food Microbiology 85 (2020) 103283

6

Table5

Leve

lsof

indicatorba

cteria

repo

rted

inch

eeses.

Stud

yCou

ntry

Milk

aChe

ese

Noof

samples

exam

ined

Bacteria

Noof

samples

with<

100CFU

/gNoof

samples

with

<10

CFU

/gOther

inform

ation

Broo

kset

al.(20

12)

USA

bovine

(R)

Che

ddar,B

lue,

Gou

da,

Gruye

reMon

tereyJack

35E.

coli

134

caprine(R

)Che

ddar,B

lue

2E.

coli

2ov

ine(R

)Rom

anoan

dothe

rs4

E.coli

13

O'Brien

etal.(20

09)

Irelan

dBo

vine

,ovine

,caprine(R

&P)

NRb,b

luemoldripe

ned

351

E.coli

NR

277

U.S

.FDA,2

016

USA

Variety

ofmilk

(R)

Variety

5698

subsam

ples

E.coli

97%

90%

82%

<3MPN

b/g

2.3%

>10

0MPN

/g0.7%

>11

00MPN

/gRan

tsiouet

al.(20

08)

Greece

NR

Feta

4E.

coli,

coliforms

13

Ayg

unet

al.(20

05)

Turkey

caprine,

bovine

(R)

Carra

50E.

coli

41NR

Bricke

ret

al.(20

05)

Mexico

Bovine

(R)

Men

nonite-style

8fecal

coliforms

00

Allha

d2-5log

Bovine

(P)

20

2Eleftheriado

uet

al.(20

02)

Cyp

rus

NR

Cyp

rusChe

ese

NR

E.coli

>18

0NR

Araùjoet

al.,20

02Brazil

Bovine

(P)

Brazilian

Soft

45fecal

coliforms

22

43ha

d>

103log 1

0MPN

/g

E.coli

NR

NR

Foun

din

97.7%

ofsamples

Men

énde

zet

al.(20

01)

Spain

Bovine

(R)

Tetilla

24E.

coli

0mean1.7

Papa

georgiou

etal.(19

98)

Greece

Cap

rine

,ovine

ormix

(R)

Pich

toga

loCha

nion

62co

liforms

NR

NR

15samples:r

ange

1–4log 1

0MPN

/g28

samples:

rang

e4–

5log 1

0MPN

/g19

samples:>

5log 1

0

MPN

/gE.

coli

NR

7ne

gative

26samples:r

ange

1–4log 1

0MPN

/g17

samples:

rang

e4–

5loglog 1

0MPN

/g12

samples:>

5log 1

0

MPN

/gKha

yatet

al.(19

88)

USA

Various

(P)

Various

256

coliforms

46%

NR

54%

2->

6log

Tzan

etak

iset

al.(19

87)

Greece

Ovine

(R)

Kop

anisti

50co

liforms

NR

3016

:1–3

log4:

3log

Martine

zMan

soan

dFe

rnan

dez-Sa

lgue

ro,19

78Sp

ain

ovine(R

)La

Serena

10co

liforms

1010

All10

werene

gative

Rosen

gren

etal.(20

10)

Swed

enNR(R

andP)

Includ

edFresh,

Che

vre

andCam

embe

rt55

(R)

E.coli

1936

sixat

103–1

05CFU

/g,o

neat

>10

5

96(P)

93ne

gative

93ne

gative

3freshch

eesesha

dE.

coli,

oneat

103-105

a R–raw;P

-pa

steu

rized.

bNR–no

trepo

rted

;MPN

–mostprob

able

numbe

r.

M. Metz, et al. Food Microbiology 85 (2020) 103283

7

Torres-Llanez et al. (2006) studied the production of artisanal MexicanFresco cheese and showed that fecal coliforms present at 1 log10 CFU/ml in milk rose to nearly 5 log10 CFU/g immediately after production.But, even after 10 days of ripening, the levels only dropped by 2 logsand were present in 100% of the products tested and therefore thecheeses were deemed to be of unhygienic quality. Similarly, Temelliet al. (2006) examined a Turkish white-brined cheese and showed thatthe finished product had mean counts of 2.84 and 3.45 log10 CFU/g ofEnterobacteriaceae and coliforms, respectively. Interestingly, this studytested the raw milk used prior to cheese-making and found it to be oflow hygienic quality as coliforms and Enterobacteriaceae levels exceeded6 log10 CFU/ml. As a result, the milk was pasteurized prior to use,which reduced the indicators levels to< 1 log10 CFU/ml. Thus, thefinding of indicators at 2 to 3 log10 CFU/g in cheeses made with pas-teurized milk was unexpected. However, follow-up inspection of theprocessing facility identified unsanitary conditions and contaminatedequipment, which were the likely causes for high levels of En-terobacteriaceae and coliforms in the finished products.

Several studies also examined the fate of pathogenic E. coli duringcheese-making and obtained similar results to that for indicator bac-teria. Vernozy-Rozand et al. (2005) examined the survival of Shigatoxin-producing E. coli (STEC) O157:H7 during the making and aging ofraw caprine milk cheese in France and showed that 10–1000 CFU/g ofO157:H7 seeded into the raw milk decreased to<1 log10 CFU/g afteraging, representing at least a 1 to 3-log reduction. Bachman and Spahr(1995) showed that many pathogens, including Campylobacter jejuni,Salmonella spp., Staphylococcus aureus, Yersinia enterocolitica and others,did not survive the processing steps used in the manufacturing of hardand semi-hard Swiss cheese varieties. Frank et al. (1978) inoculated500 CFU/ml of different enteropathogenic E. coli strains on the surfacesof Brick cheese blocks. The strains showed different growth rates thatvaried from 100 to 1000-fold and reached their highest levels 7 h afterinoculation. But the strains that showed a 1000-fold increase also hadthe fastest reduction, declining by 90–95% after 2 weeks of aging, whilethe strain that only increased by 100-fold, declined by only 50% in thesame period and remained at 4 log10 CFU/g even after 7 weeks. Similarstrain to strain variations were observed in the making of Camembertcheese (Frank et al., 1977), where some enteropathogenic E. coli as wellas generic E. coli strains seeded into milk at 2 log10 CFU/ml decreasedto< 10 CFU/g after production and ripening for one week. However,other strains took two weeks to decline to< 10 CFU/g and some eventook six and seven weeks to decrease to that level. These findings andothers (Cosciani-Cunico et al., 2014; Miszczycha et al. 2013, 2015;Schlesser et al., 2006) suggest that there are strain-to-strain variationsin growth and in bacterial persistence in cheeses and that some strainscan persist even after long ripening periods.

To summarize, indicator bacteria levels tend to increase during theinitial stages of cheese-making, but, as the pH decreases due to lactosefermentation, indicator levels decline and are generally further reducedduring aging, but the reduction levels vary with indicator bacteria andare also affected by the intrinsic characteristics of the cheese types.Exception are fresh cheeses, which are not aged or ripened, so there areno further reductions in the levels of indicator.

6. Levels of indicator bacteria found in cheeses

It is evident from the above studies that cheeses made under propersanitary conditions using raw milk of good quality and ripened ac-cordingly should be devoid of, or, at most, have low levels of indicatorbacteria. Consistent with that premise, in a report to the EuropeanCommission, Cogan and Rea (1996) compiled the results of many stu-dies on the microflora of various artisanal cheeses from Spain (Man-chego, Cabrales, La Serena, Majorero, Mahon); Italy (Fiore Sardo, CasuAxedu, Fontina, Toma, Mozzarella, Caciotta); France (Comte, Beaufort,‘Mont d’Or’) and Portugal (Serra Da Estrela, Serpa, Sao Jorge) andshowed that E. coli was seldom found in any of these cheeses.

Studies have also examined the levels of indicator bacteria in othertypes of cheeses worldwide (Table 5). Martinez Manso and Fernandez-Salguero (1978) examined the interior of La Serena cheese after it hadbeen aged for 60 days and did not find any coliforms. Aygun et al.(2005) looked at the microbiological quality of Carra, a traditionalTurkish semi-hard to hard cheese made from raw caprine milk orsometimes with raw bovine milk and ripened for three months in sealedearthenware jugs buried in the ground. Of the 50 samples analyzed,82% had E. coli counts of< 2 log10 CFU/g. Brooks et al. (2012) ex-amined 41 raw-milk cheeses made from bovine, caprine or ovine milkfrom different regions of the U.S. and found 95% (39/41) of samples tohave< 10 CFU/g of E. coli. The other two samples, a bovine and anovine milk cheese, contained E. coli at 10 and 30 CFU/g, respectively.O’Brien et al. (2009) tested two cheese samples per month for one yearfrom 15 dairy farms in Ireland. Analysis of the 351 cheeses, which weremade from both raw or pasteurized bovine, caprine or ovine milk,showed that 79% of the raw-milk cheeses had E. coli at< 10 CFU/g.Rantsiou et al. (2008) tested four Feta cheese samples made by fourdifferent Greek cheese-makers and found one sample to have<100CFU/g and the other three samples had< 10 CFU/g of coliforms and E.coli. In a review of microflora and characteristics of several greek cheesetypes, Litopoulou-Tzanetaki and Tzanetake (2011) found coliform le-vels to be low or negligible in Kasseri, a semi-hard cheese usually madewith raw ovine milk with 5–10% added caprine or bovine milk, and inMelichloro, a hard cheese made from raw ovine milk, coliforms werefound only at levels of 1 log10 CFU/g. Menéndez et al. (2001) tested 24samples of 2 - 3-week-old Tetilla, a Spanish cheese made with rawbovine milk, and found E. coli at mean levels of 1.72 log10 CFU/g.Rosengren et al. (2010) examined 151 samples (96 pasteurized-milkand 55 raw-milk cheeses) of fresh or short-time ripened cheese from 43farms in Sweden and found only 3% (3/96) of the pasteurized-milkcheeses having E. coli as compared to 34% (19/55) of the raw-milkcheeses. Some of these, including a fresh cheese made with pasteurizedmilk, had E. coli at levels of 3–5 log10 CFU/g and some of the raw milkcheeses had E. coli at> 5 log10 CFU/g. The study emphasized the im-portance of using quality raw milk and hygienic barriers to improveprocess control for raw-milk cheese production.

Most of the studies mentioned above were done using conventionalmicrobiological methods; however, others have used more sensitivemolecular methods to examine the microbial flora of cheeses. Feureret al. (2004) used 16S ribosomal DNA sequencing to study the micro-flora of French red-smeared cheeses made with raw or pasteurized milkand did not detect E. coli in either cheese type. Quigley et al. (2012)used DNA sequencing to examine the microbiota of 62 Irish artisanalcheeses, including soft, semi-hard or hard cheeses made from raw orpasteurized milk and did not detect E. coli in any of the samples.Ercolini et al. (2003) used 16S ribosomal DNA analysis to study thestructure and location of microbial communities in Stilton cheese, butE. coli was not among the microbial flora present in the 16 samplestested. Randazzo et al. (2006) used 16S ribosomal RNA analyses toexamine the bacterial diversity in artisanal and experimental PecorinoSiciliano cheese, but no E. coli was found in any of the five samplesexamined. Lastly, Martin-Platero et al. (2009) used PCR, 16S ribosomalRNA sequencing and a molecular subtyping method to examine themicrobial communities in Quesailla Arochena and Torta Arochena, twoSpanish farmhouse cheeses made from raw caprine milk, and foundHafnia and Serratia to be the predominant enterobacterial genera pre-sent in some of the cheeses, but E. coli was not found. Similar resultswere reported by Alegría et al. (2009), De Pasquale et al. (2014),Duthoit et al. (2005), Duthoit et al. (2003), Randazzo et al. (2010) andRandazzo et al. (2002). Not finding E. coli using these more sensitivemolecular methods maybe due to the low number of samples examinedby some of these studies however, others did look at larger number ofsamples and did not find E. coli therefore, the results suggest that E. coliare not normally present in cheeses and if present, are not at high le-vels.

M. Metz, et al. Food Microbiology 85 (2020) 103283

8

In some studies, cheeses were found to have high indicator counts,but most researchers attributed these findings to high indicator countsin the raw milk used to make the cheese or to insanitary processing. Forexample, Bricker et al. (2005) examined 10 samples of Mexican Men-nonite-style cheese from Chihuahua (eight made from raw milk and twofrom pasteurized milk). Fecal coliform levels of 2–5 log10 CFU/g, with amean of 3 log10 CFU/g, were found in the raw-milk cheeses, but nonewere found in the pasteurized-milk cheeses, leading the authors toconclude that eliminating the indicator flora from the raw milk wouldimprove the quality and safety of the cheeses. Guzman-Hernandez et al.(2016) analyzed 52 unpasteurized fresh Mexican cheeses for the pre-sence of pathogenic bacteria and indicators. Fresh cheeses or quesofresco are not aged, so do not benefit from further reduction of indicatorlevels via ripening therefore, have to made under hygienic conditions.The study found fecal coliforms and E. coli at levels of> 3 log10 CFU/gin 67% and 63% of the samples, respectively, and concluded that thepoor microbiological quality of these products were consistent with theunhygienic conditions they observed at the various production facilities(Guzman-Hernandez et al., 2016). Eleftheriadou et al. (2002) char-acterized the microbiological profile of 6500 dairy food samples inCyprus, including ice cream and traditional cheeses. The total numberof cheese samples tested was not stated, but over 180 cheese samplescontained E. coli at> 100 CFU/g, a level considered to be of sanitarysignificance by the authors and concluded that it was essential tomonitor for E. coli in cheese and to improve the production practices ofsmall cheese manufacturers. Tzanetakis et al. (1987) examined 50samples of Kopanisti, a soft, raw bovine milk cheese made in Greece,and did not find coliforms in 30 samples, but 16 samples had a mean of200 CFU/g and the other 4 samples had mean counts of 1500 CFU/g.Papageorgiou et al. (1998) analyzed 62 samples of Pichtogalo Chanion,a soft, spreadable Greek cheese typically made from raw ovine orcaprine milk or a mix of both. All the samples contained coliforms, with30% showing levels of 5–5.66 log10 CFU/g. E. coli was found in 88% ofthe samples, of which 46.8% had levels> 4 log10 CFU/g. The studyattributed these high indicator counts to the use of raw milk and to poorhygienic processing conditions in making the cheese. The study alsoshowed that high-quality Pichtogalo Chanion can be made with pas-teurized milk and therefore it is now being used for making this cheese.Khayat et al. (1988) tested 256 cheeses, comprised of 22 differentvarieties and purchased from different cities in California, and foundthat 46% had coliforms at< 100 CFU/g, but some of the samples hadcoliform counts that ranged from 2 to 7 log10 CFU/g. Considering thatall these cheeses were labeled as having been made with pasteurizedmilk, the authors concluded that insanitation or high incidences of postpasteurization contamination had probably occurred. Similarly, Araùjoet al. (2002) examined the levels of fecal coliforms in 45 samples ofthree pasteurized-milk soft-cheese brands sold in Rio de Janeiro, Brazil.The study found that 95% (43/45) of the samples had exceeded theBrazilian fecal coliform standard of 100 CFU/g. The levels found rangedfrom 1 to 6 log10 MPN/g. E. coli was also isolated from 97.7% of thesamples, including many enteropathogenic E. coli strains and therefore,the products were considered unfit for consumption. Since these pro-ducts were made with pasteurized milk, the authors attributed the highfecal coliform counts to improper pasteurization or to unhygienicpractices during cheese-making.

The results of these studies showed that cheeses that are made withgood quality raw milk and under sanitary conditions and properly agedwill not contain or only have low levels of indicator bacteria. Consistentwith that premise, between 2014 and 2016, the U.S. Food and DrugAdministration analyzed 1606 samples, comprised of 5698 subsamples,of raw-milk cheeses which were aged for a minimum of 60 days andfound that 90% of the subsamples had< 10 CFU/g of E. coli (U.S. FDA,2016). In the cited studies where high indicator levels were reported incheeses, most researchers have attributed these to the low quality ofraw milk used or to contamination during processing and emphasizedthe importance of maintaining proper sanitary conditions in cheese-

making and aging in order to produce a product of sanitary quality.

7. Conclusion

In summary, this paper reviewed the scientific literature on theprevalence and levels of indicator bacteria found in different types ofraw milk, showed examples of how the cheese-making and ripeningprocesses affects indicator bacteria levels and the levels of indicatorsthat have been found in various types of cheeses around the world. It isevident from these studies that indicators can be found in various typesof raw milk and E. coli are usually present at levels of< 100 CFU/g andin good-quality raw milk, the levels are at< 10 CFU/g. The studies alsoshowed that indicator bacteria found in milk will increase by severalfolds during the initial stages of cheese-making and except for freshcheeses that are not aged, the indicator levels decline by several log/gduring fermentation, aging and ripening of the cheese and often are notfound or are present at very low levels in the finished product.Consistent with these premises, many studies that surveyed the mi-crobiological quality of various cheese types worldwide showed thatindicator bacteria are either not present or found at levels that areoften<10 CFU/g or< 100 CFU/g, which are the common indicatorlimits for cheeses set by many countries. In those instances wherecheeses had high levels of indicator bacteria, most studies attributedthese to the use of poor-quality raw milk which contained high levels ofindicator flora or to unsanitary conditions, or both. The cited studiesalso showed that raw milk cheeses made from good-quality raw milkunder sanitary conditions, using good manufacturing practices andproperly aged, should not contain high levels of indicator bacteria.

Conflicts of interest

All three authors of this review works or had worked at the time forthe U.S. Food and Drug Administration and this review was prepared aspart of our official duties. None of the authors have any conflicts ofinterest with the subject of this paper.

Acknowledgements

The authors thank Mickey Parrish for critical reading of thismanuscript. The authors would also like to thank our colleagues M.Hayman and A. Datta for editorial assistance and A. Young, A. Keller, S.Trujillo, L. Batarseh, I. Son, and K. Nieves for their assistance intranslating some of the International regulations.

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