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

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DOI: 10.1016/j.fm.2019.103283

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 Food Microbiology 85 (2020) 103283

Contents lists available at ScienceDirect

Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Use of indicator bacteria for monitoring sanitary quality of raw milk cheeses T
– 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, USA
b
Division of Microbiology, Office of Regulatory Science, FDA, College Park, MD, 20740, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Many countries use Escherichia coli and coliforms as indicators of sanitary quality of foods and have set limits for
E. coli cheeses, including raw-milk cheeses. This paper reviewed the scientific literature for E. coli and coliform levels
Coliform that are found in different types of raw milk, the fate of indicators during the manufacturing and ripening of
Indicator different cheeses and the indicator levels that have been found in the finished cheeses. These studies from
Raw milk
worldwide showed that E. coli and coliforms are found in different types of raw milk but usually at < 100 CFU/
Cheese
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 often
increase in numbers, but the levels decline as the acidity from lactose fermentation decreases the pH. Except for
fresh cheeses that are not aged, indicator levels are further reduced by 2–3 log10 CFU/g or more, during the
ripening process. As a result, indicator levels in finished cheeses are often low and within the limits of < 10
or < 100 CFU/g set by many countries. The cited studies also show that raw milk cheeses that are made with
quality raw milk, under hygienic conditions and properly aged, should not contain high levels of indicator
bacteria 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 was
not identified until much later and even then, no methods were avail-
able to test for the pathogen. In 1885, Escherichia coli was identified and
found to be a normal inhabitant of the human intestine which lead to
the idea that it may be useful as an indicator of fecal contamination and
therefore, indirect evidence that pathogens may be present. The de-
tection of E. coli was initially based on fermentation of glucose but was
later changed to lactose which made it difficult to distinguish E. coli
from other enteric bacteria that also ferment lactose. This group of
lactose-fermenting bacteria, known as the coliform, is comprised of
several genera, including Enterobacter, Klebsiella, Citrobacter and
Escherichia. In the U.S. in 1914, the coliform group was adopted as an
indicator of fecal pollution to ensure the sanitary quality of drinking
water (Tortorello, 2003). However, since coliforms may not always
originate from fecal sources, the concept of fecal coliforms, which have
the ability to ferment lactose at 45 °C, was introduced to better reflect
fecal origin. Fecal coliforms are comprised mostly of E. coli, however,
some Klebsiella spp. also ferment lactose at the elevated temperature, as
a 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, fecal
coliform and E. coli all belong in the family Enterobactericeae, which is
also used by some countries as indicator for insanitation.
The initial concept of testing for indicator bacteria was for evidence
of fecal contamination, which served as an indirect evidence that pa-
thogens may be present and therefore, posed safety concerns (Smoot
and Pierson, 1997). However, numerous studies over the years showed
a lack of correlation between the presence of indicators and pathogens
(Martin et al., 2016; Miskimin et al., 1976), thereby lessening the
usefulness of indicators for assessing product safety. Furthermore, the
presence of certain coliforms in the environment has also diminished
the association of coliforms as a group as indicators of fecal con-
tamination (Boor et al., 2017; Trmčić et al., 2016). E. coli can also be
found in the environment, but its origin is most likely intestinal, and
therefore, it is still advocated as an appropriate indicator of fecal con-
tamination and a hygiene indicator (Smoot and Pierson, 1997; Trmčić
et al., 2016).
The concept of indicators has evolved over the years, from being an

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

https://doi.org/10.1016/j.fm.2019.103283
Received 26 November 2018; Received in revised form 6 June 2019; Accepted 30 July 2019
Available online 31 July 2019
0740-0020/ Published by Elsevier Ltd.
M. Metz, et al.
indicator of safety to become mostly an indicator of sanitary quality or
unsanitary conditions. As stated by the International Life Sciences
Institute (ILSI) (ILSI, 2011), “Indicator organisms are bacteria that are
used to provide evidence of poor hygiene, inadequate processing or post-
process contamination of foods … Their absence in food provides a degree of
assurance that the hygiene and food manufacturing processes have been
carried out appropriately, whereas their presence usually indicates that a
potential problem or failure in the process has occurred.“. Similar positions
have been stated by the International Commission on Microbiological
Specifications for Foods (ICMSF), which provides science-based gui-
dance to government and industry (ICMSF, 1986), by the National
Academy of Science (NAS, 1985), by a report of the National Advisory
Committee on Microbiological Criteria for Foods (NACMCF, 2018) and
by the WHO/FAO JEMRA report on “Shiga toxin-producing Escherichia
coli (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 sanitary
quality of cheeses. These hygiene indicators have been used by the
dairy industry to monitor lapses in sanitation, and post-processing
contamination 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 the
entire process, from the quality of the raw milk used, the cheese-making
process and aging, to the levels of indicators found in finished cheeses,
therefore, we conducted a comprehensive scientific literature review
on: 1) the levels of indicators found in raw milk, 2) the fate of indicators
during cheese-making, processing and aging and 3) the levels of in-
dicators reported in finished cheese products worldwide. For reference
purposes, the review also compiled tables showing the coliform and E.
coli limits, in both raw and pasteurized cheeses and dairy products, set
by countries worldwide. Some countries also use Enterobacteriaceae as
an indicator and some of the cited studies also tested for fecal coliforms
and 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 literature
search on cheeses, the same terms were used and in addition: cheese,
processing, ripening, aging, diversity, microflora and microbiota. The
citations used in this review are those that provided information on the
levels of coliforms, fecal coliforms and E. coli in raw milk, the impact of
cheese 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 of
cheeses 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 the
publications of the USDA Global Agricultural Information Network
(GAIN), performed internet searches on indicator limits for cheeses, and
communicated directly with regulators worldwide to obtain existing
microbial limits for raw milk, raw-milk cheeses or cheeses for the dif-
ferent countries. Some countries have set sampling criteria and limits
for coliforms in cheese (Table 1), which range from < 3 to 5000 colony
forming 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, range
from < 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 59
countries, which encompass 62% of the world's population (CIA World
Fact Book). Similarities in these indicator limits have also set relative
global parity and in doing so, facilitated trade and the import/export of
cheeses worldwide.
2
Food Microbiology 85 (2020) 103283
3. Prevalence and levels of coliforms in raw milk
Coliforms can be found in fecal sources, but some genera are also
present in the environment, so are often used as indicators of in-
sanitation rather than fecal contamination. Coliforms are not inherent
microflora 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). Indicator
bacteria are usually enumerated using direct methods, like a plate count
(colony forming units [CFU]/g) or indirect method, such as most
probable number (MPN), which is a statistical estimation of the num-
bers of cells present (MPN/g). Pantoja et al. (2009) examined 7275
samples of raw bovine milk from 16 farms in Wisconsin over a one year
period and found coliform levels that ranged from not found to
1520 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 bovine
milk from 5 farms in Vermont and showed that 89% had coliform levels
of < 100 CFU/ml, with 62% showing < 10 CFU/ml and < 10% with
levels at 1001–10,000 CFU/ml. Of the other milk types tested, 76% of
the 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 22
samples of ovine milk analyzed, 92% had coliforms at < 100 CFU/ml,
with 75% at < 10 CFU/ml and < 10% had coliforms in the range of
1001–10,000 CFU/ml. This Vermont study was followed up two years
later by the analysis of 85 samples from 21 farms (12 cow, 5 goat and 4
sheep) but the results were similar in that 96% of the bovine milk
samples had coliforms at < 100 CFU/ml, 69% at < 10 CFU/ml and
only < 10% exceeded 1000 CFU/ml. Analysis of caprine milk samples
showed 84% contained coliforms at < 100 CFU/ml, 51% at < 10 CFU/
ml and none had > 1000 CFU/ml. With respect to the ovine milk
samples, 93% had < 100 CFU/ml, 60% had < 10 CFU/ml and < 10%
exceeded 1000 CFU/ml (D'Amico and Donnelly, 2010). Other studies
analyzed bulk tank raw-milk samples taken from different dairy herds
in different States and found similar levels of coliforms (Boor et al.,
1988; Jayarao et al., 2004; Jayarao and Wang, 1999) (Table 3) and one
study also found a significant association between coliform levels and
herd sizes with smaller herds often showing lower counts (Jayarao
et al., 2004). Costello et al. (2003) tested 833 samples of bulk tank milk
over 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 the
geometric mean was 14 CFU/ml. From these studies, it can be surmised
that between 50 and 96% of the U.S raw-milk samples had coliforms
at < 100 CFU/ml and 10–75% of samples were at levels of < 10 CFU/
ml.
Analyses of samples from other countries showed that raw caprine
and ovine milks can sometimes have very high coliform counts. For
example, de Garnica et al. (2012) tested 751 bulk tank samples of ovine
milk in Spain, collected over one year and showed mean coliform
counts ranging from 3.42 to 4.57 log10 CFU/ml throughout the four
seasons. Compared to the study by Jayarao et al. (2004), the U.S. bulk
tank bovine milk samples only had a mean coliform count of 70 CFU/
ml, thus the ovine milk had much higher coliform counts. Differences in
milking facilities, production and practices, such as the absence of teat
washing before milking are a few of the factors that maybe contributing
to higher coliform counts in ovine milk. Morgan et al. (2003) tested raw
caprine milk sampled from reception tanks at seven small and medium
dairy plants in Greece, Portugal and France and found that samples
from Greece and Portugal had the highest coliform levels with means of
5–6 log10 CFU/ml. But, those from French dairies only had coliforms at
2 log10 CFU/ml. The study noted that France had implemented
M. Metz, et al. Food Microbiology 85 (2020) 103283

Table 1
Coliform limits set for cheeses by different countries.
Country Cheese type Sampling plana Comments

n c m M

c
Brazil Low moisture 5 2 100 500
Medium moisture 5 2 500 1000
High moisture 5 2 1000 5000
Very high moisture 5 2 500 5000
46–55% moisture 5 2 1000 5000
Dried 5 2 <3 10
Grating 5 2 100 1000
Pasteurized, processed 5 2 <3 10
Very high moisture - condiments, herbs, other ingredients 5 2 50 100
Low-medium moisture - condiments, herbs, other ingredients 5 2 100 500
Chinad 5 2 100 1000
Mexicoe Fresh 100 fecal coliforms
Aged 50
Processed NBTb
Philippinesf products, pasteurized 5 1 11 1000
Processed, Spread 5 1 10 100
g
Peru Processed 5 1 10 100
Non-matured 5 2 500 1000
Matured 5 2 200 1000
Russia and EACUb, h Absence
U.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 be
rejected 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 Microbiological
Criteria for Foods.
c
ResolucÃo-RDC Nº 12, de 02 de Janeiro de 2001, Anexo-Regulamento técnico sobre os padrÕes microbiológicos para alimenots
d
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.

intensive breeding systems, where modern techniques of livestock
breeding were used to produce large herds of improved breeds and
therefore concluded that improved farming and milking systems are
needed in Greece and Portugal to improve the hygiene of the caprine
milk produced. Foschino et al. (2002) found coliform levels in caprine
milk in Italy to be similar to those found by Morgan et al. (2003) in
France and also noted that coliform levels were significantly higher in
milk from farms which did not have refrigeration.
These studies from the various countries showed that coliforms are
found in different types of raw milk with most samples
containing < 100 CFU/ml, regardless of the lactating species or the
country. Occasionally, some samples contained coliform levels of up to
6 log10 CFU/ml, but this does not appear to be the norm. Also, the
coliform levels varied greatly, perhaps due to seasonal and geographic
variations, 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 been
used as an indicator of both insanitation and fecal contamination. Many
studies worldwide have surveyed raw milk samples for the presence
and 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 in
Finland and found E. coli in 45% of the samples and the levels ranged
from 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 direct
points of sale in Poland and found that 76% had no E. coli and the
remaining 24% of the samples had E. coli at levels ranging from 5 to
110 CFU/ml. A survey of 495 raw milk samples taken over a 24-month
period in South-East Scotland found that 96.8% had E. coli at < 100
CFU/ml (Coia et al., 2001). Desmasures and Guéguen (1997) tested
good-quality milk samples (defined by the authors as milk from farms
that met a standard of < 50,000 CFU/ml total count and < 250,000
somatic cells/ml) over a two year-period in France and found that less
than one third of the samples contained E. coli. Desmasures et al. (1997)
tested 69 raw milk samples collected from bulk tanks in the Camembert
region 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 from
Ireland showed that approximately 80% of the 386 raw milk samples
collected 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 from
bulk tanks in Belgium showed that 90.8% had E. coli at ≤100 CFU/ml
and 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 and
found that 64.5% of the samples contained E. coli. The mean counts,
which varied among the regions, ranged from 1900 - 15,000 CFU/ml
and the authors concluded that the milk samples that had high E. coli
counts were most likely produced under unsanitary conditions.
Some studies also examined the prevalence of E. coli in other milk
types. Foschino et al. (2002) tested 60 samples of raw caprine milk in
Italy and showed that 10% of the samples had no E. coli and in the other
90% of the samples, the counts ranged from 0.5 to 7.9 CFU/ml with a

3
M. Metz, et al. Food Microbiology 85 (2020) 103283

Table 2
Generic Escherichia coli limits set for dairy foods by different countries.
Country Cheese product Sampling plana Comments

n c m M

c
Argentina All types 5 0 <3
Australia and New Zealandd All cheese 5 1 10 100 Unified Standards for Australia and New Zealand
Canadae Pasteurized 5 2 100 2000
Raw 5 2 500 2000
EUb,f Pasteurized 5 2 100 1000 Process hygiene criterion
Gulf Cooperation Councilg Hard and semi-hard 5 0 0
Processed, in non-metal containers 5 0 0
Soft, pasteurized milk 5 1 10 100
Hong Kongh All cheeses excluding those ripened with Hafnia > 100
alvei or Proteus vulgaris and raw milk cheeses
Indiai Processed and spreads 0 Absent
All others 5 0 > 10
j
New Zealand Raw milk used to make raw milk products < 100 Code of Practice
Peruk Non-matured 5 1 3 10
Philippinesl Cheese products, pasteurized 5 1 11 110 MPN/g
South Africam All dairy products 0 Absence
South Korean Processed 5 2 <3 100
Natural or raw 5 1 10 100
Taiwano Cheese 100
Turkeyp 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 process
United States DOD/ Pasteurized < 10 > 100 non-routine sampling; for DOD only
NACMCFb,q

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 be
rejected 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.

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 as
high as 15,000 CFU/ml (Chye et al., 2004), but most of the studies
showed 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 levels
in 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 contains
coliforms and/or E. coli, it is typically at low levels. However, this does
not necessarily translate into a presence or corresponding levels of
coliforms 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 involved
in the production of hundreds of different raw-milk cheeses and the
great diversity of intrinsic characteristics of cheeses makes it difficult to
generalize the fate of indicator organisms in the various cheese types.
Nevertheless, from the large amount of data collected over the years
from different kinds of raw milk cheeses, a common scenario emerges.
During the initial milk ripening process there is an increase in the
numbers of the indicator organisms followed by a decline of the num-
bers as manufacturing proceeds and throughout the ripening of the

4
M. Metz, et al. Food Microbiology 85 (2020) 103283

Table 3
Levels 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

a
de Garnica et al. (2012) Spain ovine 196 (Winter) mean 4.57 log NR NR
191 (Spring) mean 3.69 log NR NR
190 (Summer) mean 3.42 log NR NR
174 (Fall) mean 3.55 log NR NR
D'Amico and Donnelly (2010) USA bovine 45 range < 1–68,336 96 69
caprine 25 range < 1–10,668 84 51
ovine 15 range 6–146 93 60
Pantoja et al. (2009) USA bovine 7275 range 0–1520 60 10
D'Amico et al. (2008) USA bovine 67 mean 343 median 6.5 89 62
caprine 49 mean 212 median 4.5 76 55
ovine 22 mean 24.6 median 2.8 92 75
Jayarao et al. (2004) USA bovine 126 range 5–4130 mean 70 50 NR
Costello et al. (2003) USA bovine 833 range 1–90,000 mean 500 88 34
Morgan et al. (2003) Greece caprine NR mean 5–6 log NR NR
Portugal caprine NR mean 6 log NR NR
France caprine NR mean 2 log NR NR
Foschino et al. (2002) Italy caprine 60 mean 910 NR NR
Jayarao and Wang (1999) USA bovine 130 range 0–50,000 73.1 43.9
Boor et al. (1988) USA bovine 855 range 14–290 77 30

a
NR – not reported.

product. The increase in cell numbers during the early stages of cheese
manufacture could be at least partially attributed to the entrapment of
the organisms during the curd-making process, but it is thought to be
due largely to the multiplication of the organisms. The following are a
few examples which depicts how the behavior of indicator bacteria
varies in different cheeses and their relationship with the load of the
indicator 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 the
raw milk. Other studies examined the fate of pathogens like Shiga toxin-
producing E. coli (STEC) that were seeded into milk prior to making the
cheese. 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, a
creamy spreadable cheese, was examined by Fernandez del Pozo et al.
(1988). The study showed that fecal coliforms present in raw ewes’ milk
initially rose during the early stages of manufacturing, but then de-
clined to < 10 CFU/g after aging for 60 days with a mean reduction of
4.71 logs from the highest level found at day 2 to the end of ripening.
Alessandria et al. (2010) studied the survival of fecal coliforms and
coliforms in Bolona, a pressed fresh cheese from the Cape Verde Island
of Sant Antao, which is made without starter culture or thermal treat-
ment. The study showed that the milk and cheese samples taken from
the two producers participating in the study had very different counts.
The milk used by producer A contained 0.57 log10 CFU/ml of fecal
coliforms and 1.55 log10 CFU/ml of coliforms and the resulting cheese
had 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 but
the resulting cheese had fecal coliform and coliform levels of 4.25 and
4.29 log10 CFU/g, respectively. Bolona and other local cheeses are
usually made in traditional stone huts with straw and matting roofs;
water is scarce and so equipment is cleaned with whey. These peculiar
production practices and conditions and the quality of raw milk used
for production are thought to have accounted for the discrepancy in
counts observed between the producers. Similarly, in the making of
Cabrales, a Spanish blue cheese, made with raw bovine milk with added
ovine and caprine milk, Nuñez (1978) found that the level of coliforms,
both in the milk and in the finished cheeses varied between two small
dairies. In the first dairy, coliform levels of 4 log10 CFU/ml found in the
raw 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 by
day 15. The more rapid microbial decline in the second cheese was
probably due to the lower pH and the higher salt content of the cheese
and it was also deemed by a taste panel to be of a “finer quality” than
the first cheese. Zárate et al. (1997) examined Tenerife, a hard, Spanish
raw caprine milk cheese traditionally made without added starter cul-
tures and showed that the raw milk used can contain 4 log10 CFU/ml of
fecal coliforms but after aging the cheese for 60 days, the fecal coliform
levels in the interior and on the surface of the cheese were at < 1 and
1.87 log10 CFU/g, respectively. Tornadijo et al. (2001) examined the
making of San Simon, a smoked, hard traditional Spanish cheese made
with raw bovine milk and showed that Enterobacteriaceae and coliform
levels of 2–3 log10 CFU/g present in the raw milk increased to the

Table 4
Levels 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

a
Pyz-Łukasik et al., 2015 Poland bovine 50 range 5–110 NR > 76
Ruusunen et al. (2013) Finland bovine 183 range 1–885 NR NR
Chye 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 46.8
Foschino et al. (2002) Italy caprine 60 mean 2.9 90 NR
Coia et al. (2001) Scotland bovine 495 NR 96.8 NR
Desmasures et al. (1997) France bovine 69 NR NR 80
Desmasures and Guéguen, 1997 France bovine 60 NR NR 66.6 (negative)
Rea et al. (1992) Ireland bovine 386 NR 95 80

a
NR – not reported.

5
M. Metz, et al.
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 interior
and 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, no
coliforms or Enterobacteriaceae were found either on the surface or in
the interior of the cheese. Log reductions for Enterobacteriaceae for the
interior and surface were 4.65 and 4.88, respectively, while for coli-
forms, the interior log reduction was 3.73 and the surface log reduction
was 4.51.
The effects of ripening on bacterial reduction have also been re-
ported for coliforms in Canestrato Pugliese, a hard Italian cheese made
from raw ovine milk (Albenzio et al., 2001; De Pasquale et al., 2014);
for fecal coliforms in Spanish Manchego, a hard cheese made from raw
ovine milk (Cabrezas et al., 2007); for Enterobacteriaceae and coliforms
in Anevato, a soft, spreadable Greek cheese usually made from raw
caprine 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 the
Canary Islands made with raw caprine milk (Fontecha et al., 1990); and
for 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 cheese
made either with no added starter culture or with thermized milk with
added starter and aged for 60 days was examined by Vassiliadis et al.
(2009). Enterobacteriaceae and coliforms reached their highest levels on
day 1 but decreased by 4.76 and 5.24 log10 CFU/g, respectively, in the
finished raw-milk Feta cheese. The decline was even more rapid in
thermized-milk Feta, where reductions of 6.19 and 6.27 log10 CFU/g,
respectively, were observed from the highest level found on day 1 of
manufacturing. 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 fecal
coliform 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 the
end of the 30-day ripening period. Dolci et al. (2008) studied the mi-
crobial dynamics of Castelmagno and observed that no coliforms were
found 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 of
Fiore Sardo, a raw ovine milk cheese, but three batches were made with
autochthonous adjunct cultures and one batch was made with com-
mercial starter to serve as control. At the onset of ripening for all four
batches, Enterobacteriaceae and E. coli were present at levels of 6–7
log10 CFU/g and 4–6 log10 CFU/g, respectively, but neither were found
after 90 days of aging. The study also noted that higher levels of E. coli
were present in the control cheese, and the high level, which persisted
during most of the maturation, may have contributed to product texture
defect (irregularly shaped eyes). Similarly, Lück and Dunkeld (1981)
found that high fecal coliform levels can be associated with texture and
flavor defects in cheeses and that the defects were more prominent in
cheeses with higher levels. Manolopoulou et al. (2003) examined Feta
cheeses produced by three different dairies in Greece and showed that
in two dairies, the highest levels of E coli were found at day 4 of aging
and at day 16 at the third dairy. The extent of reduction during aging
ranged 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 60
days, 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 in
France. 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
6
Food Microbiology 85 (2020) 103283
coliform levels of 4 log10 CFU/g, a reduction of only 1 log; but at the
center of the cheeses, a 4 to 5-log reduction was attained and only 1 to
2-log10 CFU/g was found. Several other studies have also reported dif-
ferences in indicator counts between the inside and the surfaces of
cheeses (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 during
processing, aging or both. However, since the center of the cheese is not
exposed to possible cross contamination during handling and aging,
counts from the centers of the cheeses are most likely representative of
the original indicator population in the raw milk and therefore better
reflect the fate of these bacteria during the making of cheeses.
The impact of cheese ripening on the reduction of coliforms, E. coli
and Enterobacteriaceae is dependent on many factors including usage of
starter 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, traditional
Greek cheese made from raw caprine milk and showed that initial levels
of Enterobacteriaceae and coliforms in the milk were similar at each of
the seasons. However, products made in the spring had a 6.5-log re-
duction for both group of microorganisms and none were found in the
finished product. In contrast, Batzos made in winter and summer only
had 4.5 to 5 log-reductions and both indicators were enumerated at
levels of 1.5–2 log10 CFU/g in the final product. The authors suggested
that low storage temperatures, variations in salt concentration and
antibacterial activity by lactic acid bacteria were some of the factors
that 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 spring
and summer productions and calculating from the levels of indicators
found 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 in
summer (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 higher
initial indicator levels than those made in the spring, but summer
Batzos also had faster decline in counts during aging, resulting in lower
indicator levels in the finished product than spring Batzos. Similar
seasonal effect of aging on the fate of Enterobacteriaceae and coliform
indicators have also been reported for Greek Manouri (Lioliou et al.,
2001) and Anevato cheeses (Hatzikamari et al., 1999) and for E. coli in
Italian Caprino d’Aspromonte cheese (Caridi et al., 2003).
Aging is usually effective in reducing indicator flora levels in
cheeses. Tabla et al. (2016) reported a rapid decline in E. coli levels
during the manufacturing and ripening of two raw ovine milk cheese
varieties, but also attributed the lack of finding E. coli in the finished
product to the low levels of E. coli that were present in the raw milk
used for cheese manufacture. However, some finished cheeses have
occasionally been found to contain elevated indicator levels. This can
occur if the raw milk used for production had high indicator flora le-
vels, the cheese wasn't aged properly, or the product was processed
under insanitary conditions or a combination of these factors. For ex-
ample, Prodromou et al. (2001) tested fresh Greek Orinotyri, a raw
ovine milk cheese and found initial counts of nearly 8 log10 CFU/g of
Enterobacteriaceae and coliforms. After aging for three months, the le-
vels of Enterobacteriaceae and coliforms had declined by 3.01
log10 CFU/g and 2.76 log10 CFU/g, respectively; however, counts re-
mained at nearly 5 log10 CFU/g in the finished product and therefore
the cheese was deemed to pose public health concerns that affected the
marketing of the product. The study further suggested that the higher
levels of indicators in Orinotyri cheese as compared to other fresh raw
ovine milk cheeses were due to the much higher pH of Orinotyri and
that lactic culture inoculation would have enhanced the acidifying
ability and the proteolytic and lipolytic activity, which would have
improved both the sanitary and technological qualities of the cheese.
 M. Metz, et al.

Table 5
Levels of indicator bacteria reported in cheeses.
Study Country Milka Cheese No of samples Bacteria No of samples No of samples with Other information
examined with < 100 CFU/g < 10 CFU/g

Brooks et al. (2012) USA bovine (R) Cheddar, Blue, Gouda, 35 E. coli 1 34
Gruyere Monterey Jack
caprine (R) Cheddar, Blue 2 E. coli 2
ovine (R) Romano and others 4 E. coli 1 3
O'Brien et al. (2009) Ireland Bovine, ovine, NRb, blue mold ripened 351 E. coli NR 277
caprine (R&P)
U. S. FDA, 2016 USA Variety of milk Variety 5698 subsamples E. coli 97% 90% 82% < 3 MPNb/g
(R) 2.3% > 100 MPN/g
0.7% > 1100 MPN/g
Rantsiou et al. (2008) Greece NR Feta 4 E. coli, 1 3
coliforms
Aygun et al. (2005) Turkey caprine, bovine Carra 50 E. coli 41 NR
(R)
Bricker et al. (2005) Mexico Bovine (R) Mennonite-style 8 fecal 0 0 All had 2-5 log
Bovine (P) 2 coliforms 0 2

7
Eleftheriadou et al. (2002) Cyprus NR Cyprus Cheese NR E. coli > 180 NR
Araùjo et al., 2002 Brazil Bovine (P) Brazilian Soft 45 fecal 2 2 43 had > 103 log10 MPN/g
coliforms
E. coli NR NR Found in 97.7% of samples
Menéndez et al. (2001) Spain Bovine (R) Tetilla 24 E. coli 0 mean 1.7
Papageorgiou et al. (1998) Greece Caprine, ovine or Pichtogalo Chanion 62 coliforms NR NR 15 samples: range 1–4 log10 MPN/g 28 samples:
mix (R) range 4–5 log10 MPN/g 19 samples: > 5 log10
MPN/g
E. coli NR 7 negative 26 samples: range 1–4 log10 MPN/g 17 samples:
range 4–5 log log10 MPN/g 12 samples: > 5 log10
MPN/g
Khayat et al. (1988) USA Various (P) Various 256 coliforms 46% NR 54% 2 - > 6 log
Tzanetakis et al. (1987) Greece Ovine (R) Kopanisti 50 coliforms NR 30 16: 1–3 log 4: 3 log
Martinez Manso and Spain ovine (R) La Serena 10 coliforms 10 10 All 10 were negative
Fernandez-Salguero, 1978
Rosengren et al. (2010) Sweden NR (R and P) Included Fresh, Chevre 55 (R) E. coli 19 36 six at 103–105 CFU/g, one at > 105
and Camembert 96 (P) 93 negative 93 negative 3 fresh cheeses had E. coli, one at 103 -105

a
R – raw; P - pasteurized.
b
NR – not reported; MPN – most probable number.
Food Microbiology 85 (2020) 103283
M. Metz, et al.
Torres-Llanez et al. (2006) studied the production of artisanal Mexican
Fresco 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 logs
and were present in 100% of the products tested and therefore the
cheeses were deemed to be of unhygienic quality. Similarly, Temelli
et al. (2006) examined a Turkish white-brined cheese and showed that
the finished product had mean counts of 2.84 and 3.45 log10 CFU/g of
Enterobacteriaceae and coliforms, respectively. Interestingly, this study
tested the raw milk used prior to cheese-making and found it to be of
low hygienic quality as coliforms and Enterobacteriaceae levels exceeded
6 log10 CFU/ml. As a result, the milk was pasteurized prior to use,
which reduced the indicators levels to < 1 log10 CFU/ml. Thus, the
finding of indicators at 2 to 3 log10 CFU/g in cheeses made with pas-
teurized milk was unexpected. However, follow-up inspection of the
processing facility identified unsanitary conditions and contaminated
equipment, 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 during
cheese-making and obtained similar results to that for indicator bac-
teria. Vernozy-Rozand et al. (2005) examined the survival of Shiga
toxin-producing E. coli (STEC) O157:H7 during the making and aging of
raw caprine milk cheese in France and showed that 10–1000 CFU/g of
O157:H7 seeded into the raw milk decreased to < 1 log10 CFU/g after
aging, 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 hard
and semi-hard Swiss cheese varieties. Frank et al. (1978) inoculated
500 CFU/ml of different enteropathogenic E. coli strains on the surfaces
of Brick cheese blocks. The strains showed different growth rates that
varied from 100 to 1000-fold and reached their highest levels 7 h after
inoculation. But the strains that showed a 1000-fold increase also had
the fastest reduction, declining by 90–95% after 2 weeks of aging, while
the strain that only increased by 100-fold, declined by only 50% in the
same period and remained at 4 log10 CFU/g even after 7 weeks. Similar
strain to strain variations were observed in the making of Camembert
cheese (Frank et al., 1977), where some enteropathogenic E. coli as well
as generic E. coli strains seeded into milk at 2 log10 CFU/ml decreased
to < 10 CFU/g after production and ripening for one week. However,
other strains took two weeks to decline to < 10 CFU/g and some even
took six and seven weeks to decrease to that level. These findings and
others (Cosciani-Cunico et al., 2014; Miszczycha et al. 2013, 2015;
Schlesser et al., 2006) suggest that there are strain-to-strain variations
in growth and in bacterial persistence in cheeses and that some strains
can persist even after long ripening periods.
To summarize, indicator bacteria levels tend to increase during the
initial stages of cheese-making, but, as the pH decreases due to lactose
fermentation, indicator levels decline and are generally further reduced
during aging, but the reduction levels vary with indicator bacteria and
are also affected by the intrinsic characteristics of the cheese types.
Exception are fresh cheeses, which are not aged or ripened, so there are
no 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 proper
sanitary conditions using raw milk of good quality and ripened ac-
cordingly should be devoid of, or, at most, have low levels of indicator
bacteria. Consistent with that premise, in a report to the European
Commission, 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, Casu
Axedu, Fontina, Toma, Mozzarella, Caciotta); France (Comte, Beaufort,
‘Mont d’Or’) and Portugal (Serra Da Estrela, Serpa, Sao Jorge) and
showed that E. coli was seldom found in any of these cheeses.
8
Food Microbiology 85 (2020) 103283
Studies have also examined the levels of indicator bacteria in other
types of cheeses worldwide (Table 5). Martinez Manso and Fernandez-
Salguero (1978) examined the interior of La Serena cheese after it had
been aged for 60 days and did not find any coliforms. Aygun et al.
(2005) looked at the microbiological quality of Carra, a traditional
Turkish semi-hard to hard cheese made from raw caprine milk or
sometimes with raw bovine milk and ripened for three months in sealed
earthenware 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 milk
from different regions of the U.S. and found 95% (39/41) of samples to
have < 10 CFU/g of E. coli. The other two samples, a bovine and an
ovine 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 year
from 15 dairy farms in Ireland. Analysis of the 351 cheeses, which were
made 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 four
different Greek cheese-makers and found one sample to have < 100
CFU/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 cheese
types, Litopoulou-Tzanetaki and Tzanetake (2011) found coliform le-
vels to be low or negligible in Kasseri, a semi-hard cheese usually made
with raw ovine milk with 5–10% added caprine or bovine milk, and in
Melichloro, a hard cheese made from raw ovine milk, coliforms were
found only at levels of 1 log10 CFU/g. Menéndez et al. (2001) tested 24
samples of 2 - 3-week-old Tetilla, a Spanish cheese made with raw
bovine milk, and found E. coli at mean levels of 1.72 log10 CFU/g.
Rosengren et al. (2010) examined 151 samples (96 pasteurized-milk
and 55 raw-milk cheeses) of fresh or short-time ripened cheese from 43
farms in Sweden and found only 3% (3/96) of the pasteurized-milk
cheeses having E. coli as compared to 34% (19/55) of the raw-milk
cheeses. Some of these, including a fresh cheese made with pasteurized
milk, had E. coli at levels of 3–5 log10 CFU/g and some of the raw milk
cheeses had E. coli at > 5 log10 CFU/g. The study emphasized the im-
portance of using quality raw milk and hygienic barriers to improve
process control for raw-milk cheese production.
Most of the studies mentioned above were done using conventional
microbiological methods; however, others have used more sensitive
molecular methods to examine the microbial flora of cheeses. Feurer
et al. (2004) used 16S ribosomal DNA sequencing to study the micro-
flora of French red-smeared cheeses made with raw or pasteurized milk
and did not detect E. coli in either cheese type. Quigley et al. (2012)
used DNA sequencing to examine the microbiota of 62 Irish artisanal
cheeses, including soft, semi-hard or hard cheeses made from raw or
pasteurized milk and did not detect E. coli in any of the samples.
Ercolini et al. (2003) used 16S ribosomal DNA analysis to study the
structure and location of microbial communities in Stilton cheese, but
E. coli was not among the microbial flora present in the 16 samples
tested. Randazzo et al. (2006) used 16S ribosomal RNA analyses to
examine the bacterial diversity in artisanal and experimental Pecorino
Siciliano cheese, but no E. coli was found in any of the five samples
examined. Lastly, Martin-Platero et al. (2009) used PCR, 16S ribosomal
RNA sequencing and a molecular subtyping method to examine the
microbial communities in Quesailla Arochena and Torta Arochena, two
Spanish farmhouse cheeses made from raw caprine milk, and found
Hafnia and Serratia to be the predominant enterobacterial genera pre-
sent in some of the cheeses, but E. coli was not found. Similar results
were reported by Alegría et al. (2009), De Pasquale et al. (2014),
Duthoit et al. (2005), Duthoit et al. (2003), Randazzo et al. (2010) and
Randazzo et al. (2002). Not finding E. coli using these more sensitive
molecular methods maybe due to the low number of samples examined
by some of these studies however, others did look at larger number of
samples and did not find E. coli therefore, the results suggest that E. coli
are not normally present in cheeses and if present, are not at high le-
vels.
M. Metz, et al.
In some studies, cheeses were found to have high indicator counts,
but most researchers attributed these findings to high indicator counts
in the raw milk used to make the cheese or to insanitary processing. For
example, Bricker et al. (2005) examined 10 samples of Mexican Men-
nonite-style cheese from Chihuahua (eight made from raw milk and two
from pasteurized milk). Fecal coliform levels of 2–5 log10 CFU/g, with a
mean of 3 log10 CFU/g, were found in the raw-milk cheeses, but none
were found in the pasteurized-milk cheeses, leading the authors to
conclude that eliminating the indicator flora from the raw milk would
improve 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 queso
fresco are not aged, so do not benefit from further reduction of indicator
levels via ripening therefore, have to made under hygienic conditions.
The study found fecal coliforms and E. coli at levels of > 3 log10 CFU/g
in 67% and 63% of the samples, respectively, and concluded that the
poor microbiological quality of these products were consistent with the
unhygienic 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 in
Cyprus, including ice cream and traditional cheeses. The total number
of cheese samples tested was not stated, but over 180 cheese samples
contained E. coli at > 100 CFU/g, a level considered to be of sanitary
significance by the authors and concluded that it was essential to
monitor for E. coli in cheese and to improve the production practices of
small cheese manufacturers. Tzanetakis et al. (1987) examined 50
samples 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 of
200 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 or
caprine milk or a mix of both. All the samples contained coliforms, with
30% showing levels of 5–5.66 log10 CFU/g. E. coli was found in 88% of
the samples, of which 46.8% had levels > 4 log10 CFU/g. The study
attributed these high indicator counts to the use of raw milk and to poor
hygienic processing conditions in making the cheese. The study also
showed 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 different
varieties and purchased from different cities in California, and found
that 46% had coliforms at < 100 CFU/g, but some of the samples had
coliform counts that ranged from 2 to 7 log10 CFU/g. Considering that
all these cheeses were labeled as having been made with pasteurized
milk, the authors concluded that insanitation or high incidences of post
pasteurization contamination had probably occurred. Similarly, Araùjo
et al. (2002) examined the levels of fecal coliforms in 45 samples of
three pasteurized-milk soft-cheese brands sold in Rio de Janeiro, Brazil.
The study found that 95% (43/45) of the samples had exceeded the
Brazilian fecal coliform standard of 100 CFU/g. The levels found ranged
from 1 to 6 log10 MPN/g. E. coli was also isolated from 97.7% of the
samples, 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 high
fecal coliform counts to improper pasteurization or to unhygienic
practices during cheese-making.
The results of these studies showed that cheeses that are made with
good quality raw milk and under sanitary conditions and properly aged
will not contain or only have low levels of indicator bacteria. Consistent
with that premise, between 2014 and 2016, the U.S. Food and Drug
Administration analyzed 1606 samples, comprised of 5698 subsamples,
of raw-milk cheeses which were aged for a minimum of 60 days and
found 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 in
cheeses, most researchers have attributed these to the low quality of
raw milk used or to contamination during processing and emphasized
the importance of maintaining proper sanitary conditions in cheese-
9
Food Microbiology 85 (2020) 103283
making and aging in order to produce a product of sanitary quality.
7. Conclusion
In summary, this paper reviewed the scientific literature on the
prevalence and levels of indicator bacteria found in different types of
raw milk, showed examples of how the cheese-making and ripening
processes affects indicator bacteria levels and the levels of indicators
that have been found in various types of cheeses around the world. It is
evident from these studies that indicators can be found in various types
of raw milk and E. coli are usually present at levels of < 100 CFU/g and
in good-quality raw milk, the levels are at < 10 CFU/g. The studies also
showed that indicator bacteria found in milk will increase by several
folds during the initial stages of cheese-making and except for fresh
cheeses that are not aged, the indicator levels decline by several log/g
during fermentation, aging and ripening of the cheese and often are not
found 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 that
indicator bacteria are either not present or found at levels that are
often < 10 CFU/g or < 100 CFU/g, which are the common indicator
limits for cheeses set by many countries. In those instances where
cheeses had high levels of indicator bacteria, most studies attributed
these to the use of poor-quality raw milk which contained high levels of
indicator flora or to unsanitary conditions, or both. The cited studies
also showed that raw milk cheeses made from good-quality raw milk
under sanitary conditions, using good manufacturing practices and
properly 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 for
the U.S. Food and Drug Administration and this review was prepared as
part of our official duties. None of the authors have any conflicts of
interest with the subject of this paper.
Acknowledgements
The authors thank Mickey Parrish for critical reading of this
manuscript. 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 in
translating some of the International regulations.
References
Albenzio, M., Carbo, M.R., Rehman, S.U., Fox, P.F., De Angelis, M., Corsetti, A., Sevi, A.,
Gobbetti, M., 2001. Microbiological and biochemical characteristics of Canestrato
Pugliese cheese made from raw milk, pasteurized milk or by heating the curd in hot
whey. Int. J. Food Microbiol. 67, 35–48.
Alegría, Á., Álvarez-Martín, P., Sacristán, N., Fernández, E., Delgado, S., Mayo, B., 2009.
Diversity and evolution of the microbial populations during manufacture and ri-
pening of Casín, a traditional Spanish, starter-free cheese made from cow's milk. Int.
J. Food Microbiol. 136, 44–51.
Alessandria, V., Dolci, P., Rantsiou, K., Pattono, D., Dalmasso, A., Civera, T., Cocolin, L.,
2010. Microbiota of the Planalto de bolona: an artisanal cheese produced in un-
common environmental conditions in the Cape Verde islands. World J. Microbiol.
Biotechnol. 26, 2211–2221.
Araùjo, V.S., Pagliares, V.A., Queiroz, M.L.P., Freitas-Almeida, A.C., 2002. Occurrence of
Staphylococcus and enteropathogens in soft cheese commercialized in the city of Rio
de Janeiro, Brazil. J. Appl. Microbiol. 92, 1172–1177.
Aygun, O., Aslantas, O., Oner, S., 2005. A survey on the microbiological quality of Carra,
a traditional Turkish Cheese. J. Food Eng. 66, 401–404.
Bachmann, H.P., Spahr, U., 1995. The fate of potentially pathogenic bacteria in Swiss
hard and semihard cheeses made from raw milk. J. Dairy Sci. 78, 476–483.
Boor, K.J., Brown, D.P., Murphy, S.C., Kozlowski, S.M., Bandler, D.K., 1988.
Microbiological and chemical quality of raw milk in New York State. J. Dairy Sci. 81,
1743–1748.
Boor, K.J., Wiedmann, M., Murphy, S., Alcaine, S., 2017. A 100-year review: micro-
biology and safety of milk handling. J. Dairy Sci. 100, 9933–9951.
Bricker, A.L., Van Hekken, D.L., Guerrero, V.M., Gardea, A.A., 2005. Microflora isolated
M. Metz, et al.
from Mexican Mennonite-style cheeses. Food Prot. Trends 25, 637–648.
Brooks, J.C., Martinez, B., Stratton, J., Bianchini, A., Krokstrom, R., Hutkins, R., 2012.
Survey of raw milk cheeses for microbiological quality and prevalence of foodborne
pathogens. Food Microbiol. 31, 154–158.
Cabrezas, L., Sánchez, I., Poveda, J.M., Seseña, S., Palop, M.L.L., 2007. Comparison of
microflora, chemical and sensory characteristics of artisanal Manchego cheese from
two dairies. Food Control 18, 11–17.
Caridi, A., Micari, P., Foti, F., Ramondino, D., Sarullo, V., 2003. Ripening and seasonal
changes in microbiological and chemical parameters of the artisanal cheese Caprino
d'Aspromonte produced from raw or thermized goat's milk. Food Microbiol. 20,
201–209.
Chye, F.Y., Abdullah, A., Ayob, M.K., 2004. Bacteriological quality and safety of raw milk
in Malaysia. Food Microbiol. 21, 535–541.
CIA world Fact book. https://www.cia.gov/library/publications/the-world-factbook/
rankorder/2119rank.html.
Code of Practice, March 2010. Additional Measures for Raw Milk Products. New Zealand
Food Safety Authority.
Cogan, T.M., Rea, M.C., 1996. Artisanal European Cheeses. European Commission,
Luxembourg.
Coia, J.E., Johnston, Y., Steers, N.J., Hanson, M.F., 2001. A survey of the prevalence of
Escherichia coli O157:H7 in raw meats, raw cow's milk and raw-milk cheeses in south-
east Scotland. Int. J. Food Microbiol. 66, 63–69.
Coppola, S., Fusco, V., Andolfi, R., Aponte, M., Blaiotta, G., Ercolini, D., Moschetti, G.,
2006. Evaluation of microbial diversity during the manufacture of Fior di Latte di
Agerola, a traditional raw milk pasta-filata cheese of the Naples area. J. Dairy Res. 73,
264–272.
Cosciani-Cunico, E., Dalzini, E., D'Amico, S., Sfameni, C., Bertasi, B., Losio, M.N.,
Giacometti, F., Daminelli, P., 2014. Behaviour of Escherichia coli O157:H7 during the
manufacture and ripening of an Italian traditional raw goat milk cheese. Italian J.
Food Safety 3, 2243.
Costello, M., Rhee, M.S., Bates, M.P., Clark, S., Luedecke, L.O., 2003. Eleven-year trends
of microbiological quality in bulk tank milk. Food Prot. Trends 23, 393–400.
D'Amico, D.J., Donnelly, C.W., 2010. Microbiological quality of raw milk utilized for
small scale artisan cheese production: impact of farm practices and characteristics. J.
Dairy Sci. 93, 134–147.
D'Amico, D.J., Groves, E., Donnelly, C.W., 2008. Low incidence of foodborne pathogens of
concern in raw milk utilized for farmstead cheese production. J. Food Prot. 71,
1580–1589.
de Garnica, M.L., Linage, B., Carriedo, J.A., De La Fuente, L.F., Garcia-Jimeno, M.C.,
Santos, J.A., Gonzalo, C., 2012. Relationship among specific bacterial counts and
total bacterial and somatic cell counts and factors influencing their variation in ovine
bulk tank milk. J. Dairy Sci. 96, 1021–1029.
De Pasquale, I., Calasso, M., Mancini, L., Ercolini, D., La Storia, A., de Angelis, M., Di
Cagno, R., Gobbeti, M., 2014. Causal relationship between microbial ecology dy-
namics and proteolysis during manufacture and ripening of protected designation of
origin (PDO) cheese Canestrato Pugliese. Appl. Environ. Microbiol. 80, 4085–4094.
De Reu, K., Grijspeerdt, K., Herman, L., 2004. A Belgian survey of hygiene indicator
bacteria and pathogenic bacteria in raw milk and direct marketing of raw milk farm
products. J. Food Saf. 24, 13–36.
Desmasures, N., Bazin, F., Guéguen, M., 1997. Microbiological composition of raw milk
from selected farms in the Camembert region of Normandy. J. Appl. Microbiol. 83,
53–58.
Desmasures, N., Guéguen, M., 1997. Monitoring the microbiology of high quality milk by
monthly sampling over 2 years. J. Dairy Res. 64, 271–280.
Devoyod, J.J., Bret, G., Auclair, J.E., 1968. La flore microbienne du fromage de
Roquefort. I. son evolution au cours de la fabrication et de l’affinage du fromage. Lait
48, 613–629.
Dolci, P., Alessandria, V., Rantsiou, K., Rolle, L., Zeppa, G., Cocolin, L., 2008. Microbial
dynamics of Castelmagno PDO, a traditional Italian cheese, with a focus on lactic acid
bacteria ecology. Int. J. Food Microbiol. 122, 302–311.
Duthoit, F., Callon, C., Tessier, L., Montel, M.-C., 2005. Relationship between sensorial
characteristics and microbial dynamics in “Registered designation of origin” Salers
cheese. Int. J. Food Microbiol. 103, 259–270.
Duthoit, F., Godon, J.-J., Montel, M.-C., 2003. Bacterial community dynamics during
production of registered designation of origin Salers cheese as evaluated by 16S rRNA
gene single-strand conformation polymorphism analysis. Appl. Envrion. Microbiol.
69, 3840–3848.
Eleftheriadou, M., Varnava-Tello, A., Metta-Loizidou, M., Nikolaou, A.S., Akkelidou, D.,
2002. The microbiological profile of foods in the Republic of Cyprus: 1991-2000.
Food Microbiol. 19, 463–471.
Ercolini, D., Hill, P.J., Dodd, C.E.R., 2003. Bacterial community and location in Stilton
cheese. Appl. Environ. Microbiol. 69, 3540–3548.
Fernandez del Pozo, B., Gaya, P., Medina, M., Rodríguez-Marín, A., Nuñez, M., 1988.
Changes in the microflora of La Serena ewes' milk cheese during ripening. J. Dairy
Res. 55, 449–455.
Feurer, C., Irlinger, F., Spinnler, H.E., Glaser, P., Vallaeys, T., 2004. Assessment of the
rind microbial diversity in a farmhouse-produced vs a pasteurized industrially pro-
duced soft red-smear cheese using both cultivation and rDNA-based methods. J. Appl.
Microbiol. 97, 546–556.
Fontecha, J., Peláez, Juárez, M., Requena, T., Gómez, C., Ramos, M., 1990. Biochemical
and microbial characteristics of artisanal hard goat's cheese. J. Dairy Sci. 73,
1150–1157.
Foschino, R., Invernizzi, A., Barucco, R., Stradiotto, K., 2002. Microbial composition,
including the incidence of pathogens, of goat milk from the Bergamo region of Italy
during a lactation year. J. Dairy Res. 69, 213–225.
Frank, J.F., Marth, E.H., Olson, N.F., 1977. Survival of enteropathogenic and non-
10
Food Microbiology 85 (2020) 103283
pathogenic Escherichia coli during the manufacture of Camembert cheese. J. Food
Prot. 40, 835–842.
Frank, J.F., Marth, E.H., Olson, N.F., 1978. Behavior of enteropathogenic Escherichia coli
during manufacture and ripening of Brick cheese. J. Food Prot. 41, 111–115.
Gerasi, E., Litopoulou-Tzanetaki, E., Tzanetakis, N., 2003. Microbiological study of
Manura, a hard cheese made from raw ovine milk in the Greek island Sifnos. Int. J.
Dairy Technol. 56, 117–122.
Guzman-Hernandez, R., Contreras-Rodriguez, A., Hernandez-Velez, R., Perez-Martinez, I.,
Lopez-Merino, A., Zaidi, M., Estrada-Garcia, T., 2016. Mexican unpasteurized fresh
cheeses are contaminated with Salmonella spp., non-O157:H7 Shiga toxin producing
Escherichia coli and potential uropathogenic E. coli strains: a public health risk. Int. J.
Food Microbiol. 237, 10–16.
Hatzikamari, M., Litopoulou-Tzanetaki, E., Tzanetakis, N., 1999. Microbiological char-
acteristics of Anevato: a traditional Greek cheese. J. Appl. Microbiol. 87, 595–601.
Hayes, M.C., Boor, K., 2001. Raw milk and fluid milk products. Applied Dairy
Microbiology. Marcel Dekker, New York, NY, USA, pp. 59–76.
Humphrey, T.J., Hart, R.J.C., 1986. A possible microbiological standard for raw milk.
PHLS Microbiol. Dig. 3, 8–10.
International Commission on Microbiological Specification for Foods, 1986. Sampling
plans for milk and milk products. In: Microorganisms in Foods 2 Sampling for
Microbiological Analysis: Principles and Specific Applications. University of Toronto
Press, Toronto, pp. 161–174. http://www.icmsf.org/pdf/icmsf2.pdf.
International Life Sciences Institute, 2011. The Enterobacteriaceae and Their Significance
to the Food Industry. ILSI Europe, Brussels, Belgium, pp. 9.
Jayarao, B.M., Pillai, S.R., Sawant, A.A., Wolfgang, D.R., Hegde, N.V., 2004. Guidelines
for monitoring bulk tank milk somatic cell and bacterial counts. J. Dairy Sci. 87,
3561–3573.
Jayarao, B.M., Wang, L., 1999. A study on the prevalence of gram-negative bacteria in
bulk tank milk. J. Dairy Sci. 82, 2620–2624.
Khayat, F.A., Bruhn, J.C., Richardson, G.H., 1988. A survey of coliforms and
Staphylococcus aureus in cheese using impedimetric and plate count methods. J. Food
Prot. 51, 53–55.
Lioliou, K., Litopoulou-Tzanetaki, E., Tzanetakis, N., Robinson, R.K., 2001. Changes in the
microflora of Manouri, a traditional Greek whey cheese, during storage. Int. J. Dairy
Technol. 54, 100–106.
Litopoulou-Tzanetaki, E., Tzanetakis, N., 2011. Microbiological characteristics of Greek
traditional cheeses. Small Rumin. Res. 101, 17–32.
Lück, H., Dunkeld, M., 1981. Enterobacteriaceae in cheese. S. Afr. J. Dairy Technol. 13,
9–14.
Maifreni, M., Frigo, F., Bartolomeoli, I., Innocente, N., Biasutti, M., Marino, M., 2013.
Identification of the Enterobacteriaceae in Montasio cheese and assessment of their
amino acid decarboxylase activity. J. Dairy Res. 80, 122–127.
Manolopoulou, E., Sarantinopoulos, P., Zoidou, E., Aktypis, A., Moschoupoulou, E.,
Kandarakis, I.G., Anifantakis, E.M., 2003. Evolution of microbial populations during
traditional Feta cheese manufacture and ripening. Int. J. Food Microbiol. 82,
153–161.
Marino, M., Maifreni, M., Rondinini, G., 2003. Microbiological characterization of arti-
sanal Montasio cheese: analysis of its indigenous lactic acid bacteria. FEMS
Microbiol. Lett. 229, 133–140.
Martin, N.H., Trmčić, A., Hsieh, T.-H., Boor, K.J., Wiedmann, M., 2016. The evolving role
of coliforms as indicator of unhygienic processing conditions in dairy foods. Front.
Microbiol. 7, 1549. https://doi.org/10.3389/fmicb.2016.01549.
Martin-Platero, A.M., Maqueda, M., Valdivia, E., Purswani, J., 2009. Polyphasic study of
microbial communities of two Spanish farmhouse goats' milk cheeses from Sierra de
Aracena. Food Microbiol. 26, 294–304.
Martinez Manso, P., Fernandez-Salguero, J., 1978. Estudio de algunas floras microbianas
del queso de La Serena. Arch. Zootec. 27, 93–97.
Menéndez, S., Godínez, R., Centeno, J.A., Rodríguez-Otero, J.L., 2001. Microbiological,
chemical and biochemical characteristics of ‘Tetilla” raw cows-milk cheese. Food
Microbiol. 18, 151–158.
Miskimin, D.K., Berkowitz, K.A., Solberg, M., Riha Jr., W.E., Franke, W.C., Buchanan,
R.L., O’leary, V., 1976. Relationships between indicator organisms and specific pa-
thogens in potentially hazardous foods. J. Food Sci. 41, 1001–1006.
Miszczycha, S.D., Bel, N., Gay-Perret, P., Michel, V., Montel, M.C., Sergentet-Thevenot,
D., 2015. Behavior of different Shiga toxin-producing Escherichia coli serotypes
(O26:H11, O103:H2, O145:H28, O157:H7) during the manufacture, ripening and
storage of a white mold cheese. J. Dairy Sci. 99, 5224–5229.
Miszczycha, S.D., Perrin, F., Ganet, S., Jamet, E., Tenenhaus-Aziza, F., Montel, M.,
Thevenot-Sergentet, D., 2013. Behavior of different Shiga toxin-producing Escherichia
coli serotypes in various experimentally contaminated raw-milk cheeses. Appl.
Environ. Microbiol. 79, 150–158.
Morgan, F., Massouras, T., Barbosa, M., Roseiro, L., Ravasco, F., Kandarakis, I., Bonnin,
V., Fistakoris, M., Anifantakis, E., Jaubert, G., Ranynal-Ljutovac, K., 2003.
Characteristics of goat milk collected from small and medium enterprises in Greece,
Portugal and France. Small Rumin. Res. 47, 39–49.
National Academy of Sciences (NAS), National Research Council (NCR), 1985. An
Evaluation of the Role of Microbiological Criteria for Foods and Food Ingredients.
National Academy Press, Washington, DC, pp. 121–122 1985.
National Advisory Committee on Microbiological Criteria for Foods, 2018. Response to
questions posed by the Department of defense regarding microbiological criteria as
indicators of process control or insanitary conditions. J. Food Prot. 81, 115–141.
Nikolaou, E., Tzanetakis, N., Litopoulou-Tzanetaki, E., Robinson, R.K., 2002. Changes in
the microbiological and chemical characteristics of an artisanal, low–fat cheese made
from raw ovine milk during ripening. Int. J. Dairy Technol. 55, 12–17.
Nuñez, M., 1978. Microflora of Cabrales cheese; changes during maturation. J. Dairy Res.
45, 501–508.
M. Metz, et al.
O'Brien, M., Hunt, K., McSweeney, S., Jordan, K., 2009. Occurrence of foodborne pa-
thogens in farmhouse cheese. Food Microbiol. 26, 910–914.
Ordonez, J.A., Masso, J.A., Marmol, M.P., Ramos, M., 1980. Contribution a l’etude du
fromage “Roncal”. Lait 593, 283–294.
Pantoja, J.C.F., Reinemann, D.J., Ruegg, P.L., 2009. Associations among milk quality
indicators in raw bulk milk. J. Dairy Sci. 92, 4978–4987.
Papageorgiou, D.K., Abahim, A., Bori, M., Doundounakis, S., 1998. Chemical and bac-
teriological characteristics of Pichtogalo Chanion cheese and mesophilic starter cul-
tures for its production. J. Food Prot. 61, 688–692.
Piras, C., Cesare Marincola, F., Savorani, F., Engelsen, S.B., Cosentino, S., Viale, S., Pisan,
Barbara, M., 2013. A NMR metabolomics study of the ripening process of the Fiore
Sardo cheese produced with autochthonous adjunct cultures. Food Chem. 141,
2137–2147.
Prodromou, K., Thasitou, P., Haritonidou, E., Tzanetakis, N., Litopoulou-Tzanetaki, E.,
2001. Microbiology of “Orinotyri”, an Ewe's milk cheese from the Greek mountains.
Food Microbiol. 18, 319–328.
Psoni, L., Tzanetakis, N., Litopoulou-Tzanetaki, E., 2003. Microbiological characteristics
of Batzos, a traditional Greek cheese from raw goat's milk. Food Microbiol. 20,
575–582.
Pyz-Łukasik, R., Paszkiewicz, W., Tatara, M.R., Brodzki, P., Belkot, Z., 2015.
Microbiological quality of milk sold directly from producers to consumers. J. Dairy
Sci. 98, 4294–4301.
Quigley, L., O'Sullivan, O., Beresford, T., Ross, R.P., Fitzgerald, G.F., Cotter, P.D., 2012.
High throughput sequencing for detection of subpopulations of bacteria not pre-
viously associated with artisanal cheeses. Appl. Environ. Microbiol. 78, 5717–5723.
Rantsiou, K., Urso, R., Dolci, P., Comi, G., Cocolin, L., 2008. Microflora of Feta cheese
from four Greek manufacturers. Int. J. Food Microbiol. 126, 36–42.
Randazzo, C.L., Pitino, I., Ribbera, A., Caggia, C., 2010. Pecorino Crotonese cheese: study
of bacterial population and flavor compounds. Food Microbiol. 27, 363–374.
Randazzo, C.L., Torriani, S., Akkermans, A.D.L., de Vos, W.M., Vaughan, E.E., 2002.
Diversity, dynamics and activity of bacterial communities during production of an
artisanal Sicilian cheese as evaluated by 16S rRNA analysis. Appl. Environ. Microbiol.
68, 1882–1892.
Randazzo, C., Vaughan, E.E., Caggia, C., 2006. Artisanal and experimental Pecorino
Siciliano cheese: microbial dynamics during manufacture assessed by culturing and
PCR-DGGE analyses. Int. J. Food Microbiol. 109, 1–8.
Rea, M.C., Cogan, T.M., Tobin, S., 1992. Incidence of pathogenic bacteria in raw milk in
Ireland. J. Appl. Bacteriol. 73 331-326.
Rosengren, A., Fabricius, A., Guss, B., Sylvén, S., Lindqvist, R., 2010. Occurrence of
foodborne pathogens and characterization of Staphylococcus aureus in cheese pro-
duced on farm-dairies. Int. J. Food Microbiol. 144, 263–269.
Ruusunen, M., Salonen, M., Pulkkinen, H., Huuskonen, M., Hellström, S., Revez, J.,
11
View publication stats
Food Microbiology 85 (2020) 103283
Hänninen, M.L., Fredriksson-Ahomaa, M., Linstrom, M., 2013. Pathogenic bacteria in
Finnish bulk tank milk. Foodb. Pathog. Dis. 10, 99–106.
Schlesser, J.E., Gerdes, R., Ravishankar, S., Madsen, K., Mowbray, J., Teo, A.Y.-L., 2006.
Survival of a five-strain cocktail of Escherichia coli O157:H7 during the 60-day aging
period of Cheddar cheese made from unpasteurized milk. J. Food Prot. 69, 990–998.
Smoot, L.M., Pierson, M.D., 1997. Indicator microorganisms and microbiological criteria.
In: Doyle, M.P., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology:
Fundamentals and Frontiers. ASM Press, Washington, D.C., pp. 66–80.
Tabla, R., Gómez, A., Simancas, A., Rebollo, J.E., Molina, F., Roa, I., 2016.
Enterobacteriaceae species during manufacturing and ripening of semi-hard and soft
raw Ewe's milk cheese: gas production capacity. Small Rumin. Res. 145, 123–129.
Temelli, S., Anar, Ş., Sen, C., Akyuva, P., 2006. Determination of microbiological con-
tamination sources during Turkish white cheese production. Food Control 17,
856–861.
Tornadijo, M.E., García, M.C., Fresno, J.M., Carballo, J., 2001. Study of Enterobacteriaceae
during the manufacture and ripening of san simón cheese. Food Microbiol. 18,
499–509.
Torres-Llanez, M.J., Vallejo-Cordoba, B., Díaz-Cinco, M.E., Mazorra-Manzano, M.A.,
González-Córdova, A.F., 2006. Characterization of the natural microflora of artisanal
Mexican Fresco cheese. Food Control 17, 683–690.
Tortorello, M.L., 2003. Indicator organisms for safety and quality – uses and methods for
detection: minireview. JAOAC (J. Assoc. Off. Anal. Chem.) 86, 1208–1217.
Trmčić, A., Chauhan, K., Kent, D.J., Ralyea, R.D., Martin, N.H., Boor, K.J., Wiedmann, M.,
2016. Coliform detection in cheese is associated with specific cheese characteristics,
but no association was found with pathogen detection. J. Dairy Sci. 99, 6105–6120.
https://doi.org/10.3168/jds.2016-11112.
Tzanetakis, N., Litopoulou-Tzanetaki, E., Manolkidis, K., 1987. Microbiology of Kopanisti,
a traditional Greek cheese. Food Microbiol. 4, 251–256.
U. S. FDA, July 21, 2016. FY 2014-2016 Microbiological Sampling Assignment Summary
Report: Raw Milk Cheese Aged 60 Days.
Vernozy-Rozand, C., Mazuy-Cruchaudet, C., Bavai, C., Montet, M.P., Bonin, V., Dernburg,
A., Richard, Y., 2005. Growth and survival of Escherichia coli O157:H7 during the
manufacture and ripening of raw goat milk lactic cheeses. Int. J. Food Microbiol. 105,
83–88.
Vassiliadis, A., Psoni, L., Nikolaou, S., Arvanitis, L., Tzanetakis, N., Litopoulou-Tzanetaki,
E., 2009. Changes in microbial populations and biochemical characteristics of Greek
traditional feta cheese during ripening. Int. J. Dairy Technol. 62, 39–47.
WHO/FAO JEMRA Report, 2018. Shiga Toxin-Producing Escherichia coli (STEC) and
Food: Attribution, Characterization and Monitoring. http://www.fao.org/
documents/card/en/c/CA0032EN.
Zárate, V., Belda, F., Pérez, C., Cardell, E., 1997. Changes in the microbial flora of
Tenerife goats' milk cheese during ripening. Int. Dairy J. 7, 635–641.