https://doi.org/10.1590/1808-1657000702019
SCIENTIFIC ARTICLE
Preventive Veterinary Medicine
Hygienic-sanitary quality and antimicrobial sensitivity
profile of Escherichia coli in milk and cheese sold illegally in
municipalities of northern Mato Grosso, Brazil*
Renata Henriques Ragi Pena1
Filipe Freitas1
https://orcid.org/0000-0003-0284-2888
https://orcid.org/0000-0003-3747-1562
Bruno Gomes de Castro1,**
https://orcid.org/0000-0002-0249-3326
1. Universidade Federal de Mato Grosso – Campus Universitário de Sinop – Laboratório de Diagnóstico em Sanidade Animal –
Sinop (MT), Brazil.
* This paper is part of the dissertation thesis of the first author.
** Corresponding author: castrobg@gmail.com
ABSTRACT
Products such as milk and cheese produced by hand and sold by small producers in open markets and at home are a reality in Brazil,
despite legal prohibitions. In many cases, this leads to the production of food without hygienic conditions, which may constitute an
important source of transmission of foodborne diseases and a danger to public health. This study proposes to examine the hygienicsanitary quality of milk and cheese sold illegally in municipalities of northern Mato Grosso, Brazil, to undertake a phenotypical investigation
of the presence of resistance of isolated colonies to antimicrobials and to detect the production of β-lactamase enzymes: extendedspectrum β-lactamase (ESBL), AmpC β-lactamases (AmpC) and carbapenemases. The 25 milk and 37 cheese samples analyzed were
subjected to the most probable number (MPN) test, isolation on eosin-methylene blue agar (EMB) agar and Escherichia coli identification
by biochemical tests and disk diffusion test. Results showed that 76% of the milk samples and 67.57% of the cheese samples had
thermotolerant coliform counts above the value allowed by the legislation. The milk and cheese isolates showed 15.79 and 5.88%
resistance, respectively, to at least one of the tested antimicrobials. No β-lactamase enzyme production was observed in the isolates.
Keywords: foodborne diseases; MPN; phenotypic tests; β-lactamases.
INTRODUCTION
According to the World Health Organization, foodborne diseases affect one in ten inhabitants every year in the world
and, from farm to table, the food can be contaminated in many ways (WHO, 2017b). The increasing consumption of food
in public places and the consumption of organic and artisanal foods are a current trend and if good manufacturing practices
(GMP) are not complied with throughout the production chain, products can become a danger to consumers (WHO, 2016).
Artisanal milk and cheese produced by small farmers are a reality in Brazil, where these products are sold in open
markets and at home, despite prohibitions by the national legislation (BRAZIL, 2017) and evidence of low hygienic-sanitary
quality and high rates of Escherichia coli in them (WHO, 2018b).
Escherichia coli is considered an indicator microorganism (FORSYTHE, 2013) and its presence in the food provides
information on contaminations of fecal origin, the probable presence of pathogens and spoilage microorganisms, as well
as the sanitary conditions that the product undergoes during its production (SILVA et al., 2010). It is also considered a
sensor in the examination of resistance because it is the most widely studied agent, in addition to having a diversified
ecology and various resistance mechanisms (VILA et al., 2016).
Therefore, this study proposes to investigate the hygienic-sanitary quality of raw milk and its by-products sold
illegally in the northern region of Mato Grosso, Brazil, as well as to determine the antimicrobial sensitivity profile of
E. coli isolated from the samples and detect multidrug resistance and production of β-lactamases enzymes in order to
highlight the impact that the consumption of these products can have on the health of consumers, due to the possibility
of pathogenic and/or resistant microorganism dissemination.
Received: Sep 18, 2019. Accepted: Nov 24, 2020
Associate Editor: Silvia Galleti
Peer Review History: Double-blind Peer Review.
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R.H.R. Pena et al.
METHODS
The study involved 25 raw milk and 37 cheese samples acquired directly from farmers in open markets and at home in
11 municipalities in the northern region of Mato Grosso.
Hygienic-sanitary quality was evaluated by the most probable number (MPN) test, following the American Public
Health Association (APHA), as recommended by SILVA et al. (2010). From one of the positive tubes of the EC broth
used in the thermotolerant-coliform investigation, culture was performed on eosin-methylene blue agar (EMB). After
incubation at 35 °C for 18–24 h, five typical colonies (black colonies with or without greenish metallic luster around them)
were collected individually and inoculated in tubes containing brain heart infusion broth (BHI), in which biochemical tests
were subsequently performed following the methodology proposed by RIBEIRO (2011).
Phenotypic detection of antimicrobial resistance was carried out using disk diffusion test as recommended by the
Clinical and Laboratory Standards Institute (CLSI, 2017). After the inhibition halos were read, the strains were classified
as sensitive, intermediate or resistant to the antimicrobials (CLSI, 2017; 2018).
The antimicrobial disks used were selected according to CLSI (2017; 2018) guidelines and also based on the study
of SANTIAGO (2013). These consisted of amoxicillin + clavulanic acid 30 µg, ampicillin 10 µg, amoxicillin 10 µg,
penicillin 10 IU, oxacillin 1 µg, cefepime 30 µg, ceftriaxone 30 µg, cefoxitin 30 µg, ceftazidime 30 µg, cefalotin 30 µg,
aztreonam 30 µg, imipenem 10 µg, gentamicin 10 µg, neomycin 30 µg, ciprofloxacin 5 µg and sulfamethoxazole +
trimethoprim 25 µg.
Results were also examined for the determination of multidrug resistance to antimicrobials. Strains that showed resistance
to at least one agent in three or more distinct categories of antimicrobials were considered multidrug-resistant (RIBEIRO
et al., 2016) and were subsequently subjected to the (a/b) x 100 formula to determine their percentage, where a corresponds
to the number of antimicrobials to which the isolate was resistant and b to the number of antimicrobials to which the isolate
was exposed (BARRETO et al., 2012).
To detect the extended-spectrum β-lactamase enzyme (ESBL), isolates that showed resistance to a third-generation
cephalosporin (ceftazidime or ceftriaxone) and sensitivity to clavulanic acid were suspected of producing the enzyme
classified as ESBL (2be). To confirm the production of this enzyme, the disk approximation test, the minimum inhibitory
concentration (MIC) test (using the Etest strip) and the three-dimensional extract (TET) test were carried out as described
by SANTIAGO (2013).
For the detection of the AmpC enzyme, isolates that showed resistance to cefoxitin were suspected of producing
AmpC-type enzymes. To confirm, the TET test was carried out, as described by SANTIAGO (2013).
To detect the carbapenemase enzyme, isolated colonies resistant to imipenem were subjected to disk diffusion test,
using the ertapenem disk (SANTIAGO, 2013). Suspected isolates were also subjected to the modified Hodge test, according
to CLSI guidelines (CLSI, 2017).
RESULTS AND DISCUSSION
The minimum and maximum reference values allowed by Brazilian legislation, the corresponding regulations, the
number of colonies characteristic of E. coli isolated from the samples and the results obtained in the MPN tests performed
on the raw milk samples that participated in this study to measure the hygienic-sanitary quality of the analyzed products
are specified in Table 1.
Table 1. Results of MPN test of raw milk samples.
N of samples
Sample
Total coliforms
(MPN/mL)
above standards
Thermotolerant
coliforms (MPN/mL)
above standards
Isolated E. coli
colonies
25
Raw milk
96% (24/25)
76% (19/25)
60,80% (76/125)
m = 2 M = 4 MPN/mL
m = 2 M = 4 MPN/mL
-
NI No. 62/2011
RDC 12/2001
-
Reference values
Reference
Source: research data. M – maximum allowed value; m – minimum allowed value; NI – Normative Instruction; RDC – Collegiate Board Resolution
(Resolução da Diretoria Colegiada).
2
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Hygienic-sanitary quality and antimicrobial sensitivity profile of Escherichia coli in milk and cheese sold illegally in municipalities of northern Mato Grosso, Brazil
Acceptable values for both total (TC) and thermotolerant coliforms (TTC) for milk intended for consumption
are at least two and at most four coliforms at 35 or 45 °C/mL (BRAZIL, 2001; 2011). Of the 25 milk samples analyzed,
96% (24/25) were above the limits required by legislation for TC and 76% (19/25) were above the acceptable limits for TTC.
Of these 25 samples, 125 colonies were initially isolated and 60.8% (76/125) were classified as E. coli after confirmation
with biochemical tests.
Based on the information contained in the Collegiate Board Resolution (CBR, Resolução da Diretoria Colegiada)
No. 12 of 2001, the 37 cheese samples analyzed were classified into cheese of medium moisture (36% < moisture < 46%)
and very high moisture (moisture = 55%) (BRAZIL, 2001).
The results obtained in the MPN tests for the cheese samples of medium and very high moisture, as well as the reference
values and their corresponding regulations, are shown in Tables 2 and 3, respectively.
Table 2. Results of MPN test of medium-moisture cheese samples.
N of samples
02
Sample
Total coliforms
(MPN/mL)
above standards
Thermotolerant
coliforms (MPN/mL)
above standards
Isolated E. coli
colonies
Medium-moisture
cheese
0% (0/2)
0% (0/2)
100% (10/10)
m = 1,000 M = 5,000 MPN/g
m = 500 M = 1,000 MPN/g
-
Ord. No. 146/1996
RDC 12/2001
-
Reference values
Reference
Source: research data. M – maximum allowed value; m – minimum allowed value; Ord. – ordinance; RDC – Collegiate Board Resolution (Resolução
da Diretoria Colegiada).
Table 3. Results of the MPN test of very high moisture cheese samples.
N of samples
35
Reference values
Reference
Sample
Total coliforms
(MPN/mL)
above standards
Thermotolerant
coliforms (MPN/mL)
above standards
Isolated E. coli
colonies
Very high
moisture cheese
100% (35/35)
71.43% (25/35)
71.71%
(136/175)
m = 10 M = 1,000 MPN/g
m = 50 M = 500 MPN/g
-
Ord. No. 146/1996
RDC 12/2001
-
Source: research data. M – maximum allowed value; m – minimum allowed value; Ord. – ordinance; RDC – Collegiate Board Resolution (Resolução
da Diretoria Colegiada).
Among the medium-moisture cheeses, represented in this study by the mozzarella cheeses, the minimum and maximum
values allowed in the legislation range from 500 to 1,000 coliforms at 45 °C/g for TTC (BRAZIL, 2001) and from 1,000 to
5,000 coliforms at 35 °C/g for TC (BRAZIL, 1996). The two samples classified in this category were within the standards
required by the legislation for both TC and TTC. From these two samples, 10 colonies were initially isolated (five from
each sample) and, after biochemical tests, 100% (10/10) were classified as E. coli.
As for the cheese samples classified in the category of very high moisture, represented in this study by “minas frescal”
cheese, the tolerable threshold values range from a minimum of 10 to a maximum of 1,000 coliforms at 35 °C/g for TC
(BRAZIL, 1996) and from 50 to 500 coliforms at 45 °C/g for TTC (BRAZIL, 2001). Of the total 35 very high moisture
cheese samples, 100% (35/35) were above the acceptable limits for TC and 71.43% (25/35) were above the limits required
by legislation for TTC. Of the 35 samples, 175 colonies were initially isolated, 71.71% (136/175) of which were classified
as E. coli after biochemical tests.
There are four ways in which milk can be contaminated with pathogenic microorganisms: by fecal contamination during
or after milking; through contaminated humans; by direct passage of blood into milk from systemic infection; or through
mastitis (LUCEY, 2015). It is due to these factors that, among other specifications, decree No. 9013 of 2017 determines
that milk destined for the direct consumption of the population and/or used for the production of cheeses must undergo a
previous adequate heat treatment process to ensure product safety. An exception is granted in the case of cheeses that ripen
at a temperature above 5 °C for at least 60 days, for which the heat treatment is not mandatory (BRAZIL, 2017).
Cross contamination plays an important role in food contamination and occurs when food is contaminated directly
or indirectly by pathogenic microorganisms. There are several stages at which this contamination can occur and, from
Arq. Inst. Biol., v.88, 1-8, e0702019, 2021
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R.H.R. Pena et al.
the field to the table, there are several possibilities of the food becoming contaminated. If good agricultural practices and
good manufacturing, storage, transport and distribution practices are not adopted in their production, they can become a
danger to consumers (WHO, 2016; 2017b).
Among other specifications, decree No. 9013 of 2017 determines that milk intended for direct consumption by the
population and milk used for the production of cheese must originate from healthy animals and undergo an appropriate heat
treatment process to ensure the safety of the products, since pasteurization eliminates pathogenic microorganisms (BRAZIL,
2017). However, the quality of a product cannot be recovered with the process, given that even after pasteurization, milk
retains a viable microbial population of around 0.1 to 0.5% of the initial count (LUZ et al., 2011). Therefore, it is essential
that milk — whether for direct consumption or used in the manufacture of by-products — have good microbiological
quality, observing all production, storage, transport and distribution requirements.
The raw milk samples analyzed in this study mostly reused 1 or 2 L polyethylene terephthalate (PET) bottles or 1 L
plastic bag packages similar to those used for pasteurized milk, which were tied at one end. The cheeses, however, were
packed in transparent plastic bags tied at the end and some were also laid on Styrofoam trays.
In terms of packaging, decree No. 9013 of 2017 determines that it must ensure protection according to their
characteristics, as well as storage and transport conditions and that it must be intact and properly sanitized following
the criteria established by the Federal Inspection Service (Serviço de Inspeção Federal, SIF). In the specific case of milk,
automatic filling must be done in a closed circuit and the equipment responsible for this process must contain devices
that ensure the antiseptic conditions of the packaging. Packaging for the products must also be authorized by health
regulatory agencies (BRAZIL, 2017).
With respect to the antimicrobial resistance of the isolated samples of the 212 colonies characteristic of E. coli in the
samples analyzed in this study, 76 belonged to milk and 136 to cheese. The results obtained for the antimicrobial susceptibility
test in raw milk and cheese, as well as the antimicrobials used, are shown in Figures 1 and 2, respectively.
According to the results of the susceptibility tests carried out on the 76 colonies characteristic of E. coli isolated from
raw milk samples (Fig. 1), 84.21% (64/76) of them exhibited sensitivity to all tested antimicrobials. Despite the high levels
of sensitivity, resistance was observed in approximately 15.79% (12/76), whereas 13.16% (10/76) showed intermediate
sensitivity to at least one of the antimicrobials used in this study.
100
90
80
70
60
50
40
30
20
10
0
S
AMC
AMO
AMP
ATM
IPM
CFL
CFP
CFO
CAZ
CRO
CIP
GEN
NEO
SUT
96.01
88.16
88.16
100
100
96.05
98.68
97.37
100
97.37
93.42
97.37
97.37
88.15
IS
1.32
6.58
6.58
0
0
0
1.32
0
0
2.63
0
0
0
1.32
R
2.67
5.26
5.26
0
0
3.95
0
2.63
0
0
6.58
2.63
2.63
10.53
R
IS
S
Figure 1. Results of antimicrobial susceptibility tests in Escherichia coli colonies isolated from samples
of raw milk sold illegally in municipalities in the northern region of Mato Grosso, Brazil.
Source: research data. AMC - amoxicillin + clavulanic acid, AMO - amoxicillin, AMP - ampicillin, ATM - aztreonam,
IPM - imipenem, CFL - cefalotin, CFO - cefoxitin, CAZ - ceftazidime, CRO - ceftriaxone, CFP - cefepime, CIP - ciprofloxacin,
NEO - neomycin, SUT - sulfamethoxazole + trimethoprim. R - resistance, IS - intermediate sensitivity, S - sensitivity.
Of the 136 colonies isolated from the cheese samples, 94.12% (128/136) showed sensitivity to all tested antimicrobials.
However, in 5.88% of the isolated strains, resistance and intermediate sensitivity to at least one of the tested drugs were
observed (Fig. 2).
Despite occurring naturally, misuse, indiscriminate sale and prescription without criteria in some countries contribute
to the increase and appearance of new resistance mechanisms in bacteria (WHO, 2018a). For this reason, entities such as
the World Health Organization (WHO), the World Organization for Animal Health (OIE) and the United Nations Food
and Agriculture Organization (FAO) have attempted to combat the global threat of resistance to antimicrobials (OIE, 2018)
by promoting actions such as enhancing antimicrobials of medical importance in animals to safeguard the effectiveness
4
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Hygienic-sanitary quality and antimicrobial sensitivity profile of Escherichia coli in milk and cheese sold illegally in municipalities of northern Mato Grosso, Brazil
of antimicrobials of importance to human health (WHO, 2017a) and implementing an international standard to increase
awareness and knowledge, as well as training professionals working in the production area (OIE, 2018). In this way, space is
created for the One Health concept to prevent and control emergency diseases that stem from the interrelationship between
animals, humans and ecosystems (OIE, 2019).
100
90
80
70
60
50
40
30
20
10
0
S
AMC
AMO
AMP
ATM
IPM
CFL
CFP
CFO
CAZ
CRO
CIP
GEN
NEO
SUT
97.79
93.38
93.38
99.26
100
97.79
100
98.53
98.53
99.26
100
99.26
100
98.53
IS
0.74
2.94
3.68
0
0
0.74
0
0
0
0.74
0
0
0
0
R
1.47
3.68
2.94
0.74
0
1.47
0
1.47
1.47
0
0
0.74
0
1.47
R
IS
S
Figure 2. Results of antimicrobial susceptibility tests in Escherichia coli colonies isolated from samples
of cheese sold illegally in municipalities in the northern region of Mato Grosso, Brazil.
Source: research data. AMC - amoxicillin + clavulanic acid, AMO - amoxicillin, AMP - ampicillin, ATM - aztreonam,
IPM - imipenem, CFL - cefalotin, CFO - cefoxitin, CAZ - ceftazidime, CRO - ceftriaxone, CFP - cefepime, CIP - ciprofloxacin,
NEO - neomycin, SUT - sulfamethoxazole + trimethoprim. R - resistance, IS - intermediate sensitivity, S - sensitivity.
Transmission of resistant microorganisms by animals can occur directly (humans or animals) or indirectly (via the
food chain or the environment) and because many of them cause disease in humans, control over the spread of resistant
agents is extremely important for global public health (WHO, 2017a).
Normative Instruction No. 62 of 2011 determines that food-processing companies must register their suppliers with the
Brazilian Ministry of Agriculture, Livestock and Supply and implement a raw material quality program associated with a
continuing education program whose effectiveness must be proven through product quality analysis (BRAZIL, 2011). On this
basis, the obligation to control the quality of the raw material most likely causes producers who deliver their products to processing
companies to endeavor in order to achieve greater control over mastitis and one form of controlling it is through antibiotic therapy.
This fact can be corroborated by an epidemiological survey and bacteriological study carried out on dairy farms in the
southern region of the state of Rio de Janeiro, which proved that 80% of the surveyed farms had unsatisfactory hygienic
conditions in the milking line and regarding the water supply. For these reasons, the treatment of mastitis was excessively
dependent on the use of antimicrobials (ALENCAR et al., 2014).
As can be seen in the present study, the vast majority of raw milk and cheese samples showed low hygienic-sanitary quality
and the colonies isolated from these samples exhibited high levels of sensitivity to the tested antimicrobials. This suggests that
these strains most likely come from commensal and/or environmental strains and, consequently, the microorganisms did
not suffer selective pressure due to the non-use of antimicrobials. In domestic environments and in subsistence agriculture,
antimicrobials are used mainly for the treatment of the animal rather than for the promotion of growth or as a prophylactic
measure. Thus, the incidence of antimicrobial resistance is low (ROUSHAM et al., 2018).
Lack of good milking and hygiene practices during the production process on the part of employees will result in a
contamination cycle that will originate contaminated products, constituting a source of transmission of resistant and potentially
pathogenic bacteria. Besides the fact that the loss of sensitivity directly influences the control of animal health, it is also
reflected in public health, due to the similar treatment protocols between species (animals and humans) (SANTIAGO, 2013).
Loss of sensitivity, associated with the fact that E. coli was included in a WHO list as one of the main causes of diseases in
different contexts (communities, hospitals or food chain) (VILA et al., 2016; LEKSHMI et al., 2017), increases the danger of
spreading resistant microorganisms. Therefore, in addition to pathogenic microorganisms, the consumption of products with low
hygienic-sanitary quality can transmit antimicrobial-resistant bacteria, making it difficult to treat infections (BARRETO et al., 2012).
Multidrug resistance to antimicrobials (MRA) is defined as the non-susceptibility or acquisition of resistance by a
microorganism to at least one agent in three or more different antimicrobial categories (RIBEIRO et al., 2016). The results
obtained from the samples in this study show that only 5.26% (4/76) of the colonies from raw milk and 1.47% (2/136) of
the colonies from cheese had MRA, which is an extremely low average of multidrug resistance among the investigated
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R.H.R. Pena et al.
isolates. As for the total isolates, the average MRA value was 2.83% (6/212). However, due to the presence of intermediate
sensitivity in some isolates, these data may change in the near future. Additionally, resistance genes can be transferred by
conjugation (plasmid or transposon), transduction (bacteriophages) or transformation (direct transfer of DNA between
compatible bacteria) (BAPTISTA, 2013).
Regarding the production of β-lactamase enzymes, only one isolate was suspected of producing ESBL (2be), as it
showed resistance to ceftazidime and sensitivity to clavulanic acid, for which the disk approximation, MIC and TET
tests were performed, as mentioned in the methodology. The result was negative for the three procedures, indicating
that the isolate in question is not a producer of the enzyme β-lactamase ESBL (2be). Thus, the resistance shown to
the tested drugs aztreonam and ceftazidime is most likely due to other resistance mechanisms, such as changes in
porins or efflux pumps.
Four isolates were suspected of producing AmpC β-lactamases enzymes, as they showed resistance to cefoxitin. Although
the Clinical and Laboratory Standards Institute (CLSI) does not have standardized tests for the determination of AmpC
enzymes, research has shown satisfactory results. Therefore, to check for the existence of AmpC enzymes, the TET test was
performed (SANTIAGO, 2013). All samples showed negative results for the production of the enzyme.
None of the isolates showed resistance to imipenem; thus, there were no suspects of carbapenemase enzyme production,
so the modified Hodge test is not justified.
CONCLUSIONS
The present study demonstrated that the great majority of the samples of raw milk and cheese analyzed have low
hygienic-sanitary quality due to their total and thermotolerant coliforms indices being above the standards required by
the legislation. It also demonstrated that the E. coli colonies isolated from the samples showed high levels of sensitivity to
the tested antimicrobials and low rates of multidrug resistance to the antimicrobials, none of them showed production of
β-lactamase enzymes (ESBL, AmpC and carbapenemase) based on phenotypic tests performed. Nonetheless, despite the
satisfactory results found regarding this, resistant colonies were still isolated; therefore, the foods in question can function
as disseminators of resistance. In both cases (low hygienic-sanitary quality of the products and presence of resistant
microorganisms), the products constitute a problem for public health because they can carry pathogenic microorganisms
resistant to antimicrobials.
AUTHORS’ CONTRIBUTIONS
Conceptualization: Pena, R. H. R.; Castro, B. G. Formal analysis: Pena, R. H. R.; Castro, B. G. Investigation: Pena, R. H. R.; Freitas, F.
Writing – review & editing: Pena, R. H. R.; Castro, B.G. Supervision: Castro, B.G. Funding acquisition: Pena, R. H. R.; Castro, B. G.
AVAILABILITY OF DATA AND MATERIAL
All data generated or analyzed during this study are included in this published article.
FUNDING
Fundação de Amparo à Pesquisa do Estado de Mato Grosso
Grant No. 041/2016
https://doi.org/10.13039/501100005286
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Finance Code 001
https://doi.org/10.13039/501100002322
CONFLICTS OF INTEREST
The authors declare that they have no conflict of interest.
ETHICAL APPROVAL
Not applicable.
ACKNOWLEDGEMENTS
Not applicable.
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