Introduction

The serrano artisanal cheese is a traditional product from the highland fields in the South region of Brazil. The production is characterized by the use of raw milk from dairy cattle that grazed on natural pastures of its own rural property (PEREIRA et al., 2014). Besides the possibility of raw milk to contain potentially harmful microorganisms to human health (NOBILI et al., 2016; NTULI et al., 2016; RANJBAR et al., 2018), serrano artisanal cheese is often produced under poorly hygienic conditions, posing at risk the health of consumers (MELO et al., 2013; PONTAROLO et al., 2017; SOUZA et al., 2003; ZAFFARI et al., 2007). Previous studies showed Escherichia coli is one of the major contaminants found in serrano artisanal cheese, produced in the Serrana region of Santa Catarina state, with a contamination rate superior to 45% (MELO et al., 2013; PONTAROLO et al., 2017). Although several strains of E. coli are considered commensal, there are pathogenic strains that cause serious gastrointestinal infections in humans and are called diarrheagenic E. coli (YANG; WANG, 2014).

The diarrheagenic strains of E. coli exhibit have specific virulence factors that ease their interaction with the host. For that reason, it is important to distinguish pathogenic E. coli types, such as: enteropatogenic E. coli (EPEC), shiga toxin-producing E. coli (STEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and enteroaggregative E. coli (EAEC), from those nonpathogenic (GYLES; FAIRBROTHER, 2010; YANG; WANG, 2014). This distinction of E. coli strains has not only epidemiological implications, but also clinical consequences to prevent and control of diarrheal diseases. Detection of virulence factors is a way to identify the different types of this pathogen, since they are considered exclusive to each pathotype (GIRÃO et al., 2006).

Eight diarrheagenic E. coli (DEC) groups are known: shiga toxin-producing E. coli (STEC), which includes the subgroup enterohemorrhagic E. coli (EHEC); typical and atypical enteropathogenic E. coli (EPECt and EPECa); enterotoxigenic E. coli (ETEC); enteroinvasive E. coli (EIEC); enteroaggregative E. coli (EAEC); diffusely adherent E. coli (DAEC); and adherent invasive E. coli (AIEC). Enteroaggregative hemorrhagic E. coli (EAHEC) O104:H4 is an emerging E. coli pathotype associated with an outbreak that occurred in Germany in May 2011. The genes used for genetic characterization of these groups are stx1 and stx2 (STEC); stx1, stx2, and eae (EHEC); eae (EPECa); eae and bfp (EPECt); enterotoxin genes LT and ST and colonization factors (ETEC); ipaH and ial (EIEC); and aggR, aap, and AA probe (EAEC) (CROXEN et al., 2013). Genes for the genetic characterization of AIEC have not yet been characterized and there is no agreement among authors for the genetic characterization of DAEC (NATARO; KAPER, 1998; CROXEN et al., 2013). EAHEC O104:H4 is an enteroaggregative E. coli with the stx2a encoding gene (BEUTIN; MARTIN, 2012).

Another factor that can contribute to virulence is the ability of different microorganisms to form biofilms (KRAGH et al., 2016). Several studies have recognized the potential of E. coli to form biofilms, which represents an important issue not only for the treatment of infections caused by this pathogen but also for food processing environments (CULLER et al., 2014; PONNUSAMY et al., 2012; SILAGYI et al., 2009).

Not only the identification of virulence factors in E. coli, but also the investigation of antimicrobial susceptibility profile, are of utmost importance, since these microorganisms are often involved in serious human infections (SLAMA et al., 2010) and, bacterial dissemination is considered highly associated with processing production, transformation, manufacturing and trade of food (KIRBIS; KRIZMAN, 2015). Studies worldwide have demonstrated serious concern about the alarming antimicrobial resistance index, and the number of multi-drug resistant E. coli strains isolated from raw and pasteurized milk (NTULI et al., 2016), dairy products (GUILLÉN et al., 2014; NOBILI et al., 2016) and other types of food (GÓMEZ-ALDAPA et al., 2016; WANG et al., 2017). One of the major resistance mechanisms developed by E. coli to adapt to antibiotic is the hydrolysis of the β-lactam ring through enzymes, defined as extended-spectrum beta-lactamases (ESBL), that confer resistance to antimicrobials from β-lactam class, including penicillins, cephalosporins and monobactams (ECDC, 2013; PFEIFER et al., 2010). However, these enzymes are not able to hydrolyse cephamycins and carbapenems, and could be inactivated by the action of beta-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) (LEE et al., 2012; PATERSON; BONOMO, 2005).

The ESBLs are classified into three major distinct structural and evolutionary families such as TEM, SHV and CTX-M (BUSH; JACOBY, 2010). Most genes encoding this type of antimicrobial resistance are located in conjugative plasmids or integron systems, and could be exchanged readily between enterobacteria species, facilitating their dissemination (CARATTOLI, 2013; WOODFORD et al., 2011). Current studies have reported the presence of ESBL producing E. coli in food, such as meat, milk and different types of cheese (GUNDOGAN; AVCI, 2013; SU et al., 2016; TEKINER; OZPINAR, 2016; VRABEC et al., 2015). Once beta-lactam resistant E. coli strains have become a global emerging problem in the last few years, carbapenems have been one of the main therapy options against human infections, including those caused by ESBL-producing bacteria (GENC et al., 2016; SPELLBERG et al., 2011). However, there are already reports of carbapenem resistance in isolates of clinical samples due to occurrence of enzymes named as carbapenemases (THOMSON, 2010). Although rare, some studies have isolated enterobacteria producing carbapenemase from foods such as seafood and vegetables (SINGH et al., 2016; ZURFLUH et al., 2015). Several types of carbapenemases were already described among Enterobacteriaceae, such as Klebsiella pneumoniae carbapenemase (KPC); zinc cofactor dependent carbapenemases known as metallo-beta-lactamases (MBL); oxacilinases (OXAs) (NORDMANN et al., 2011; TZOUVELEKIS et al., 2012); and cephalosporinases (AmpC) associated with the loss of porin (NORDMANN et al., 2012). The objective of this study was to evaluate the presence of virulence genes, antimicrobial susceptibility profiles and bifilm-production ability in E. coli isolates obtained from raw milk and artisanal cheese produced in Southern Brazil.

Material and Methods

Bacterial isolates

A total of 117 strains of Escherichia coli isolated from raw milk (n=8) and serrano artisanal cheese (n=109) were evaluated. These isolates were obtained from previous studies carried out by our research group (MELO et al., 2013; PONTAROLO et al., 2017; DALMINA, 2018) at the Centro de Diagnóstico Microbiológico Animal (CEDIMA) at Universidade do Estado de Santa Catarina (UDESC), in cooperation with serrano artisanal cheese producers from Santa Catarina, South region of Brazil.

Bacterial DNA extraction

DNA extraction of isolates and reference strains (to assure quality control) was performed according to protocol described by Doyle and Doyle (1987) with some modifications. Bacterial isolates were cultivated in Brain Heart Infusion broth (BD Difco, USA) at 37ºC for 24h, and consequently stored at -20ºC. An aliquot of 200 µL of each bacterial inoculum was transferred to a sterile microtube, and 500µl of chloroform: isoamyl alcohol (24:1) were added. The microtubes were placed in a water-bath at 56ºC for 30 min. After incubation period, microtubes were centrifuged for 10 min at 12,000 rpm. The supernatant of each sample was transferred to another sterile microtube, and supplemented with 600µL of 70% alcohol. The samples were further centrifuged for 20 min at 13,500 rpm. Then, supernatant was discarded by inversion and the pellet was dried off at room temperature. Finally, DNA samples were resuspended in 200µL of sterile Milli-Q water.

Detection of virulence genes

Detection of E. coli virulence genes was performed through an adapted Multiplex PCR based on protocols described by Toma et al. (2003) and López-Saucedo et al. (2003), using the primers listed in table 1. PCR amplifications were conducted in a 25µL final volume containing PCR buffer (Tris-HCl - 20mM, KCl - 50mM), MgCl₂ (2mM), dNTP (200mM of each), Taq DNA polymerase (0.5U), primers (4 pmol of each) and 2µl (15-60ng) bacterial DNA. All reagents were purchased from Invitrogen^® (Carlsbad, USA) and reactions were carried out in Thermal Cycler Applied Biosystem (model MJ96, Thermo Fisher, USA). The PCR program was carried as follows: initial denaturation at 95 °C for 5 min, 40 cycles of denaturation at 95 °C for 45s, annealing at 50 °C for 45s, extension at 72 °C for 45s, and final extension at 72 °C for 10 min. Amplification products were analyzed by electrophoresis (100V, 300mA) for 1h using 2% agarose gel. PCR products were stained with GelRed^TM and visualized on transilluminator (Kasvi, model K33-312, Brazil). The reference strains Escherichia coli INCQS 00170 (ATCC 43893) - EIEC; Escherichia coli INCQS 00171 (ATCC 43895) - EHEC; Escherichia coli INCQS 00180 (CDC O111ab) - EPEC (Fundação Oswaldo Cruz - Fiocruz); Escherichia coli ATCC 35401 - ETEC; Escherichia coli 3929 - L0815 - EAEC (Instituto Adolfo Lutz) were used as positive controls to assure quality control of the assays. Escherichia coli ATCC 25922 reference strain was used as a negative control.

Determination of antimicrobial susceptibility

Susceptibility of isolates to antimicrobial agents was defined by disk diffusion method, following the guidelines of Clinical and Standards Institute (CLSI), which recommend the use of plates containing Muller-Hinton Agar (BD Difco, USA) (CLSI, 2015). The plates were incubated for approximately 16-18h at 35+ 2ºC. All isolates were examined for susceptibility to the following antimicrobial agents: amoxicillin-clavulanate - AMC (20/10µg), aztreonam - ATM (30µg), cefepime - CPM (30µg), ceftazidime - CAZ (30µg); ceftriaxone - CRO (30µg), cefotaxime - CTX (30µg), meropenem - MER (10µg), imipenem - IPM (10µg), cefoxitin - CFO (30µg), ampicillin - AMP (10µg), tetracycline - TET (30µg), doxycycline - DOX (10µg), ciprofloxacin - CIP (5µg), norfloxacin - NOR (10µg), levofloxacin - LVX (5µg), tobramycin - TOB (10µg), gentamicin - GEN (10µg), amikacin - AMI (30µg), streptomycin - EST (10µg), chloramphenicol - CLO (30µg), trimethoprim/sulfamethoxazole - SUT (1.25/23.75µg) and nitrofurantoin - NIT (300µg). Inhibition zone diameters of microbial growth were measured and interpreted according to the breakpoints recommended by CLSI (2018). Isolates reported with an intermediate resistance for certain antimicrobial agents were considered resistant for statistical purposes, since the use of such antibiotics are not advisable for clinical treatment. Escherichia coli ATCC 25922 strain was used as quality control to determine susceptibility to antimicrobial agents.

Phenotypic and genotypic characterization of isolates producing extended-spectrum beta-lactamases (ESBL)

Screening for ESBL-producing E. coli isolates was carried out by the disk diffusion test as recommended by CLSI (2018). To perform this test, one amoxicillin-clavulanate disk was placed in the center of a Mueller-Hinton agar plate, and at a distance of 30 mm from other beta-lactam disks (ceftazidime, cefotaxime, ceftriaxone, cefepime, and aztreonam). The plate was incubated for 24h at 37ºC. After incubation, it was considered an indicative that a sample was ESBL-producer if there was an increase of the inhibition zone or the formation of a phantom zone, followed by distortion of the inhibition zone around the β-lactam disk (DRIEUX et al., 2008). The isolates that showed a phenotypic ESBL-producing profile were further investigated by multiplex PCR as described by Dallenne et al. (2010) with some modifications. One PCR approach was to detect the presence of the genes bla_TEM, bla_SHV, bla_OXA-1, whereas the other focused on the genes of groups bla_CTX-M-1, bla_CTX-M-2 e bla_CTX-M-9, using the primers listed in table 2.

To detect these genes, PCR mix contained PCR buffer (Tris-HCl - 20mM, KCl - 50mM), MgCl2 (1.5mM), dNTP (200mM of each), primers (4 pmol of each) and bacterial DNA (15-60ng) to result in a final volume of 25 µL. Thermal cycling parameters for the genes bla_TEM and bla_SHV, as well as for bla_CTX-M1, bla_CTX-M2 and bla_CTX-M9 were as follows: initial denaturation step at 94ºC for 10 min, followed by 40 cycles of 94ºC for 40s, 56ºC for 40s and 72ºC for 1 min, and a final extension at 72ºC for 7 min. Amplified fragments were separated electrophoretically (100V, 300mA) for 1h using 2% agarose gel. Amplicons were stained with GelRed^TM and visualized on transilluminator. Klebsiella pneumoniae CCBH5991 and Klebsiella pneumoniae CCBH15948 (both kindly donated by the Fundação Oswaldo Cruz - Fiocruz) were used as positive quality control to detect the genes bla_TEM+, bla_SHV+, bla_CTX-M+ and bla_OXA+. Escherichia coli ATCC 25922 reference strain was used as a negative control.

Phenotypic and genotypic characterization of carbapenemase-producing isolates

The isolates that exhibited resistance or intermediate resistance to carbapenems were further investigated. We placed meropenem and imipenem disks on Mueller Hinton agar petri dishes (non-supplemented) and, we also supplemented ones with EDTA, cloxacillin (CLOX) and phenylboronic acid (PhBA). Non-supplemented disks were used for comparison to supplemented disks. The interpretation consisted on checking if: a) isolates with an inhibition zone difference ≥ 5mm for imipenem or meropenem supplemented with EDTA (compared to non-supplemented) were considered potential metallo-beta-lactamase (MBL); b) isolates with an inhibition zone difference ≥ 5mm for carbapenem supplemented with phenylboronic acid in comparison to any substrate (imipenem or meropenem) were considered potential Klebsiella pneumoniae carbapenemase (KPC) producers; c) Inhibition zone difference > 5mm for antimicrobial disks supplemented with CLOXA and AFB (compared to non-supplemented substrate) were classified as plasmid-mediated AmpC; d) If zone difference (< 5mm) for non-supplemented and supplemented antimicrobial disks was observed, isolates were possibly considered producers of other β-lactamase (ex. OXA-48) or exhibited porin loss (ANVISA, 2013).

After phenotypic screening, PCR was carried out to detect the presence of the gene bla_OXA-48-LIKE using the following pair of primers: forward (5’ GCTTGATCGCCCTCGATT 3’) and reverse (5’ GATTTGCTCCGTGGCCGAAA 3’) (DALLENNE et al., 2010). Klebsiella pneumoniae CCBH23559 (Fundação Oswaldo Cruz - Fiocruz) and Escherichia coli ATCC 25922 reference strains was used as positive and negative quality control, respectively.

Evaluation of biofilm-production ability by Congo red agar

Biofilm formation of E. coli isolates was performed by the inoculation method in Congo red agar as described by Freeman et al. (1989). The isolates were cultivated into plates containing Congo red agar and incubated for 24h at 37ºC under aerobic conditions. Isolates considered biofilm producer formed black colonies with a dry crystalline consistency, while the non-biofilm producer ones formed red colonies with a smooth and darkened appearance in the center.

Results and Discussion

The virulence genes were identified in 25 out of the 117 studied isolates (21.37%) and three pathotypes of E. coli were found. EPEC was the most prevalent pathotype, since 15 isolates (12.82%) exhibited the presence of gene eae. ETEC was detected in 8 isolates (6.84%) by the presence of of gene st and/or lt, while EAEC was identified in 2 isolates (1.71%) that were positive for gene aggR (table 3). EAEC encondes genes that codify adhesin proteins, aggregative adherence factors, regulated by the expression of aggR gene. This transcriptional activator is responsible for the expression of plasmid-borne and chromosomal virulence factors. For this reason, detection of aggR gene can be used to characterize typical EAEC (BRÜSSOW, 2014; HARRIGTON et al., 2006).

These results were similar to several previous studies, like the one conducted by Holko et al. (2006). They found virulence gene in 9.47% of the E. coli isolates obtained from cheese produced by raw milk in Slovakia, being EPEC the most prevalent pathotype. Altalhi and Hassan (2009) detected the presence of gene eae, typically found in EPEC strains, in 9.1% of samples of raw milk and raw dairy products analyzed in Saudi Arabia. Further, Canizalez-Roman et al. (2013) investigated the frequency of diarrheagenic E. coli strains isolated in different types of food in Mexico, and among the analyzed dairy products, EPEC was the most prevalent (9.1%) pathogenic type, whereas EAEC and ETEC were detected in 1.4% and 0.7% of strains, respectively. On the other hand, de Campos et al. (2018) detected only the presence of a single diarrheagenic E. coli isolate (2.56%), characterized as EPEC, in different types of cheese produced with raw milk in Brazil. Ombarak et al. (2016) observed a high bacterial contamination rate of raw milk and raw cheese by potentially pathogenic E. coli isolates in Egypt due to the presence of virulence genes, being EPEC detected in only one sample (0.90%).

The presence of EPEC, ETEC and EAEC in samples of raw milk and artisanal cheese points major flaws in hygienic and sanitary measures adopted by the producers, not only at the milk collection but also during the processing (manufacture) of cheese, since the isolation of these microorganisms is reported in a wide range of animals used for food production (HERNANDES et al., 2009), such as dairy cattle (MONAGHAN et al., 2013; TÓTH et al., 2009), and humans are considered the main hosts/reservoirs for these pathotypes (HU; TORRES, 2015; YANG; WANG, 2014). Such types of food could be directly or indirectly contaminated by faeces of humans and/or infected animals, which become carriers and promote transmission of diarrheagenic E. coli isolates to consumers. Consequently, the consumption of such products might result in foodborne illness, an important public health issues, since subclinical, severe or fatal infections could be caused/developed in children and adults (DUBREUIL, 2014; YANG; WANG, 2014).

The antimicrobial susceptibility profile of 117 E. coli isolates, using 23 antibiotics, was phenotypically defined by disk diffusion method. In general, 62 isolates (52.99%) were considered susceptible to the tested antimicrobial agents. The results indicated that all isolates were susceptible to meropenem, norfloxacin, levofloxacin, tobramycin, amikacin and chloramphenicol (table 4). A total of 10 isolates (8.55%), all originally from cheese samples, exhibited a multi-drug resistance profile, which means they were resistant to three or more antimicrobial classes. On the other hand, 21 isolates (17.95%), two obtained from milk samples and the others from cheese, were resistant to two antimicrobial classes. These results were similar to the findings described by Guillén et al. (2014), which detected multiresistance in 11.1% of E. coli isolates obtained from artisanal dairy products manufactured in Venezuela. Ribeiro et al. (2016) and Ombarak et al. (2018) reported multiresistance rates lower than 20% in E. coli isolates collected from raw milk and/or cheese produced with raw milk in Brazil and Egypt, respectively. In contrast, de Campos et al. (2018) disclosed that 33.3% of E. coli strains, originally isolated from raw milk cheese produced in Brazil, were resistant to at least one antimicrobial agent, and only a single isolate (2.56%) was multidrug-resistant.

On the subject of susceptibility profile of 25 E. coli isolates potentially pathogenic (EPEC, ETEC and EAEC) found in this study, we observed: 11 (44%) were resistant to two or more tested antimicrobial agents, six (24%) were resistant to two antimicrobial classes (6C, 7C, 9C, 10C, 16C, 21C), and three isolates (12%) were multi-drug resistant (4C, 5C and 20C) (table 3). Our results showed consistency with those presented by Canizalez-Roman et al. (2013), which demonstrated that 39.2% of diarrheagenic E. coli strains isolated in food items manufactured in Mexico, including dairy products, were resistant to two or more antibiotics. In addition, Wang et al. (2017) verified that 61% of diarrheagenic E. coli isolates, recovered from different types of food commercialized in local Japanese retail markets, were resistant to at least one of the test antimicrobial agents, and 70% of them were resistant to two or more antibiotics. Moreover, Gómez-Aldapa et al. (2016) recently detected multiresistance in all diarrheagenic isolates recovered from coriander samples in Mexico.

Transmission of virulence genes and resistance by E. coli to other intestinal pathogenic bacteria is often plasmid-associated. Therefore, industrialized food and animal source food are potential reservoirs for antimicrobial resistant and virulent bacteria (KIRBIS; KRIZMAN, 2015). Since we recovered pathogenic antibiotic-resistant E. coli isolates from raw milk and serrano artisanal cheese, it is of utmost importance to point out questions related to food safety regulations, regarding aspects of quality raw material, primary production, processing, retailing and consumer-handling, as well as education in hygienic handling of food for individuals involved in the manufacturing process.

An investigation of ESBL-producing isolates was also carried out, and we found out that four (3.42%) samples, one from milk and the others from cheese, were positive for ESBL phenotype and harbored the bla_TEM gene. One isolate showed multiresistance profile (C); whereas another one (D), characterized as EPEC, exhibited resistance to 80% of the tested β-lactams (table 5). Likewise, studies worldwide also reported the presence of ESBL-producing E. coli isolates in raw milk and/or cheese manufactured in Turkey (TEKINER; OZPINAR, 2016; TEPELI; ZORBA, 2018), Egypt (OMBARAK et al., 2018), Czech Republic (SKOCKOVÁ et al., 2015), Brazil (RIBEIRO et al., 2016) and Slovakia (VRABEC et al., 2015), being the bla_TEM gene the most prevalent among the recovered samples. ESBL-producing microorganisms represent a serious global health concern because they are frequently isolated (BOUCHILLON et al., 2004), and the group of β-lactams is still the most commonly worldwide used antimicrobial agent against Gram-negative bacterial infections (OCA et al., 2015), which lately leads to complicated treatment strategies.

Since three isolates showed intermediate resistance to imipenem, a phenotypic screening of carbapenemase-producing E. coli was performed. However, all isolates were negative for this resistance profile. Based on this result, we carried out PCRs to detect the presence of OXA-48 gene, but none of the mentioned isolates were positive for it. In this case, intermediate resistance might be due to apparent loss or reduction in the expression level of porins present in these isolates (ANVISA, 2013).

Additionally, we verified the ability of E. coli isolates to potential form biofilm by the Congo red method. More than half of E. coli isolates (67.52% out of 117 isolates) were considered potential biofilm producers. It is important to mention that there are other well-established methods to define and quantify biofilm-producer strains (AZEREDO et al., 2017). However, previous studies have demonstrated that Congo red agar method results well-correlated with the ones obtained by tissue culture plate and tube methods (PONNUSAMY et al., 2012) and by microtiter plate method (CHAGAS et al., 2017). For this reason, this method could be further performed in order to investigate the potential ability of E. coli isolates to form biofilm.

Out of the 79 isolates positive for Congo red agar test, 42 (53.16%) were resistant to at least one of the tested antimicrobial agents. The biofilm formation profile of diarrheagenic E. coli isolates is shown in table 3 and revealed that 75% of them were considered potential biofilm-producers and resistant to at least one antibiotic. Analyses of the ten multi-drug resistant isolates found in the current study revealed that eight (80%) were considered potential biofilm-producers. This finding is in line with the results obtained by Ponnusamy et al. (2012), emphasizing a possible association between these factors.

Biofilm formation in food processing environments depends on several factors, and dairy products are very susceptible to contamination especially due to unsuitable hygiene of machines and utensils (SREY et al., 2013). Biofilms are microbial communities embedded in an extracellular matrix composed mainly by polysaccharides and proteins adhered to the surface, and they remarkable underpin bacteria against effects of antibacterial treatments (COSTERTON et al, 1987; HOBLEY et al., 2015). This fact emphasizes even more the demand for actions to improve serrano artisanal cheese manufacturing processes in order to guarantee the quality of these foodstuffs for consumers.

Conclusions

This study reveals the presence of diarrheagenic E. coli isolates (EPEC, ETEC and EAEC) recovered from raw milk and artisanal cheese produced in Santa Catarina, South region of Brazil. We identified isolates that exhibited multi-drug resistance and ESBL-producing profiles, and the potential ability to form biofilms. These results warn of a serious public health issue, since these contaminated foods offer risks to consumers’ health. For that reason, they underpin the need to intensify hygienic-sanitary controls in all steps of production, and also to implement measures that allow epidemiological control of potential pathogenic bacterial strains in artisanal cheese produced in the Serrana region of Santa Catarina, South of Brazil.

Acknowledgements

We thank the Fundação Oswaldo Cruz (Fiocruz) and the Instituto Adolfo Lutz for providing the reference strains.

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Author notes

leandro.parussolo@ifsc.edu.br