1148 Journal of Food Protection, Vol. 77, No. 7, 2014, Pages 1148-1152 doi: 10.4315/0362-028X.JFP-13-434 C o p y rig h t© , International A sso ciation fo r Food Protection
Low Occurrence of Listeria monocytogenes on Bovine Hides and Carcasses in Minas Gerais State, Brazil: Molecular Characterization and Antimicrobial Resistance ANDERSON CARLOS CAMARGO, ANDREA LAFISCA, MARCUS VINICIUS COUTINHO COSSI, FREDERICO GERMANO PISCITELLI ALVARENGA LANNA, MARIANE REZENDE DIAS, PAULO SERGIO d e ARRUDA PINTO, a n d LUIS AUGUSTO NERO* Universidade Federal de Vigosa, Departamento de Veterinaria, Campus UFV, 36570 000, Vigosa, Minas Gerais, Brazil MS 13-434: Received 9 October 2013/Accepted 28 February 2014 A BSTR A CT Listeria monocytogenes occurrence was assessed in three slaughterhouses located in Minas Gerais state, Brazil, by analysis of 209 bovine carcasses. Four sponge samples were obtained from each carcass in different steps (A, from hide, before bleeding; B, after hide removal; C, after evisceration; and D, after end washing), resulting in a total of 836 samples. The samples were tested for the presence of L. monocytogenes according to the International Organization for Standardization 11290-1, and positive results were recorded in steps A (1 of 209) and D (1 of 209) from slaughterhouse 03. L. monocytogenes isolates (n — 5) were identified by multiplex PCR as belonging to serogroup lie (representing serotypes l/2c or 3c) and presented identical pulsed-field gel electrophoresis profiles; in addition, the isolates harbored the virulence genes inlA, inlB, inlC, inU, plcA, hlyA, actA, and iap and were sensitive to ampicillin, vancomycin, gentamicin, erythromycin, tetracycline, rifampin, chloramphenicol, trimethoprim, and sulfamethoxazole. The obtained data indicated a low occurrence of L. monocytogenes on bovine hides and carcasses from slaughterhouses located in Minas Gerais state, Brazil, and the presence of distinct virulence makers and susceptibility to a variety of antimicrobials by the obtained isolates.
Listeria monocytogenes is a foodbome pathogen that is frequently associated with meat and ready-to-eat products, and it is the causative agent of listeriosis, a severe disease that is of particularly high risk for immune compromised persons, children, the elderly, and pregnant women (2, 31). The invasive form of listeriosis is characterized by septicemia, stillbirths and abortions, encephalitis, and meningitis, which lead to mortality rates between 20 and 30%, highlighting its significance for food safety (33). The different steps of bovine slaughtering and beef processing can be considered relevant sources of L. monocytogenes contamination (11, 14, 35). The slaughter house is naturally contaminated with L. monocytogenes, and the animal hides are often associated with the continuous introduction of novel strains in such an environment (26, 34). The characterization of the genetic profiles of isolates by DNA enzymatic macrorestriction, followed by pulsedfield gel electrophoresis (PFGE), and the serogroup identification are relevant tools to demonstrate this contin uous contamination of L. monocytogenes in the beef processing chain (13, 24). Serogrouping is important to predict the possible risks to consumers: strains from serotypes l/2a, l/2b, and 4b are considered the most vimlent, being frequently associated
with human listeriosis cases and outbreaks (24). Detection and sequencing of vimlence genes in L. monocytogenes isolates are also important to identify the risk and possible pathogenesis in consumers (19). A variety of proteins are described as associated with the virulence activity of L. monocytogenes, such as intemalins, listeriolysins, and phospholipases (6). In addition, the characterization of the susceptibility of a range of antimicrobials is relevant to identify the resistance profiles of strains associated with foods and listeriosis cases and outbreaks (18). This study aimed to identify the occurrence of L. monocytogenes on bovine hides and carcasses during slaughtering in Minas Gerais state, Brazil, and to conduct further analysis with the obtained isolates to characterize their genetic profiles, main virulence genes, and antimicro bials resistance profiles.
* Author for correspondence. Tel: + 55 31 3899 1463; Fax: + 55 31 3899 1457; E-mail: [email protected].
Slaughterhouse 01 (SOI): daily slaughtering of 150 to 180 bovines, conducted by approximately 50 employees, and absence
M A TER IA LS A N D M E TH O D S Slaughtering facilities and sampling. Three bovine slaugh terhouses were selected for the present study, all of them located in Minas Gerais state, Brazil, and subjected to Brazilian official inspection services. All selected slaughterhouses have hazard analysis and critical control point programs, and their key characteristics are described in the following:
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of a physical division between dirty and clean areas. SOI exports only viscera, and beef cuts are distributed for sale in different Brazilian states. Slaughterhouse 02 (S02): bovine slaughtering occurs two or three times a week, with 90 to 100 bovines being processed by approximately 25 employees, and absence of a physical division between dirty and clean areas. S02 is not allowed to export any beef product; consequently, its production is limited exclusively to the Brazilian market. Slaughterhouse 03 (S03): daily slaughtering of 130 to 150 bovines, conducted by approximately 50 employees, and absence of a physical division between dirty and clean areas. S03 is allowed to export a diversity of beef products, which are also distributed for sale in the Brazilian market. The selected slaughterhouses were visited at least seven times each (SOI, 8; S02, 7; and S03, 14) at intervals of 2 to 3 weeks to obtain 836 surface samples from 209 animals and carcasses (SOI, 70; S02, 70; and S03, 69) at four stages of the slaughtering process (A, from hide, before bleeding; B, after hide removal; C, after evisceration; and D, after end washing). At each stage, four sterile plastic templates of 100 cm2 (10 by 10 cm) were placed on the shoulder and chest areas on both sides of each bovine carcass (steps A and B) or in the same areas in the inner and outer regions of both half carcasses (steps C and D), following the recommen dations of the International Organization for Standardization 17604 (16) and EC 1441 (9). The delimited areas were then swabbed using sterile sponges (3M Microbiology, St. Paul, MN) previously moistened with 10 ml of buffered peptone (1%, wt/vol) saline (0.85%, wt/vol) solution (Oxoid Ltd., Basingstoke, UK) and kept under refrigeration for a maximum of 4 h until processing. In sterile conditions, each set of four sponges from each slaughtering step was transferred to a sterile bag, and 160 ml of buffered peptone saline solution was added. The samples were then homogenized for 60 s (Stomacher 400 Circulator, Seward Ltd., Easting Close, Sussex, UK) and subjected to microbiological analysis. Detection of Listeria spp. and L. monocytogenes. All collected samples were subjected to Listeria spp. analysis according to the International Organization for Standardization 11290-1 (15, 17). Aliquots of 40 ml (corresponding to 80 cm2) from each sample were centrifuged at 4°C for 15 min at 1,000 x g, and after discarding the supernatant, the obtained pellet was resuspended in 10 ml of half Fraser broth, followed by incubation at 30°C for 24 h. Then, 0.1 ml of the cultures were transferred to 10 ml of Fraser broth and incubated at 35°C for 48 h. Aliquots were streaked on chromogenic Listeria agar and modified Oxford agar plates and incubated at 35°C for 24 to 48 h. All culture media were from Oxoid. Up to five presumptive-positive Listeria spp. colonies from both culture media, per sample, were streaked on tryptone soya agar (Oxoid), incubated at 30°C for 24 h, and isolated colonies were subject to biochemical identification according to the International Organization for Standardization 11290-1 (15): catalase production, hemolysis in horse blood agar, motility at 25 °C, and fermentation pattern of dextrose, xylose, rhamnose, and mannitol. Isolates identified as Listeria spp. were transferred to tryptone soya broth (Oxoid), incubated at 30°C for 24 h, subjected to DNA extraction and purification using the Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI) and subjected to molecular serogrouping according to Doumith et al. (7), with modifications. Reactions of 25 pi consisted of 12.5 pi of GoTaq Green Master Mix (Promega), 2.0 pi of the extracted DNA, 8.4 pi
1149
of the five pairs of primers, and 2.1 pi of ultrapure PCR water (Promega). PCR products were subjected to electrophoresis on 2.0% (wt/vol) agarose gels in 0.5 x Tris-borate-EDTA buffer, stained with GelRed (Biotium Inc., Hayward, CA), and visualized in a transilluminator. L. monocytogenes ATCC 7644 was tested in parallel as a positive control for isolation procedure and molecular serogrouping. DNA macrorestriction and PFGE. All isolates identified as L. monocytogenes were subjected to DNA macrorestriction, according to the protocol described by Graves and Swaminathan (13), as recommended by PulseNet (available at http://www.cdc. gov/pulsenet/pathogens/listeria.html, Centers for Disease Control and Prevention, Atlanta, GA). AscI and Apal restriction enzymes (New England Biolabs Inc., Ipswich, MA) were used to digest the DNA of the isolate cultures, and the macrorestriction products were separated in agarose gels at 1% (wt/vol) by PFGE using a CHEF-DR II Variable Angle System (Bio-Rad Lab., Hercules, CA) adjusted using the following parameters: 20 h, 6 V/cm gradient, and 4 to 40 s interpooled pulses. Macrorestriction profiles were analyzed using BioNumerics 6.6 (Applied Maths, SintMartens-Latem, Ghent, Belgium) based on 1.5% optimization and 1.5% Dice similarity of bands. The unweighted pair group method using averages was considered to cluster the obtained patterns. Pulse Marker 50 (1,000 kb; Sigma-Aldrich Corp., St. Louis, MO) was used as a reference. Virulence genes. The DNA of all L. monocytogenes isolates was subjected to PCR reactions to detect the following virulence genes: inlA, M B, inlC, inlJ, plcA, hlyA, actA, and iap. The protocols for each PCR set (multiplex or simplex) were performed according to Liu et al. (20) and Rawool et al. (28), with modifications. Reactions of 25 pi composed of 12.5 pi of GoTaq Green Master Mix (Promega), 2.0 pi of the extracted DNA, 1.0 pi of each primer, and ultrapure PCR water (Promega) to make up the final volume. Primers sequences, PCR conditions, and expected fragment sizes were previously described and considered in the present study (10, 20, 23, 25, 30). PCR products were separated and visualized, as described above. L. monocytogenes ATCC 7644 was tested in parallel as a positive control for the tested virulence genes. Antimicrobial resistance. L. monocytogenes strains were subjected to phenotypical analysis to detect their resistance against nine total antimicrobials using Etest (bioMerieux, 1'Etoile, France) (rifampin, chloramphenicol, trimethoprim, and sulfamethoxazole) and M.I.C. Evaluator Strips (Oxoid) (ampicillin, gentamicin, erythromycin, tetracycline, and vancomycin). Cultures were transferred to brain heart infusion (Oxoid), incubated at 35°C overnight, and diluted in 0.85% NaCl (wt/vol) until turbidity similar to 0.5 MacFarland. Diluted cultures were swabbed onto the surface of Mueller-Hinton agar (Oxoid), and the antimicrobial strips were added. After incubation at 35°C for 18 and 24 h, the MIC obtained for each antimicrobial agent tested was recorded (micrograms per milliliter), and their resistance profiles were classified as sensitive, intermediate, and resistant, as described by the manufacturers for gram-positive aerobic organisms (bioMer ieux and Oxoid).
RESULTS AND DISCUSSION The occurrence of Listeria spp. and L. monocytogenes at different stages of slaughtering of bovines is presented in Table 1. SOI produced no positive results for Listeria spp. at
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TABLE 1. Occurrence o f Listeria spp. and L. monocytogenes on bovine carcasses sampled in four steps o f slaughtering from three slaughterhouses located in Minas Gerais state, Brazila Listeria spp JL. monocytogenes occurrence Slaughterhouse
n
A
B
c
D
SOI S02 S03
70 70 69
0/0 0/0 3/1
0/0 I/O 0/0
0/0 0/0 4/0
Total
209
3/1
1/0
4/0
0/0 3/0 1/1 4/1
“ A, hide, before bleeding; B, after hide removal; C, after evisceration; D, after end washing. any stage, and S02 produced positive results for Listeria innocua at low frequencies at stages B (1 of 70) and D (3 of 70). Only S03 gave positive results for L. monocytogenes at stages A (1 of 69) and D (1 of 69), as well as positive results for L. innocua at stage A (2 of 69) and C (4 of 69), and Listeria welshimeri at stage C (1 of 69). Similar results for Listeria spp. and L. monocytogenes occurrence on bovine slaughtering have been previously reported. Vanderline et al. (32) detected low frequencies (0.59%) of L. monocyto genes on bovine carcasses from slaughterhouses in Australia that process beef for exporting and only after the end washing step. In a study conducted in the United States, Rivera-Betancourt et al. (29) identified different frequencies of L. monocytogenes on bovine hides and carcasses, only before evisceration. Guerini et al. (14) found variable frequencies (0.0 to 47.9%) of L. monocytogenes positive hides and carcasses, before and after evisceration from different slaughterhouses located in the United States. The same study demonstrated that Listeria species were more prevalent on hides during the winter and spring. In a study conducted in Ireland, L. monocytogenes was detected in 10 (4.76%) bovine feces before slaughtering (21). As observed in the present study (Table 1), the bovine carcasses were usually contaminated by L. monocytogenes at the initial or the end stages of slaughtering. In a study conducted in three slaughterhouses located in China, Zhu et al. (36) observed low prevalence of L. monocytogenes on bovine hides and carcasses from two facilities, and high prevalence in the other one, including the presence of the pathogen in the end cuts, with isolates identified as belonging to serotype l/2c. Thirty isolated colonies were confinned as Listeria spp. by biochemical analysis. The molecular serogrouping
100 ___ i
AscI
Apa\
, 1 lit i lit i l l [ill .4
SIH1S«
li'h
confirmed all isolates as belonging to genus Listeria based on the positive results for the prs gene (7), and the five isolates identified as L. monocytogenes were classified as belonging to serogroup lie, which includes serotypes l/2c and 3c (8). According to Orsi et al. (24), serotype 3c belongs to lineage I and serotype l/2c belongs to lineage II. L. monocytogenes serotype l/2c is usually isolated from foods and industry environment (7, 24) and has also been isolated from bovine hides and carcasses before and after eviscer ation (14, 34), as observed in the present study. Even identifying the low frequency of positive results for L. monocytogenes, and the identification of only five isolates (named as LM01, from a sample collected at stage A, visit 7, and LM02, LM03, LM04, and LM05, from samples collected at stage D, visit 12), PFGE was performed to characterize their genetic profiles. PFGE analysis demonstrated that all L. monocytogenes isolates presented an identical genetic profile (Fig. 1). This result suggests that isolates with identical genetic profiles are being constantly introduced in the slaughtering environment or the mainte nance of this specific strain in S03. A number of different risk factors that are strain based (adherence potential, sanitizer tolerance, and stress tolerance) and facility based (presence of harborage sites) have been hypothesized to facilitate the persistence of L. monocytogenes in the processing environment for extend periods of time to explain this finding (2). The L. monocytogenes isolates presented positive results for all virulence genes tested by PCR. These genes play important roles in the virulence mechanisms of L. monocytogenes and are involved in different stages of its pathogenesis (1, 24). The intemalin-related genes (iniA, inlB, inlC, and inlJ) are involved in cell adhesion and invasion, and plcA is associated with phospholipase C production (33). After the invasion of host cells, additional L. monocytogenes virulence genes are expressed, including hlyA, which is involved in the production of listeriolysin-O; actA, which is responsible for the production of ActA protein; and iap, which is associated with the expression of the p60 protein that plays an important role in intestinal cell invasion and survival (33). Similar results were observed by Liu et al. (20), who detected the same intemalin genes (inlA, inlB, inlC, and inlJ) in L. monocytogenes isolates belonging to serotypes l/2c or 3c. L. monocytogenes isolates that represent a potential risk to humans express a full-length isolate
serogroup
step
LM01
lie
A
LM02
lie
D
LM03
lie
D
LM04
lie
D
LM05
lie
D
FIGURE 1. PFGE patterns after the macrorestriction (ApaI and Asc/) o f five Listeria monocytogenes isolates from bovine hides and carcasses in distinct steps o f slaughtering (A, before bleeding; D, after end washing) in visits in a slaughterhouse (S03) located in Minas Gerais state, Brazil. Similarities between the identified PFGE pulsotypes were estimated using the Dice coefficient (1.5% tolerance).
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functional InlA, demanding the sequencing of the ini genes of obtained isolates to identify properly their vimlence potential (27). The analysis for resistance profiles indicated that all L. monocytogenes isolates presented sensitivity to all tested antimicrobials. Similar results were observed by Wieczorek et al. (34). Most isolates recovered from raw meat or food processing environments are susceptible to the majority of antimicrobials considered important in listeriosis treatment (4,18). Antimicrobial resistance by microorganisms can be developed as a consequence of endogenous and exogenous factors, and the environment plays an important role allowing for interactions with other bacteria and consequent gene or plasmid transfer (3,5). The absence of antimicrobial resistance identified in the present study suggests that L. monocytogenes isolates did not harbor the related genes or these genetic regions are not being expressed, demanding complementary analysis for a proper characterization of this activity (12, 22). In conclusion, the obtained results demonstrated the low frequencies of L. monocytogenes on bovine hides and carcasses during slaughtering in facilities located in Minas Gerais state, Brazil. The obtained L. monocytogenes isolates were susceptible to different antimicrobials and harbored different virulence genes, demanding further analysis to assess their pathogenic potential.
10.
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ACKNOWLEDGMENTS The authors thank Conselho Nacional de Desenvolvimento Cientffico e Tecnologico (CNPq), Coordenajao de Aperfeijoamento de Pessoal de Nivel Superior (CAPES), and Fundafao de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG).
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Low Occurrence of Listeria monocytogenes on Bovine Hides and Carcasses in Minas Gerais State, Brazil: Molecular Characterization and Antimicrobial Resistance ANDERSON CARLOS CAMARGO, ANDREA LAFISCA, MARCUS VINICIUS COUTINHO COSSI, FREDERICO GERMANO PISCITELLI ALVARENGA LANNA, MARIANE REZENDE DIAS, PAULO SERGIO d e ARRUDA PINTO, a n d LUIS AUGUSTO NERO* Universidade Federal de Vigosa, Departamento de Veterinaria, Campus UFV, 36570 000, Vigosa, Minas Gerais, Brazil MS 13-434: Received 9 October 2013/Accepted 28 February 2014 A BSTR A CT Listeria monocytogenes occurrence was assessed in three slaughterhouses located in Minas Gerais state, Brazil, by analysis of 209 bovine carcasses. Four sponge samples were obtained from each carcass in different steps (A, from hide, before bleeding; B, after hide removal; C, after evisceration; and D, after end washing), resulting in a total of 836 samples. The samples were tested for the presence of L. monocytogenes according to the International Organization for Standardization 11290-1, and positive results were recorded in steps A (1 of 209) and D (1 of 209) from slaughterhouse 03. L. monocytogenes isolates (n — 5) were identified by multiplex PCR as belonging to serogroup lie (representing serotypes l/2c or 3c) and presented identical pulsed-field gel electrophoresis profiles; in addition, the isolates harbored the virulence genes inlA, inlB, inlC, inU, plcA, hlyA, actA, and iap and were sensitive to ampicillin, vancomycin, gentamicin, erythromycin, tetracycline, rifampin, chloramphenicol, trimethoprim, and sulfamethoxazole. The obtained data indicated a low occurrence of L. monocytogenes on bovine hides and carcasses from slaughterhouses located in Minas Gerais state, Brazil, and the presence of distinct virulence makers and susceptibility to a variety of antimicrobials by the obtained isolates.
Listeria monocytogenes is a foodbome pathogen that is frequently associated with meat and ready-to-eat products, and it is the causative agent of listeriosis, a severe disease that is of particularly high risk for immune compromised persons, children, the elderly, and pregnant women (2, 31). The invasive form of listeriosis is characterized by septicemia, stillbirths and abortions, encephalitis, and meningitis, which lead to mortality rates between 20 and 30%, highlighting its significance for food safety (33). The different steps of bovine slaughtering and beef processing can be considered relevant sources of L. monocytogenes contamination (11, 14, 35). The slaughter house is naturally contaminated with L. monocytogenes, and the animal hides are often associated with the continuous introduction of novel strains in such an environment (26, 34). The characterization of the genetic profiles of isolates by DNA enzymatic macrorestriction, followed by pulsedfield gel electrophoresis (PFGE), and the serogroup identification are relevant tools to demonstrate this contin uous contamination of L. monocytogenes in the beef processing chain (13, 24). Serogrouping is important to predict the possible risks to consumers: strains from serotypes l/2a, l/2b, and 4b are considered the most vimlent, being frequently associated
with human listeriosis cases and outbreaks (24). Detection and sequencing of vimlence genes in L. monocytogenes isolates are also important to identify the risk and possible pathogenesis in consumers (19). A variety of proteins are described as associated with the virulence activity of L. monocytogenes, such as intemalins, listeriolysins, and phospholipases (6). In addition, the characterization of the susceptibility of a range of antimicrobials is relevant to identify the resistance profiles of strains associated with foods and listeriosis cases and outbreaks (18). This study aimed to identify the occurrence of L. monocytogenes on bovine hides and carcasses during slaughtering in Minas Gerais state, Brazil, and to conduct further analysis with the obtained isolates to characterize their genetic profiles, main virulence genes, and antimicro bials resistance profiles.
* Author for correspondence. Tel: + 55 31 3899 1463; Fax: + 55 31 3899 1457; E-mail: [email protected].
Slaughterhouse 01 (SOI): daily slaughtering of 150 to 180 bovines, conducted by approximately 50 employees, and absence
M A TER IA LS A N D M E TH O D S Slaughtering facilities and sampling. Three bovine slaugh terhouses were selected for the present study, all of them located in Minas Gerais state, Brazil, and subjected to Brazilian official inspection services. All selected slaughterhouses have hazard analysis and critical control point programs, and their key characteristics are described in the following:
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of a physical division between dirty and clean areas. SOI exports only viscera, and beef cuts are distributed for sale in different Brazilian states. Slaughterhouse 02 (S02): bovine slaughtering occurs two or three times a week, with 90 to 100 bovines being processed by approximately 25 employees, and absence of a physical division between dirty and clean areas. S02 is not allowed to export any beef product; consequently, its production is limited exclusively to the Brazilian market. Slaughterhouse 03 (S03): daily slaughtering of 130 to 150 bovines, conducted by approximately 50 employees, and absence of a physical division between dirty and clean areas. S03 is allowed to export a diversity of beef products, which are also distributed for sale in the Brazilian market. The selected slaughterhouses were visited at least seven times each (SOI, 8; S02, 7; and S03, 14) at intervals of 2 to 3 weeks to obtain 836 surface samples from 209 animals and carcasses (SOI, 70; S02, 70; and S03, 69) at four stages of the slaughtering process (A, from hide, before bleeding; B, after hide removal; C, after evisceration; and D, after end washing). At each stage, four sterile plastic templates of 100 cm2 (10 by 10 cm) were placed on the shoulder and chest areas on both sides of each bovine carcass (steps A and B) or in the same areas in the inner and outer regions of both half carcasses (steps C and D), following the recommen dations of the International Organization for Standardization 17604 (16) and EC 1441 (9). The delimited areas were then swabbed using sterile sponges (3M Microbiology, St. Paul, MN) previously moistened with 10 ml of buffered peptone (1%, wt/vol) saline (0.85%, wt/vol) solution (Oxoid Ltd., Basingstoke, UK) and kept under refrigeration for a maximum of 4 h until processing. In sterile conditions, each set of four sponges from each slaughtering step was transferred to a sterile bag, and 160 ml of buffered peptone saline solution was added. The samples were then homogenized for 60 s (Stomacher 400 Circulator, Seward Ltd., Easting Close, Sussex, UK) and subjected to microbiological analysis. Detection of Listeria spp. and L. monocytogenes. All collected samples were subjected to Listeria spp. analysis according to the International Organization for Standardization 11290-1 (15, 17). Aliquots of 40 ml (corresponding to 80 cm2) from each sample were centrifuged at 4°C for 15 min at 1,000 x g, and after discarding the supernatant, the obtained pellet was resuspended in 10 ml of half Fraser broth, followed by incubation at 30°C for 24 h. Then, 0.1 ml of the cultures were transferred to 10 ml of Fraser broth and incubated at 35°C for 48 h. Aliquots were streaked on chromogenic Listeria agar and modified Oxford agar plates and incubated at 35°C for 24 to 48 h. All culture media were from Oxoid. Up to five presumptive-positive Listeria spp. colonies from both culture media, per sample, were streaked on tryptone soya agar (Oxoid), incubated at 30°C for 24 h, and isolated colonies were subject to biochemical identification according to the International Organization for Standardization 11290-1 (15): catalase production, hemolysis in horse blood agar, motility at 25 °C, and fermentation pattern of dextrose, xylose, rhamnose, and mannitol. Isolates identified as Listeria spp. were transferred to tryptone soya broth (Oxoid), incubated at 30°C for 24 h, subjected to DNA extraction and purification using the Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI) and subjected to molecular serogrouping according to Doumith et al. (7), with modifications. Reactions of 25 pi consisted of 12.5 pi of GoTaq Green Master Mix (Promega), 2.0 pi of the extracted DNA, 8.4 pi
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of the five pairs of primers, and 2.1 pi of ultrapure PCR water (Promega). PCR products were subjected to electrophoresis on 2.0% (wt/vol) agarose gels in 0.5 x Tris-borate-EDTA buffer, stained with GelRed (Biotium Inc., Hayward, CA), and visualized in a transilluminator. L. monocytogenes ATCC 7644 was tested in parallel as a positive control for isolation procedure and molecular serogrouping. DNA macrorestriction and PFGE. All isolates identified as L. monocytogenes were subjected to DNA macrorestriction, according to the protocol described by Graves and Swaminathan (13), as recommended by PulseNet (available at http://www.cdc. gov/pulsenet/pathogens/listeria.html, Centers for Disease Control and Prevention, Atlanta, GA). AscI and Apal restriction enzymes (New England Biolabs Inc., Ipswich, MA) were used to digest the DNA of the isolate cultures, and the macrorestriction products were separated in agarose gels at 1% (wt/vol) by PFGE using a CHEF-DR II Variable Angle System (Bio-Rad Lab., Hercules, CA) adjusted using the following parameters: 20 h, 6 V/cm gradient, and 4 to 40 s interpooled pulses. Macrorestriction profiles were analyzed using BioNumerics 6.6 (Applied Maths, SintMartens-Latem, Ghent, Belgium) based on 1.5% optimization and 1.5% Dice similarity of bands. The unweighted pair group method using averages was considered to cluster the obtained patterns. Pulse Marker 50 (1,000 kb; Sigma-Aldrich Corp., St. Louis, MO) was used as a reference. Virulence genes. The DNA of all L. monocytogenes isolates was subjected to PCR reactions to detect the following virulence genes: inlA, M B, inlC, inlJ, plcA, hlyA, actA, and iap. The protocols for each PCR set (multiplex or simplex) were performed according to Liu et al. (20) and Rawool et al. (28), with modifications. Reactions of 25 pi composed of 12.5 pi of GoTaq Green Master Mix (Promega), 2.0 pi of the extracted DNA, 1.0 pi of each primer, and ultrapure PCR water (Promega) to make up the final volume. Primers sequences, PCR conditions, and expected fragment sizes were previously described and considered in the present study (10, 20, 23, 25, 30). PCR products were separated and visualized, as described above. L. monocytogenes ATCC 7644 was tested in parallel as a positive control for the tested virulence genes. Antimicrobial resistance. L. monocytogenes strains were subjected to phenotypical analysis to detect their resistance against nine total antimicrobials using Etest (bioMerieux, 1'Etoile, France) (rifampin, chloramphenicol, trimethoprim, and sulfamethoxazole) and M.I.C. Evaluator Strips (Oxoid) (ampicillin, gentamicin, erythromycin, tetracycline, and vancomycin). Cultures were transferred to brain heart infusion (Oxoid), incubated at 35°C overnight, and diluted in 0.85% NaCl (wt/vol) until turbidity similar to 0.5 MacFarland. Diluted cultures were swabbed onto the surface of Mueller-Hinton agar (Oxoid), and the antimicrobial strips were added. After incubation at 35°C for 18 and 24 h, the MIC obtained for each antimicrobial agent tested was recorded (micrograms per milliliter), and their resistance profiles were classified as sensitive, intermediate, and resistant, as described by the manufacturers for gram-positive aerobic organisms (bioMer ieux and Oxoid).
RESULTS AND DISCUSSION The occurrence of Listeria spp. and L. monocytogenes at different stages of slaughtering of bovines is presented in Table 1. SOI produced no positive results for Listeria spp. at
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TABLE 1. Occurrence o f Listeria spp. and L. monocytogenes on bovine carcasses sampled in four steps o f slaughtering from three slaughterhouses located in Minas Gerais state, Brazila Listeria spp JL. monocytogenes occurrence Slaughterhouse
n
A
B
c
D
SOI S02 S03
70 70 69
0/0 0/0 3/1
0/0 I/O 0/0
0/0 0/0 4/0
Total
209
3/1
1/0
4/0
0/0 3/0 1/1 4/1
“ A, hide, before bleeding; B, after hide removal; C, after evisceration; D, after end washing. any stage, and S02 produced positive results for Listeria innocua at low frequencies at stages B (1 of 70) and D (3 of 70). Only S03 gave positive results for L. monocytogenes at stages A (1 of 69) and D (1 of 69), as well as positive results for L. innocua at stage A (2 of 69) and C (4 of 69), and Listeria welshimeri at stage C (1 of 69). Similar results for Listeria spp. and L. monocytogenes occurrence on bovine slaughtering have been previously reported. Vanderline et al. (32) detected low frequencies (0.59%) of L. monocyto genes on bovine carcasses from slaughterhouses in Australia that process beef for exporting and only after the end washing step. In a study conducted in the United States, Rivera-Betancourt et al. (29) identified different frequencies of L. monocytogenes on bovine hides and carcasses, only before evisceration. Guerini et al. (14) found variable frequencies (0.0 to 47.9%) of L. monocytogenes positive hides and carcasses, before and after evisceration from different slaughterhouses located in the United States. The same study demonstrated that Listeria species were more prevalent on hides during the winter and spring. In a study conducted in Ireland, L. monocytogenes was detected in 10 (4.76%) bovine feces before slaughtering (21). As observed in the present study (Table 1), the bovine carcasses were usually contaminated by L. monocytogenes at the initial or the end stages of slaughtering. In a study conducted in three slaughterhouses located in China, Zhu et al. (36) observed low prevalence of L. monocytogenes on bovine hides and carcasses from two facilities, and high prevalence in the other one, including the presence of the pathogen in the end cuts, with isolates identified as belonging to serotype l/2c. Thirty isolated colonies were confinned as Listeria spp. by biochemical analysis. The molecular serogrouping
100 ___ i
AscI
Apa\
, 1 lit i lit i l l [ill .4
SIH1S«
li'h
confirmed all isolates as belonging to genus Listeria based on the positive results for the prs gene (7), and the five isolates identified as L. monocytogenes were classified as belonging to serogroup lie, which includes serotypes l/2c and 3c (8). According to Orsi et al. (24), serotype 3c belongs to lineage I and serotype l/2c belongs to lineage II. L. monocytogenes serotype l/2c is usually isolated from foods and industry environment (7, 24) and has also been isolated from bovine hides and carcasses before and after eviscer ation (14, 34), as observed in the present study. Even identifying the low frequency of positive results for L. monocytogenes, and the identification of only five isolates (named as LM01, from a sample collected at stage A, visit 7, and LM02, LM03, LM04, and LM05, from samples collected at stage D, visit 12), PFGE was performed to characterize their genetic profiles. PFGE analysis demonstrated that all L. monocytogenes isolates presented an identical genetic profile (Fig. 1). This result suggests that isolates with identical genetic profiles are being constantly introduced in the slaughtering environment or the mainte nance of this specific strain in S03. A number of different risk factors that are strain based (adherence potential, sanitizer tolerance, and stress tolerance) and facility based (presence of harborage sites) have been hypothesized to facilitate the persistence of L. monocytogenes in the processing environment for extend periods of time to explain this finding (2). The L. monocytogenes isolates presented positive results for all virulence genes tested by PCR. These genes play important roles in the virulence mechanisms of L. monocytogenes and are involved in different stages of its pathogenesis (1, 24). The intemalin-related genes (iniA, inlB, inlC, and inlJ) are involved in cell adhesion and invasion, and plcA is associated with phospholipase C production (33). After the invasion of host cells, additional L. monocytogenes virulence genes are expressed, including hlyA, which is involved in the production of listeriolysin-O; actA, which is responsible for the production of ActA protein; and iap, which is associated with the expression of the p60 protein that plays an important role in intestinal cell invasion and survival (33). Similar results were observed by Liu et al. (20), who detected the same intemalin genes (inlA, inlB, inlC, and inlJ) in L. monocytogenes isolates belonging to serotypes l/2c or 3c. L. monocytogenes isolates that represent a potential risk to humans express a full-length isolate
serogroup
step
LM01
lie
A
LM02
lie
D
LM03
lie
D
LM04
lie
D
LM05
lie
D
FIGURE 1. PFGE patterns after the macrorestriction (ApaI and Asc/) o f five Listeria monocytogenes isolates from bovine hides and carcasses in distinct steps o f slaughtering (A, before bleeding; D, after end washing) in visits in a slaughterhouse (S03) located in Minas Gerais state, Brazil. Similarities between the identified PFGE pulsotypes were estimated using the Dice coefficient (1.5% tolerance).
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functional InlA, demanding the sequencing of the ini genes of obtained isolates to identify properly their vimlence potential (27). The analysis for resistance profiles indicated that all L. monocytogenes isolates presented sensitivity to all tested antimicrobials. Similar results were observed by Wieczorek et al. (34). Most isolates recovered from raw meat or food processing environments are susceptible to the majority of antimicrobials considered important in listeriosis treatment (4,18). Antimicrobial resistance by microorganisms can be developed as a consequence of endogenous and exogenous factors, and the environment plays an important role allowing for interactions with other bacteria and consequent gene or plasmid transfer (3,5). The absence of antimicrobial resistance identified in the present study suggests that L. monocytogenes isolates did not harbor the related genes or these genetic regions are not being expressed, demanding complementary analysis for a proper characterization of this activity (12, 22). In conclusion, the obtained results demonstrated the low frequencies of L. monocytogenes on bovine hides and carcasses during slaughtering in facilities located in Minas Gerais state, Brazil. The obtained L. monocytogenes isolates were susceptible to different antimicrobials and harbored different virulence genes, demanding further analysis to assess their pathogenic potential.
10.
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ACKNOWLEDGMENTS The authors thank Conselho Nacional de Desenvolvimento Cientffico e Tecnologico (CNPq), Coordenajao de Aperfeijoamento de Pessoal de Nivel Superior (CAPES), and Fundafao de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG).
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