2053

Journal of Food Protection, Vol. 84, No. 12, 2021, Pages 2053–2058
https://doi.org/10.4315/JFP-21-140
Copyright Ó, International Association for Food Protection

Research Paper

Multidrug-Resistant and Extended-Spectrum

b-Lactamase–Producing Salmonella enterica Serotype Heidelberg
Is Widespread in a Poultry Processing Facility in Southern Brazil
LUCIANO DOS SANTOS BERSOT https://orcid.org/0000-0001-7013-5574,1* NEILA RITA CARBONERA,1
CAROLINA DIAS RODRIGUES VALCANAIA,1 CIBELI VIANA https://orcid.org/0000-0002-5917-5783,2 AND
LUÍS AUGUSTO NERO https://orcid.org/0000-0002-4954-58242

1LACOMA - Laboratório de Inspeção e Controle de Qualidade de Alimentos e Água, Universidade Federal do Paraná, Setor Palotina, Departamento de
Ciências Veterinárias, Rua Pioneiro, 2153, Jardim Dallas, 85950-000, Palotina, PR, Brazil; and 2InsPOA - Laboratório de Inspeção de Produtos de
Origem Animal, Universidade Federal de Viçosa, Departamento de Veterinária, Avenida PH Rolfs, s/n, Campus Universitário, 36570-900,
Viçosa, MG, Brazil
MS 21-140: Received 31 March 2021/Accepted 16 July 2021/Published Online 29 July 2021

ABSTRACT
This study was conducted to characterize the distribution of Salmonella isolates in a poultry processing facility and to
identify their antibiotic resistance profiles. Salmonella enterica was detected in 146 samples (66.7%), and 125 isolates were
identified as Salmonella Heidelberg (n ¼ 123), Salmonella Abony (n ¼ 1), and Salmonella O:4,5 (n ¼ 1). Salmonella Heidelberg
isolates were subjected to XbaI macrorestriction analysis and pulsed-field gel electrophoresis. The 66 pulsotypes obtained were
grouped into four major clusters, indicating cross-contamination and persistence of this serotype in the processing facility.
Selected S. enterica isolates were characterized by their antibiotic resistance, and most (n ¼ 122, 97.6%) were multidrug
resistant. Resistance to third-generation cephalosporins ceftazidime (84 isolates, 67.2%) and cefotaxime and ceftriaxone (91
isolates, 72.8%) was particularly prevalent. Production of extended-spectrum b-lactamases (ESBL) was identified in 24 isolates
(19.2%), and ESBL-producing isolates were resistant to at least eight antibiotics. This study revealed the high prevalence of
Salmonella Heidelberg in the poultry chain, providing insight into the ecology of this pathogen in this facility. The high
prevalence of multidrug-resistant S. enterica is a concern due to the potential consequences for public health.

HIGHLIGHTS
 Wide distribution of Salmonella enterica Heidelberg was found in a poultry processing facility.
 Most S. enterica isolates in this facility were multidrug resistant.
 High prevalence (19.2%) was found of ESBL-producing S. enterica.

Key words: Multidrug resistance; Poultry; Pulsed-field gel electrophoresis; Salmonella

Salmonella is a foodborne pathogen associated with The control of S. enterica contamination is highly
outbreaks worldwide (10). Poultry and its products are dependent on understanding the pathogen ecology and
considered important sources of Salmonella enterica, often distribution in the production chain and pathogen physiol-
associated with human salmonellosis in various countries ogy and resistance and adequate hygienic measures (42).
(1). This pathogen is widely distributed, and humans and The development of effective control programs is highly
dependent on characterization of S. enterica isolates and
animals are its main natural reservoirs (16). Once the
their distribution, diversity, and main contamination points
poultry gut is colonized by S. enterica, these animals may
in the production chain (22, 42). Control programs are
spread this pathogen during their lives and become a important to assure safe international trade of poultry
potential source of contamination during slaughtering and because S. enterica contamination is a worldwide concern
processing (1, 34). Control of S. enterica contamination in (22). In Brazil, official plans based on various strategies
the production chain is a challenge for the poultry industry, have been established by the Brazilian Ministry of
leading to the adoption of various strategies from farm to Agriculture and have been adopted to allow proper
slaughterhouse. surveillance and control of Salmonella in the poultry
production chain (2).
* Author for correspondence. Tel: þ55 44 3211-8513; S. enterica is often subjected to selective pressure from
E-mail: [email protected]. the use of antibiotics in the poultry production (10). As a
2054 BERSOT ET AL. J. Food Prot., Vol. 84, No. 12

consequence, S. enterica isolates are becoming increasingly incubated at 37 and 42.58C, respectively, for 24 h. Cultures were
resistant to various antibiotics and can act as important then streaked onto plates containing xylose lysine deoxycholate
agents for dispersal of antibiotic resistance genes (23, 24, agar (XLD; Oxoid) and brilliant green agar (BG; Oxoid) and
29). The increasing prevalence of multidrug-resistant S. incubated at 378C for 24 h. Suspect Salmonella colonies (up to
enterica and strains capable of producing extended- three colonies per sample; red to pink with red background and
black center on XLD; pink to white surrounded by a red zone on
spectrum b-lactamase (ESBL) are particular concerns in
BG) were transferred to triple iron sugar agar (Oxoid), lysine iron
the poultry industry due to the limitations of therapeutic
agar (Oxoid), SIM agar (Oxoid) and urea broth (Oxoid) and
strategies for addressing the potential spread of these strains
incubated at 378C for 24 h. Isolates with reactions consistent with
(9, 28, 38, 47). Salmonella were confirmed through slide agglutination assays
Detailed and accurate knowledge of the distribution of with somatic and flagellar polyvalent antisera (Probac do Brasil,
S. enterica strains and their antibiotic resistance profiles is São Paulo, SP, Brasil), resulting in a collection of 146 isolates (1
relevant to understanding the ecology of this pathogen and isolate per sample). Samples were characterized as positive or
consequently its control in the poultry production chain. negative for Salmonella, and the recorded prevalences were
Such information is needed to design strategies based on compared by chi-square analysis and the Marascuilo procedure
various approaches to control, prevent, and/or minimize (XLSTAT 19.01, Addinsoft, New York, NY) to identify significant
Salmonella contamination and assure safe products for differences (P , 0.05) in Salmonella prevalence among
consumers. This study was conducted to characterize the processing steps.
distribution of Salmonella isolates in a poultry production
chain in Brazil and to describe the antibiotic resistance Serotyping and PFGE of Salmonella isolates. Of the 146
profiles of these isolates. confirmed Salmonella isolates, 125 were available for serotyping
via agglutination assays with diverse antisera to identify key
MATERIALS AND METHODS somatic and flagellar antigens (Laboratory of Enterobacteriaceae,
Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brazil). Isolates
Poultry processing facility and sampling. The poultry serotyped as Salmonella Heidelberg were subjected to DNA
processing facility selected for the study is located in Paraná state macrorestriction with XbaI, as recommended by PulseNet (Centers
in southern Brazil and is inspected by the Brazilian Ministry of for Disease Control and Prevention, Atlanta, GA) and described
Agriculture. The facility slaughters 330,000 chickens per day, with by Ribot et al. (35), with some modifications. Isolates were
mechanized lines for evisceration and processing and a control cultured in brain heart infusion broth (BHI; Oxoid), incubated at
program for end products destined for national and international 378C for 24 h, and diluted to an optical density at 610 nm of 0.9
trade. The facility was visited 10 times in 1 year, and 219 total for making plugs with low-melting-point agarose (1%, w/v; Bio-
samples were obtained from the reception area (transport box, n ¼ Rad, Hercules, CA). Plugs were subjected to enzymatic digestion
10), slaughtering area (carcass after defeathering, n ¼ 50; carcass in 200 μL of buffer and 50 U of XbaI (New England Biolabs,
after evisceration, n ¼ 50; carcass after prechilling, n ¼ 50), Ipswich, MA) at 378C for 2 h. CHEF-DRIII (Bio-Rad) was used
processing area (knife, n ¼ 9; cutting board, n ¼ 10; hands of for pulsed-field gel electrophoresis (PFGE) with the following
employees, n ¼ 10), and end products (leg, n ¼ 10; wing, n ¼ 10; parameters: initial switch time of 2.2 s, finish switch time of 63.8
breast, n ¼ 10). Reception and processing samples were obtained s, voltage of 6 V/cm, 1208 angle, and 16-h running time.
by swabbing four areas (four samples per area, each swabbing 10 Salmonella Braenderup (ATCC BAA-664TM) was subjected to
by 10 cm ¼ 100 cm2) with sterile sponges (3M Microbiology, St. the same digestion protocol and used as a molecular mass marker.
Paul, MN). One sponge was used per area, and each sponge was Gels were stained with UniSafe dye (Biotium, Fremont, CA) and
premoistened with 10 mL of phosphate-buffered saline (PBS; visualized with a transilluminator and digitally recorded (LTB-HE,
Oxoid, Basingstoke, England) at pH 7.2. After swabbing, each Loccus, São Paulo, SP, Brazil). The restriction results were
sponge set (n ¼ 4) was packed in a sterile sample bag (Whirl-Pak, analyzed with the software BioNumerics version 7.6 (Applied-
Nasco, Fort Atkinson, WI), and 160 mL of PBS was added for a Maths, Kortrijk, Belgium) with optimization and a tolerance of
final volume of 200 mL corresponding to a 400-cm2 area sampled. 1.5%. A dendrogram was generated based on the Dice similarity
Samples from the slaughtering area and from end products were coefficient and cluster analysis and the unweighted pair group
packed in sterile sample bags (Whirl-Pak), 400 mL of PBS was method with arithmetic means.
added to each bag and massaged as recommended by the U.S.
Department of Agriculture, Food Safety and Inspection Service Antibiotic resistance. Isolates identified as S. enterica were
(USDA-FSIS) (41), and the homogenates were transferred to characterized according to their resistance to 19 antibiotics from
sterile sample bags. All samples were refrigerated for up to 4 h nine classes determined with disk diffusion assays as described by
until analysis. the Clinical and Laboratory Standards Institute (CLSI) (6). Isolate
cultures were obtained in BHI, incubated overnight at 378C,
Salmonella detection. Samples were analyzed for the diluted in BHI up to an optical density at 610 nm of 0.9, and
presence of Salmonella based on the USDA-FSIS method (41) spread onto the surface of Muller-Hinton agar (Oxoid). After
and ISO 6579 (17). For reception and processing samples, 25 mL drying, disks impregnated with antibiotics from the nine classes
of the homogenate was transferred to a plastic bag with 225 mL of (Centro de Controle e Produtos para Diagnóstico, São Paulo, SP,
1% (w/v) buffered peptone water (BPW; Oxoid) and incubated at Brazil) were placed on the plates: the b-lactams (penicillins)
378C for 18 h (17). For slaughtering and end-product samples, 30 ampicillin (10 μg) and amoxicillin–clavulanic acid (20, 10 μg); the
mL of the homogenate was transferred to a plastic bag with 30 mL cephems ceftazidime (30 μg), cefotaxime (30 μg), cefepime (30
of 4% (w/v) BPW and incubated at 378C for 18 h (41). After μg), and ceftriaxone (30 μg); the monobactam aztreonam (30 μg);
incubation, aliquots of the obtained cultures were transferred to the phenicol chloramphenicol (30 μg); the quinolones ciproflox-
Muller-Kauffmann tetrathionate broth supplemented with novobi- acin (5 μg) and nalidixic acid (30 μg); the carbapenems imipenem
ocin (Oxoid) and to Rappaport-Vassiliadis broth (Oxoid) and (10 μg), ertapenem (10 μg), and meropenem (10 μg); the
J. Food Prot., Vol. 84, No. 12 RESISTANT S. ENTERICA IN A POULTRY PROCESSING PLANT 2055

TABLE 1. Prevalence of Salmonella-positive samples obtained through the poultry slaughtering process indicates flaws in
from a poultry processing facility in Paraná state, southern Brazil the hygiene system in this facility, because a decrease of
No. (%) of Salmonella prevalence expected during such processing
Processing Sample type positive samplesa steps in a controlled system (18, 36). Salmonella prevalence
was high in the end products in this facility (Table 1), and
Reception Transport box 9 (90.0) A the isolates recovered had macrorestriction patterns similar
Slaughtering Carcass after defeathering 28 (56.0) AB
or identical to those of isolates from previous steps,
Carcass after evisceration 38 (76.0) A
indicating the spread of the pathogen in the facility (Fig. 1).
Carcass after prechilling 41 (82.0) A
Serologic evaluation confirmed all 125 tested isolates
Processing Knife 3 (33.3) AB
as S. enterica, and the identified Salmonella serotypes were
Cutting board 3 (30.0) AB
Employee hand 1 (10.0) B
Heidelberg (n ¼ 123, 98.4%), Abony (n ¼ 1, 0.8%), and
End product Leg 8 (80.0) A
O:4,5 (n ¼ 1, 0.8%). Salmonella Heidelberg was the main
Wing 8 (80.0) A serotype identified among these isolates, and this serotype is
Breast 7 (70.0) AB being increasingly described as relevant in the poultry chain
Total 146 (66.7) from various countries, including Brazil (7, 15, 25). This
specific trend has also been observed in various Brazilian
a
Values followed by different letters are significantly different (P states, including Paraná (27, 28), which is the location of the
, 0.05). χ2 ¼ 38.9, df ¼ 9, P , 0.001. studied facility and the main producer and exporter of
poultry products in Brazil. Isolates obtained from retail sale
aminoglycosides gentamicin (10 μg), amikacin (30 μg), and samples produced in the same facility previously were
tobramycin (10 μg); the tetracyclines tetracycline (30 μg) and identified as Salmonella Heidelberg (28), as found in the
doxycycline (30 μg); and the sulfonamide trimethoprim-sulfa- end products in the present study. The presence of
methoxazole (23.75, 1.25 μg). Plates were incubated at 358C for Salmonella Heidelberg has increased in comparison to that
16 to 18 h, inhibition zones were measured, and isolates were of other serotypes probably due to some particular
characterized as resistant or not resistant based on established
characteristics, such as efficient adaptation to the environ-
CLSI breakpoints. Escherichia coli ATCC 25922 was used as a
ment, invasive ability, biofilm formation, and/or resistance
pansusceptible quality control.
Isolates resistant to ceftazidime and/or cefotaxime were
to extreme pH (32, 37, 39). The high prevalence of
evaluated phenotypically for ESBL production according to the Salmonella Heidelberg also can be a consequence of the
method of European Committee on Antimicrobial Susceptibility initial strategy in a Salmonella control plan developed by
Testing (11). Cultures of the selected isolates were obtained in BHI the Brazilian Ministry of Agriculture (2), which focused on
and spread onto Muller-Hinton agar plates. After drying, disks elimination of flocks contaminated by Salmonella Gallina-
containing cefotaxime (30 μg), ceftazidime (30 μg), and cefepime rum and Salmonella Pullorum and vaccination against
(30 μg) were applied next to a disk with amoxicillin–clavulanic Salmonella Enteritidis. This control protocol for these
acid (20, 10 μg). Isolates were characterized as ESBL producers specific serotypes could have allowed the emergence of
when the inhibition zones around any of the cephem class disks other serotypes, such as Salmonella Heidelberg (13).
had confluent extension in the direction of the disk containing Macrorestriction patterns of the Salmonella Heidelberg
amoxicillin–clavulanic acid. isolates are presented in Figure 1; a digestion pattern was
not obtained from one of the isolates. The 66 pulsotypes
RESULTS AND DISCUSSION were grouped into four major clusters: A, with 4 isolates
The prevalence of Salmonella among samples exam- sharing 83.3% band similarity; B, with 8 isolates sharing
ined in this study are presented in Table 1. Salmonella was 85.2% band similarity; C, with 70 isolates sharing 86.1%
detected in 146 (66.7%) of the 219 samples collected at band similarity; and D, with 39 isolates sharing 80.8% band
different stages of poultry processing. Salmonella was more similarity. Only one isolate was not included in any of these
prevalent in transport boxes, carcasses after evisceration, groups. All clusters contained isolates with identical
carcasses after prechilling, chicken legs, and chicken breasts pulsotypes or high band match similarity (.90.0%)
than on the hands of employees (P , 0.05) (Table 1). obtained from various samples collected in the poultry
Transport boxes appear to be a relevant entry source of S. processing facility, indicating cross-contamination with
enterica in the this processing facility. Reiter et al. (33) Salmonella Heidelberg (Fig. 1). This clear relationship
described the poultry transport boxes as relevant sources of among isolates from all processing steps of this poultry
Salmonella contamination, and this contamination is chain also indicates potential routes of contamination. The
associated with the presence of the pathogen in the poultry present PFGE findings are in accordance with those of other
farms (4). studies in which Salmonella contamination routes were
The prevalence of Salmonella increased somewhat (P tracked in the poultry production chain. Dias et al. (8)
. 0.05) along the slaughtering line, from 56% in poultry reported that isolates obtained from slaughtering and
carcasses after defeathering to 76% in carcasses after processing had the same XbaI macrorestriction pattern as
evisceration and 82% in carcasses after prechilling (Table did those isolated in the chicken reception area. The clonal
1). Similar patterns of increasing Salmonella contamination relationship between Salmonella Heidelberg isolates has
during processing of poultry carcasses also was reported by been observed in various regions of Paraná state (28) and
Vinueza-Burgos et al. (45) and Villagómez Estrada et al. could be evidence of circulation of a particular Salmonella
(44) in Ecuador. The increasing prevalence of the pathogen Heidelberg strain, probably from a common source of
2056 BERSOT ET AL. J. Food Prot., Vol. 84, No. 12

TABLE 2. Prevalence of antibiotic resistance among Salmonella

enterica isolates recovered from a poultry processing facility in
Paraná state, southern Brazil
No. (%) of
Antibiotic class Antibiotic resistant isolates

b-Lactams Ampicillin 108 (86.4)

Amoxicillin-clavulanate 94 (75.2)
Cephems Ceftazidime 84 (67.2)
Cefotaxime 92 (73.6)
Cefepime 17 (13.6)
Ceftriaxone 91 (72.8)
Monobactams Aztreonam 67 (53.6)
Phenicols Chloramphenicol 8 (6.4)
Quinolones Ciprofloxacin 3 (2.4)
Nalidixic acid 125 (100.0)
Carbapenems Imipenem 0 (0.0)
Ertapenem 0 (0.0)
Meropenem 4 (3.2)
Aminoglycosides Gentamicin 47 (37.6)
Amikacin 1 (0.8)
Tobramycin 49 (39.2)
Tetracyclines Tetracycline 125 (100.0)
Doxycycline 125 (100.0)
Sulfonamides Trimethoprim- 21 (16.8)
sulfamethoxazole

chicks, feed, and/or other products used in the poultry

production by different farms belonging to the same
cooperative (28). The poultry production chain in this
Brazilian state is characterized by high output and high
complexity due to the presence of cooperatives that
integrate the production from multiple farms; therefore,
circulation of a persistent strain is highly probable and
deserves special attention to control the spread (5, 26, 28).
The persistence of these Salmonella Heidelberg pulsotypes
in the environment may be associated with their ability to
form biofilms (7).
Table 2 presents the prevalence of antibiotic resistance
among the recovered S. enterica isolates. All isolates were
resistant to tetracyclines and nalidixic acid and susceptible
to imipenem and ertapenem. Resistance to ceftazidime and/
or cefotaxime was identified in 93 isolates, of which 24
were characterized as ESBL producers. Patterns of
resistance to seven antibiotic classes are presented in Table
3; 122 (97.6%) isolates were resistant to three or more
antibiotic classes and thus were characterized as multidrug
resistant. The emergence of multidrug resistant Salmonella
strains has been considered one of the main concerns related
to global health (20). High prevalences of multidrug-
resistant Salmonella strains have been observed in poultry
chains and poultry products (12, 28, 46). The same pattern
FIGURE 1. Numbers of isolates, sampling sites, and 66 unique was observed in our study, with 97.6% multidrug resistance
macrorestriction banding patterns (XbaI) generated by pulsed-
among the S. enterica isolates. Salmonella Heidelberg is an
field gel electrophoresis from Salmonella enterica Heidelberg
important serotype associated with multidrug resistance and
isolates recovered from a poultry slaughtering facility in Paraná
state, southern Brazil. Identity was estimated using the Dice thus is a significant threat to public health (9, 14, 25). All
coefficient (1.5% tolerance). Uppercase letters indicate the four isolates obtained in our study were resistant to tetracyclines
major clusters of isolates based on the similarity indexes. and nalidixic acid (Table 2). Both antibiotics were widely
Sampling sites: r, reception; s, slaughtering; p, processing; ep, used in livestock as growth promoters until they were
end products. banned in Brazil in 2009 (31), but their use until that time
may have increased the selection and spread of antibiotic-
J. Food Prot., Vol. 84, No. 12 RESISTANT S. ENTERICA IN A POULTRY PROCESSING PLANT 2057

TABLE 3. Simultaneous resistance to multiple classes of delberg strain in the studied poultry chain. This information
antibiotics among Salmonella enterica isolates recovered from a extends the knowledge base of S. enterica ecology and
poultry processing facility in Paraná state, southern Brazil spread over time, confirming the urgent need for Salmonella
No. of No. (%) of surveillance in the poultry production chain.
Antibiotic classesa classes resistant isolates
ACKNOWLEDGMENTS
PEN-CEP-AMI-TET-QUI-SUL-PHE 7 1 (0.8)
This work was supported in part by Conselho Nacional de
PEN-AMI-TET-QUI-SUL-PHE 6 3 (2.4)
Desenvolvimento Científico e Tecnológico (Brasília, DF, Brazil) and
PEN-CEP-AMI-TET-QUI-PHE 6 1 (0.8)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brasília,
PEN-CEP-AMI-TET-QUI-SUL 6 5 (4.0) DF, Brazil; financial code 001).
PEN-AMI-TET-QUI-PHE 5 2 (1.6)
PEN-AMI-TET-QUI-SUL 5 4 (3.2)
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