Antimicrobial resistance profiles

of Salmonella serovars isolated

from dressed chicken meat at
slaughter in Kaduna, Nigeria

■ SANTÉ ANIMALE ET EPIDEMIOLOGIE

Michael Agbaje1* Antonia Anna Lettini2 Olufemi Ernest Ojo1
Alessandra Longo2 Elisa Marafin2 Keti Antonello2
Paola Zavagnin2 Babafela Babalola Oluwasile1
Ezekiel O. Omoshaba1 Morenike Atinuke Dipeolu1

Keywords Summary
Salmonella, resistance to antibiotic, Invasive non-typhoidal salmonellosis characterized by gastroenteritis and bac-
chicken meat, slaughtering, Nigeria teremia is endemic in sub-Sahara Africa. Most infections are foodborne with
animals serving as asymptomatic carriers. We investigated Salmonella serovars
Submitted: 14 December 2017 and associated resistance genes in chicken meat using culture, minimum inhibi-
Accepted: 4 September 2019
tory concentrations and PCR amplification of resistance genes. Of 100 samples
Published: 15 November 2019
DOI: 10.19182/remvt.31484
examined, 28 (28%) were Salmonella positive and spread across six serovars:
Haifa (71.4%), Chomedey (7.1%), Saintpaul (7.1%), Kainji (7.1%), Derby
(3.6%), and Blockley (3.6%). Antimicrobial resistance was observed to cipro-
floxacin (85.7%), nalidixic acid (75.0%), sulfamethoxazole (67.8%), tetracycline
(89.3%), trimethoprim (42.9%), gentamicin (35.7%), streptomycin (32.1%),
ampicillin (10.7%), chloramphenicol (10.7%), kanamycin (7.1%) and florfeni-
col (3.6%). All isolates were susceptible to cefotaxime, ceftazidime and colis-
tin, whereas 19 (67.9%) showed multidrug resistance to at least three antimicro-
bials. The predominant resistance type was Cip-Gen-Nal-Smx-Tet-Tmp detected
in six (21%) isolates. Multidrug resistance of Salmonella serovars was high in

Revue d’élevage et de médecine vétérinaire des pays tropicaux, 2019, 72 (4) : 173-179
the sampled chicken meat with resistance most observed against ciprofloxacin.
This suggests possible horizontal transfer of plasmid-mediated quinolone resis-
tance genes, which may compromise the clinical use of fluoroquinolones. Thus,
improved hygiene and provision of adequate facilities at meat processing cen-
ters could help limit meat contamination and foodborne transmission of multi-
resistant non-typhoidal Salmonella serovars from chickens to humans.
■ How to quote this article: Agbaje M., Lettini A.A., Ojo O.E., Longo A., Marafin E., Antonello K., Zava-
gnin P., Oluwasile B.B., Omoshaba E.O., Dipeolu M.A., 2019. Antimicrobial resistance profiles of Salmo-
nella serovars isolated from dressed chicken meat at slaughter in Kaduna, Nigeria. Rev. Elev. Med. Vet. Pays
Trop., 72 (4): 173-179, doi: 10.19182/remvt.31484

■ INTRODUCTION million cases of gastroenteritis and 155,000 deaths worldwide, with over
85% of Salmonella gastroenteritis attributed to foodborne transmission
Salmonella is an enteric bacterial pathogen of the family Enterobac- (Majowicz et al., 2010). Food-producing animals have been implicated
teriaceae and has the potential to invade tissue and cause bacteremia as major reservoirs and sources of zoonotic transmission of non-typhoi-
(de Jong et al., 2012). Globally, Salmonella serovars are among the dal salmonellosis (Kagambèga et al., 2013). In particular, poultry and
leading cause of foodborne gastroenteritis and constitute health and poultry products play significant roles in the transmission of salmonel-
socioeconomic burdens in both developed and developing countries. losis to humans. Apparently, healthy chickens may harbor non-typhoi-
Non-typhoidal Salmonella enterica causes an annual estimate of 93.8 dal zoonotic S. enterica serovars in their gastrointestinal tracts, which
are transmitted to humans through contact with carrier birds, feces-
1. College of Veterinary Medicine, Federal University of Agriculture, PMB 2240, contaminated environment and consumption of contaminated food and
Abeokuta, Nigeria. water (Kagambèga et al., 2011; 2013).
2. Istituto Zooprofilattico Sperimentale delle Venezie, National and OIE Reference
Laboratory for Salmonellosis, Legnaro, Italy. In developing countries, cases of salmonellosis are often misdiagnosed
* Corresponding author because of inadequate laboratory investigations (Feasey et al., 2012).
Tel.: +234 70 15 74 40 22; Email: [email protected] Moreover, unhygienic practices in food processing and in distribution
chains, low-level awareness of non-typhoidal salmonellosis, malnutri-
https://creativecommons.org/licenses/by/4.0/ tion and concurrent infections may contribute to spread and severity of
173
 Molecular typing of Salmonella in chicken meat

foodborne infections. Self-medication, delayed or lack of access to good buffered peptone water (BPW, Oxoid CM0509, UK), then incubated at
health care, inappropriate prescriptions and prevailing cultural practices 37°C for 18–24 hours. From the BPW culture, 0.1 ml was transferred
are additional factors that may contribute to complications, widespread into 9.9 ml of modified semi-solid Rapport-Vassiliadis (MSRV) selec-
dissemination, increased morbidity and case fatality of Salmonella gas- tive enrichment broth (Oxoid CM0910) supplemented with novobiocin
troenteritis (Morpeth et al., 2009). Generally, the incidence of foodborne (Oxoid SR0161), then incubated at 42°C for 24–48 hours. Afterwards,
infection in developing countries is grossly underestimated because of MSRV was observed for the typical migration pattern of Salmonella
poor record keeping, inadequate disease reporting and lack of coordi- enterica (≥ 2.0 mm migration) every 24 hours until 72 hours. A loopful
nated monitoring and surveillance of important foodborne pathogens. of observable bacterial spread was taken from the periphery of MSRV
medium and streaked simultaneously onto plates of brilliant green agar
■ SANTÉ ANIMALE ET EPIDEMIOLOGIE

The increasing incidence of antimicrobial resistance by pathogenic and (BGA, Oxoid CM0263) supplemented with sulphamandelate (Oxoid
commensal bacteria is of public health concern. In sub-Saharan Africa, SR0087) and onto xylose lysine desoxycholate agar (XLD, Oxoid). The
antimicrobial chemotherapy is an integral part of the management of inoculated plates were incubated at 37°C for 18–24 hours, then exami-
invasive non-typhoidal Salmonella infections (Feasey et al., 2012). ned for bacterial colonies. Typical Salmonella colonies were picked and
However, widespread acquisition and dissemination of antimicrobial subjected to biochemical and serological tests.
resistance traits are continuously narrowing the antimicrobial therapeu-
tic options available for the treatment of bacterial infections. Antimicro- Serotyping of Salmonella isolates
bial-resistant Salmonella spp. have been isolated from different foods
of animal origin around the world (Garedew et al., 2015; Löfström et Presumptive Salmonella isolates were shipped to the Office Internatio-
al., 2015; Saravanan et al., 2015). Extensive use of antimicrobial agents nal des Epizooties (OIE) Reference Laboratory for Salmonella, Istituto
in animal production is common and selects for multidrug resistant Zooprofilattico Sperimentale delle Venezie (IZSVe, Italy). All strains
(MDR) pathogens (Marshall and Levy, 2011; Oluwasile et al., 2014). were serotyped by agglutination tests with specific O and H antisera and
Fluoroquinolones are broad-spectrum antimicrobials effective in the classified according to Kauffman-White’s scheme as previously descri-
treatment of a wide variety of clinical and veterinary infections, parti- bed (Grimont and Weill, 2007).
cularly for life-threatening salmonellosis treatment. Phenotypic antimicrobial susceptibility testing
Quinolone resistance in Salmonella is mainly attributed to point muta- Susceptibility to 14 antimicrobials was carried out to determine the
tions in chromosomal genes encoding gyrase subunits A (gyrA) and B minimum inhibitory concentrations by means of broth microdilution fol-
(gyrB), and topoisomerase subunits C (parC) and E (parE) (Hopkins et lowing the Clinical and Laboratory Standards Institute guidelines (CLSI,
al., 2005). The fluoroquinolone resistance phenotype might be further 2009). The CMV2AGNF (sensititre, Trek Diagnostic Systems, USA)
modulated with the appearance of several plasmid-mediated quinolone susceptibility plates of the National Antimicrobial Monitoring System
resistance mechanisms (PMQR) (Luo et al., 2011; Wasyl et al., 2014) containing 14 antimicrobials was used. The antimicrobials tested were
encoded by qnrA, qnrB, qnrS, qepA genes. ampicillin, cefotaxime, ceftazidime, chloramphenicol, ciprofloxacin,
Factors responsible for the endemicity of non-typhoidal Salmonella colistin, florfenicol, gentamicin, kanamycin, nalidixic acid, streptomy-
infections in Africa are poorly understood (Morpeth et al., 2009). There cin, sulfamethoxazole, tetracycline, and trimethoprim. The breakpoints
is a dearth of information on the dynamics of Salmonella serovars and used for sulfamethoxazole and kanamycin were CLSI (2009) break-
their antimicrobial resistance properties in livestock and food of animal points, whereas for other antimicrobials, the European Committee on
Revue d’élevage et de médecine vétérinaire des pays tropicaux, 2019, 72 (4) : 173-179

origin. To prevent the continuous spread of antimicrobial resistance and Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off
zoonotic transmission of foodborne pathogens from animals to humans, was used (Aarestrup and McDermott, 2007; EUCAST, 2012). Escheri-
there is a need for regular monitoring and reporting of resistant bacte- chia coli (ATCC 25922) was used as the quality control strain.
ria in food of animal origin. The present study investigated the occur- Determination of antimicrobial resistance genes
rence of Salmonella serovars as well as their phenotypic and genotypic
antimicrobial resistance properties in freshly dressed chicken meat in DNA templates used for PCR were prepared by boiling as described by
Kaduna in Northern Nigeria. Barco et al. (2011). Molecular characterization of genetic antimicrobial
resistance determinants in all isolates was undertaken by PCR ampli-
fication according to Beutlich et al. (2011). The isolates were analyzed
■ MATERIALS AND METHODS for the presence of resistance genes corresponding to their resistance
phenotypes. The resistance-associated genes investigated included
Sample collection those encoding resistance to beta-lactam antibiotics (blaPSE1, blaTEM1-
One hundred neck skin samples were collected from freshly dressed like, blaOXA1-like), chloramphenicol and florfenicol (catA1, cmlA, floR),
chicken meat sold to consumers at two chicken processing facilities fluoroquinolones (gyrA, qnrA, qnrB, qnrS, qepA), gentamicin [aacC2,
in Kaduna, capital of Kaduna State, Nigeria. Neck skin sampling was aacC4, aac(3)-Ie (aacC5), aadB, armA], kanamycin [aphA1, aphA2,
selected in this study since it provides higher Salmonella yields than aac(6)-1b], streptomycin (aadA1-like, strA, strB), sulfamethoxazole
whole carcass rinse and other skin parts (Diezhang et al., 2014). Fifty (sul1, sul2, sul3), tetracycline [tet(A), tet(B), tet(G)], and trimethoprim
samples were collected from each facility, and sampling was carried (dfrA1-like, dfrA5-14, dfrA7-17, dfrA12). In particular, PCR amplifica-
out three times daily (morning, afternoon and evening) for two weeks tion of gyrA gene and sequencing of purified amplicons were performed
in March 2012. In each center, one and sometimes two samples per on both nucleotide strands to detect substitutions using ABI 100 DNA
visit were collected from different processors selling dressed chickens. sequencer (Applied Biosystems, USA) as previously reported (Capoor
Samples were collected in pre-labeled sample bags and preserved with et al., 2009). In each case, the gyrA sequence obtained was compared
ice pack in sterile containers before transport to the laboratory for with wild-type sequences (GenBank accession number AE006468.1).
microbiological analysis.
■ RESULTS
Isolation and presumptive identification of Salmonella
Prevalence of Salmonella serovars in chicken meat
Salmonella isolation was performed according to ISO 6579 standard
(2002) with minor modifications. In particular, each neck sample (10 g) Culture and serology confirmed almost one third (28%, 28/100) of the
was pre-enriched after thorough homogenization in 90 ml of sterile samples as Salmonella positive; they were spread across six serovars:
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 Typage moléculaire de Salmonella dans la viande de poulet

S. Haifa, S. Chomedey, S. Saintpaul, S. Derby, S. Blockley and S. resistant strains) genes. Sulfamethoxazole resistance was mostly asso-
Kainji (Table I). ciated with sul1 (15/19), however, sul2 (2/19) and sul3 (2/19) were also
detected. Tetracycline resistance was uniquely encoded by tet(A) in 25
Phenotypic antimicrobial resistance of S. isolates isolates. Trimethoprim resistance was uniquely encoded by dfrA5-14
(12 isolates). Substitutions in the codons of gyrA were responsible for
The highest antimicrobial resistance was to ciprofloxacin (85.7%, fluoroquinolone resistance in 21 isolates. Sequence analysis revealed
24/28), followed by nalidixic acid (75%), sulfamethoxazole (67.8%) and mutation of amino acid at position 83 (TCC→TAC, Ser83→Tyr) in 17
tetracycline (89.3%) (Table I). About half of Salmonella isolates showed isolates and (TCC→TTC, Ser83→Phe) in four isolates. Three fluoro-
resistance to trimethoprim, gentamicin and streptomycin, whereas quinolone-resistant isolates with similar R-type (Cip-Gen-Nal-Smx-
small numbers of isolates were resistant to ampicillin, chloramphenicol, Str-Tet-Tmp) had double point mutations in their gyrA gene. The first
kanamycin and florfenicol. All isolates were susceptible to cefotaxime, mutation occurred at position 83 (TCC→TTC, Ser83→Phe), the second
ceftazidime and colistin, whereas the two representatives of S. Kainji at position 87 (GAC→GGC, Asp87→Gly). Three (10.7%) of the Sal-
were susceptible to all tested antimicrobial agents (Table I). Of the 26 monella isolates carried a qnr gene. These isolates were characterized
resistant isolates, nineteen (67.9%) showed multidrug resistance to at by a reduced level of fluoroquinolone resistance (0.5–1 mg/l) and no
least three different classes of antimicrobials. The most prevalent resis- simultaneous chromosomal mutations occurred in gyrase or topoisom-
tance type (R-type) was Cip-Gen-Nal-Smx-Tet-Tmp observed in six erase. Two isolates (S. Chomedey) carried qnrB gene and one isolate (S.
(21%) isolates, followed by Cip-Nal-Tet in four (14%) isolates (Table II). Derby) qnrS gene.

Antimicrobial resistance genes in Salmonella isolates ■ DISCUSSION

The blaTEM gene coding for beta-lactam resistance was detected in
all three ampicillin-resistant isolates (Table I). The floR gene encoded The results showed high contamination of poultry meat during pro-
resistance to chloramphenicol and florfenicol in one isolate. Two chlor- cessing with potentially zoonotic non-typhoidal Salmonella serovars.
amphenicol-resistant isolates carried cmlA gene. Streptomycin was The serovars detected have been associated throughout the world with
mainly encoded by strB (6 isolates) and/or strA (1 isolate) genes except foodborne infections in humans. For instance, Kaibu et al. (2005) report
for two isolates that showed only aad1-like gene. Gentamicin resistance fatal cases of food intoxication by Salmonella Haifa in an older per-
was mediated by aacC2 (7/10 resistant isolates) and acc(3)-le (3/10 son and in a child. S. Derby was reported in an outbreak of foodborne

Table I
Distribution of resistant isolates and resistance genes among Salmonella serovars
isolated from fresh chicken meat in Kaduna, Nigeria

Antimicrobial agent Resistance Salmonella serovars* Total % (95% CI)

gene (n = 28)

Revue d’élevage et de médecine vétérinaire des pays tropicaux, 2019, 72 (4) : 173-179
S. Haifa S. Derby S. Chomedey S. Blockley S. Saintpaul
(n = 20) (n = 1) (n = 2) (n = 1) (n = 2)
Ampicillin blaTEM 1 – – – 2 3 10.7 (2.9–28.0)
Cefotaxime – – – – – – – –
Ceftazidime – – – – – – – –
Chloramphenicol cmlA1 – – – – 2 2 7.1 (0.9–23.7)
Chloramphenicol/ floR 1 – – – 1 3.6 (0.0–19.2)
florfenicol
Colistin – – – – – – – –
Fluoroquinolone gyrA mutation 20 – – 1 – 21 75 (56.4–87.6)
(ciprofloxacin)
Fluoroquinolone qnrB – – 2 – – 2 7.1 (0.9–23.7)
(ciprofloxacin)
Fluoroquinolone qnrS – 1 – – – 1 3.6 (0.0–19.2)
(ciprofloxacin)
Gentamicin aacC2 7 – – – – 7 25 (12.4–43.6)
Gentamicin aac(3)-Ie 3 – – – – 3 10.7 (2.9–28.0)
Kanamycin aphA1 – – – 1 1 2 7.1 (0.9–23.7)
Nalidixic acid – 20 – – 1 – 21 75 (56.4-87.6)
Streptomycin strA 1 – – – – 1 3.6 (0.0–19.2)
Streptomycin strB 3 – 2 1 – 6 21.4 (9.9–39.9)
Streptomycin aad-1like – – – – 2 2 7.1 (0.9–23.7)
Sulfamethoxazole sul1 15 – – – – 15 53.6 (35.8–70.5)
Sulfamethoxazole sul2 – – 2 – – 2 7.1 (0.9–23.7)
Sulfamethoxazole sul3 – – – – 2 2 7.1 (0.9–23.7)
Tetracycline tet(A) 19 1 2 1 2 25 89.3 (72.0–97.1)
Trimethoprim dfrA5-14 12 – – – – 12 42.9 (26.5–61.0)
* No resistant isolates were found for S. Kainji (n = 2)
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 Molecular typing of Salmonella in chicken meat

Table II
Phenotypic and genotypic resistance patterns of Salmonella serovars isolated
from fresh chicken meat in Kaduna, Nigeria

Serial Num. Serovar Phenotypic resistance pattern Resistance genes (mutations)

1 S. Haifa CIP-NAL gyrA (Ser83→Tyr)

2 S. Derby CIP-TET qnrS, tet(A)
■ SANTÉ ANIMALE ET EPIDEMIOLOGIE

3 S. Haifa CIP-NAL-TET gyrA (Ser83→Tyr), tet(A)

4 S. Haifa CIP-NAL-TET gyrA (Ser83→Tyr), tet(A)
5 S. Haifa CIP-NAL-TET gyrA (Ser83→Tyr), tet(A)
6 S. Haifa CIP-NAL-TET gyrA (Ser83→Tyr), tet(A)
7 S. Chomedey CIP-SMX-STR-TET qnrB, sul2, strB, tet(A)
8 S. Chomedey CIP-SMX-STR-TET qnrB, sul2, strB, tet(A)
9 S. Blockley CIP-KAN-NAL-STR-TET gyrA (Ser83→phe), aphA1, strA, strB, tet(A)
10 S. Haifa CIP-NAL-SMX-TET gyrA (Ser83→Tyr), sul1, tet(A)
11 S. Haifa CIP-NAL-SMX-TET gyrA (Ser83→Tyr), sul1, tet(A)
12 S. Haifa CIP-NAL-SMX-TET gyrA (Ser83→Tyr), sul1, tet(A)
13 S. Haifa CIP-NAL-SMX-TET-TMP gyrA (Ser83→Tyr), sul1, tet(A), dfrA5-14
14 S. Saintpaul AMP-CHL-FFN-SMX-STR-TET blaTEM, floR, sul3, aad1-like, tet(A)
15 S. Saintpaul AMP-CHL-KAN- SMX-STR-TET blaTEM, cmlA, aphA1, sul3, aad1-like, tet(A)
16 S. Haifa CIP-GEN-NAL-SMX-TET-TMP gyrA (Ser83→Tyr), aacC2, sul1, tet(A), dfrA5-14
17 S. Haifa CIP-GEN-NAL-SMX-TET-TMP gyrA (Ser83→Tyr), aacC2, sul1, tet(A), dfrA5-14
18 S. Haifa CIP-GEN-NAL-SMX-TET-TMP gyrA (Ser83→Tyr), aacC2, sul1, tet(A), dfrA5-14
19 S. Haifa CIP-GEN-NAL-SMX-TET-TMP gyrA (Ser83→Tyr), aacC2, sul1, tet(A), dfrA5-14
20 S. Haifa CIP-GEN-NAL-SMX-TET-TMP gyrA (Ser83→Tyr), aacC2, sul1, tet(A), dfrA5-14
21 S. Haifa CIP-GEN-NAL-SMX-TET-TMP gyrA (Ser83→Tyr), aacC2, sul1, tet(A), dfrA5-14
22 S. Haifa AMP-CIP-GEN-NAL-SMX-TET-TMP blaTEM, gyrA (Ser83→Tyr), aacC2, sul1, tet(A), dfrA5-14
23 S. Haifa CIP-CHL-NAL-SMX-STR-TET-TMP gyrA (Ser83→Tyr), cmlA, aad1-like, sul1, tet(A), dfrA5-14
24 S. Haifa CIP-GEN-NAL-SMX-STR-TET-TMP gyrA (Ser83→phe, Asp87→Gly), aac(3)Ie, sul1, strB, tet(A), dfrA5-14
25 S. Haifa CIP-GEN-NAL-SMX-STR-TET-TMP gyrA (Ser83→phe, Asp87→Gly), aac(3)Ie, sul1, strB, tet(A), dfrA5-14
26 S. Haifa CIP-GEN-NAL-SMX-STR-TET-TMP gyrA (Ser83→phe, Asp87→Gly), aac(3)Ie, sul1, strA, strB, tet(A), dfrA5-14
Revue d’élevage et de médecine vétérinaire des pays tropicaux, 2019, 72 (4) : 173-179

AMP: ampicillin; CHL: chloramphenicol; CIP: ciprofloxacin; FFN: florfenicol; GEN: gentamicin; KAN: kanamycin; NAL: nalidixic acid; STR: streptomycin; SMX: sulfa-
methoxazole; TET: tetracycline; TMP: trimethoprim
Florfenicol: floR; fluoroquinolones: gyrA, qnrB, qnrS; gentamicin: aacC2, aac(3)-Ie; kanamycin: aphA1; streptomycin: aadA1-like, strA, strB; sulfamethoxazole: sul1, sul2,
sul3; tetracycline: tet(A); trimethoprim: dfrA5-14

salmonellosis following the consumption of grilled meat in Fukuka stress usually associated with slaughterhouses (Olsen et al., 2003;
City in Japan (Ebuchi et al., 2006). S. Saintpaul was responsible for a Rasschaert et al., 2007).
large foodborne outbreak of salmonellosis involving 1500 cases with
S. Haifa was the predominant (71.4%) serovar detected in the present
21% hospitalizations and two deaths in the United States (Barton et al., study. Five other serovars were also detected suggesting that chic-
2011). Gonose et al. (2012) reported S. Blockley as the etiology of food- ken meat may act as a vehicle for the transmission of diverse ranges
borne illness characterized by diarrhea, stomach cramp and headaches of Salmonella serovars. Fashae et al. (2010) detected eight S. serovars
in three adult males in South Africa. (Virchow, Bredeney, Derby, Haifa, Havana, Muenster, Mbandaka and
The 28% overall prevalence rate of Salmonella observed in dressed Onireke) in the feces of on-farm chickens in Ibadan, and S. Virchow
chicken in our study was higher than those of 11% reported in the feces accounted for 71% of all isolates. In Maiduguri, S. Hiduddify was the
of on-farm chickens in Ibadan (Fashae et al., 2010), and of 2.3–5.2% in main (95%) serovar isolated from chicken feces and tissues (Raufu et
feces and intestinal samples of chickens in Maiduguri, Nigeria (Raufu al., 2009). Differences in geographical location, sampling site and type
et al., 2009). Unhygienic processing including lack of proper separa- may account for the observed disparities betwen these studies. Although
tion of dirty and clean areas during evisceration in the slaughterhouse, the S. serovars detected in this study could originate from the intestinal
unhygienic processors and equipment contributed to the contamination content of chickens, they may have originated from extraneous sources
of carcasses during processing. In addition, the extent of colonization including the persons involved in processing chicken meat.
of the crop due to feed withdrawal during transportation and resting Fluoroquinolones, especially ciprofloxacin, are the drugs of choice in
exposes carcasses to possible contamination (Ramirez et al., 1997). the treatment of human salmonellosis (Akinyemi et al., 2007) and they
Salmonella contamination of the neck skin has been linked to coloni- are also used frequently in livestock medicine. Resistance to ciprofloxa-
zation of the crop since this organ is more likely to be ruptured during cin was found in over 85% of all isolates. Most fluoroquinolone resis-
processing than other parts of the gastrointestinal tract such as the ceca tance (87.5%) was mediated by chromosomal mutations on gyrA gene.
(Hargis et al., 1995). Finally, successful contamination of carcasses and Resistance mutations occurring on gyrA gene, corresponding to amino
slaughter lines may also depend on the toughness of the contaminating acid changes at Ser-83 (to Phe or Tyr) or at Asp-87 (to Gly), are the
Salmonella strain, considering that such strains survive environmental most frequently observed in fluoroquinolone resistant strains (Piddock,
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 Typage moléculaire de Salmonella dans la viande de poulet

2002; Hopkins et al., 2005). In addition to these site mutations, there one S. Haifa showed resistance to ampicillin and possessed blaTEM gene.
were occurrence and dissemination of qnr genes in Salmonella isolates Resistance to streptomycin was detected in nine isolates belonging to
in this study, even though they were quite low (10.7%). This suggests four different serovars. Three streptomycin-associated genes were detec-
horizontal spread of resistance plasmid determinants in Kaduna State ted: strA and strB in S. Haifa, strB in S. Chomedey and Blockley, and
as reported by Fortini et al. (2011). However, it is notable that wide aadA-like in S. Saintpaul. The Salmonella isolates were completely
dissemination of PMQR genes mainly in commensal or pathogenic susceptible to cefotaxime, ceftazidime and colistin. These drugs are not
Gram-negative bacteria is mostly associated with defined successful known to be used in poultry production. Susceptibility to cefotaxime and
plasmids which could be positively selected by other drugs because of ceftazidime indicated that cephalosporins including highly important
the co-location of multiple resistance determinants (Fortini et al., 2011). third and fourth generation antimicrobials will be very effective in the
In general, the acquisition of PMQR genes decreases susceptibility to treatment of life-threatening and complicated salmonelloses in humans.
fluoroquinolones and may accelerate selection of fluoroquinolone-resis- These findings are subject to at least two limitations. Firstly, these data
tant mutants (Martínez-Martínez et al., 2003; Rodríguez-Martínez et al., were mainly collected from chickens originating from Kaduna State and
2007). Given their transferability and the possibility that they may cause other parts of Northern Nigeria which produce live chickens, and secondly,
increases in resistance thereby affecting the clinical response to treat- the relatively small sample size, which depended on budgetary constraints
ment, data on the occurrence of PMQR genes are important for the sur- since whole chickens had to be purchased to allow for sampling. Notwith-
veillance of quinolone resistance in humans and animals. In developing standing these limitations, this study offers some insight into the circula-
countries where poor facilities hamper accurate and timely diagnosis, ting non-typhoidal Salmonella serovars and their antibiogram.
antimicrobial agents are administered empirically for the treatment of
suspected cases of salmonellosis without recourse to laboratory inves-
tigation. Resistance may complicate Salmonella infections as resistant ■ CONCLUSION
strains are more likely to be associated with tissue invasion, severe ill-
ness and death (Helms et al., 2004). The study showed that chicken meat may contribute to the transmission
of non-typhoidal Salmonella serovars in the area. In addition, the Sal-
In this study there was a high resistance to (fluoro)quinolones, sulfo- monella observed might have been attributed to unhygienic handling
namide and tetracycline, a moderate resistance to trimethoprim and during processing. This included observed poor personal hygiene
aminoglycosides, and a low resistance to ampicillin and phenicol anti- among processors during processing as well as poor slaughterhouse
microbials. This could be a reflection of the pattern of antimicrobial sanitation due to inadequate infrastructure and unclean running water.
use in the poultry industry. Usage of any particular antimicrobial agent Improved hygiene, provision of slaughter facilities, as well as adequate
could exert selective pressure leading to the emergence, proliferation inspection at meat processing centers may limit meat contamination and
and spread of bacterium strains showing resistance to the antimicrobial reduce the risk of zoonotic transmission of non-typhoidal salmonellosis
agent. Previous studies have shown that fluoroquinolones, sulfonamides to humans. Furthermore, the study revealed circulation of S. serovars
and tetracyclines are the most widely used antimicrobial agents in the that are predominantly multidrug resistant. Preventive measures against
Nigerian poultry industry (Ogunleye et al., 2008; Oluwasile et al., 2014). misuse of antibiotics, including strict monitoring of antibiotics use both
In our study resistance to tetracycline was found in five out of six sero- for prophylaxis and growth stimulation in poultry farms will mitigate
vars and was uniquely associated with tet(A) gene. Tetracycline is used antibiotic resistance development.
as an additive in water and feed to improve performance, whereas cipro-

Revue d’élevage et de médecine vétérinaire des pays tropicaux, 2019, 72 (4) : 173-179
floxacin is the ready choice for the treatment of bacterial infections and Acknowledgments
sometimes for prophylaxis (Oluwasile et al., 2014). The authors appreciate the laboratory scientists in the Department
Although three serovar types (Haifa, Saintpaul and Chomedey) showed of Veterinary Microbiology, College of Veterinary Medicine, Fede-
resistance to sulfamethoxazole, the resistance was mediated by different ral University of Agriculture, Abeokuta, Nigeria, and OIE Reference
genes. The sul1 gene was responsible for resistance in all isolates belon- Laboratory for Salmonella, IZSVe, Legnaro, Italy. This project was
ging to serovar Haifa. In S. Chomedey, sulfamethoxazole resistance was partly supported by the International Livestock Research Institute
due to sul2, whereas in S. Saintpaul it was due to sul3. Two isolates of (ILRI), Kenya.
S. Saintpaul possessed cmlA, which accounted for resistance to chlo-
ramphenicol, whereas in S. Haifa resistance to chloramphenicol and its Author contributions statement
derivative (florfenicol) was mediated by floR. All gentamicin-resistant MA, AAL, OEO, MAD designed and planned the study; MA, AL,
Salmonella isolates belonged to serovar Haifa and resistance was attri- EM, KA, PZ, BBO, EOO performed experiments and collected data;
butable mainly to aacC2 (seven isolates) but also to aac(3)-Ie (three iso- MA, AAL, OEO analyzed and interpreted data; MA, AAL, OEO,
lates). Similarly, trimethoprim resistance was observed only in S. Haifa BBO, AL, EM, KA, PZ, EOO drafted the first version of the manus-
and was encoded solely by dfrA5-14 gene. Two S. Saintpaul isolates and cript; MA, AL, OEO, MAD critically reviewed the manuscript.

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Résumé Resumen
Agbaje M., Lettini A.A., Ojo O.E., Longo A., Marafin E., Agbaje M., Lettini A.A., Ojo O.E., Longo A., Marafin E.,
Antonello K., Zavagnin P., Oluwasile B.B., Omoshaba E.O., Antonello K., Zavagnin P., Oluwasile B.B., Omoshaba E.O.,
Dipeolu M.A. Profils de résistance antimicrobienne de séro- Dipeolu M.A. Perfiles de resistencia antimicrobiana a serova-
types de salmonelles isolés à partir de viande de poulet apprê- res de Salmonella aislados de carne de pollo en mataderos de
tée à l’abattoir à Kaduna, Nigeria Kaduna, Nigeria

La salmonellose invasive non typhique, caractérisée par une La salmonelosis invasiva no tifoidea, caracterizada por gastroen-
gastro-entérite et une bactériémie, est endémique en Afrique teritis y bacteriemia es endémica en África subsahariana. La
subsaharienne. La plupart des infections sont d’origine alimen- mayoría de las infecciones son de origen alimenticio, con ani-
taire, les animaux servant de porteurs asymptomatiques. Nous males actuando como portadores asintomáticos. Investigamos
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 Typage moléculaire de Salmonella dans la viande de poulet

avons étudié les sérotypes de Salmonella et les gènes de résis- serovares de Salmonella y los genes de resistencia asociados en
tance associés dans la viande de poulet à l’aide de cultures, carne de pollo, mediante cultivo, concentraciones mínimas inhi-
de concentrations minimales inhibitrices et de l’amplification bitorias y amplificación de PCR de los genes de resistencia. De
par PCR des gènes de résistance. Sur 100 échantillons exami- las 100 muestras examinadas, 28 (28%) fueron positivas a Sal-
nés, 28 (28 %) étaient positifs pour Salmonella et répartis dans monella y se distribuyeron en seis serovares: Haifa (71,4%), Cho-
six sérotypes : Haïfa (71,4 %), Chomedey (7,1 %), Saintpaul medey (7,1%), Saintpaul (7,1%), Kainji (7,1%), Derby (3,6%),
(7,1 %), Kainji (7,1 %), Derby (3,6 %) et Blockley (3,6 %). y Blockley (3,6%). La resistencia antimicrobiana se observó a
Une résistance antimicrobienne à la ciprofloxacine (85,7 %), ciprofloxacina (85,7%), ácido nalidíxico (75,0%), sulfametoxa-
à l’acide nalidixique (75,0 %), au sulfaméthoxazole (67,8 %), zol (67,8%), tetraciclina (89,3%), trimetoprima (42,9%), genta-
à la tétracycline (89,3 %), au triméthoprime (42,9 %), à la micina (35,7%), estreptomicina (32,1%), ampicilina (10,7%),
gentamicine (35,7 %), à la streptomycine (32,1 %), à l’ampi- cloranfenicol (10,7%), kanamicina (7,1%) y florfenicol (3,6%).
cilline (10,7 %), au chloramphenicol (10,7 %), à la kanamy- Todos los aislamientos fueron susceptibles a cefotaxima, ceftazi-
cine (7,1 %) et au florfenicol (3,6 %) a été observée. Tous les dima y colistina, mientras que 19 (67,9%) mostraron resistencia
isolats ont été sensibles à la céfotaxime, à la ceftazidime et à múltiple a por lo menos tres antimicrobianos. El tipo de resis-
la colistine ; 19 (67,9 %) ont présenté une multirésistance à tencia predominante fue Cip-Gen-Nal-Smx-Tet-Tmp, detectado
au moins trois antimicrobiens. Le type de résistance prédomi- en seis (21%) aislamientos. La resistencia múltiple a fármacos
nant était Cip-Gen-Nal-Smx-Tet-Tmp, détecté dans six isolats de serovares de Salmonella fue elevada en las muestras de carne
(21%). La multirésistance des sérovars de Salmonella était éle- de pollo, con una mayor resistencia observada a la ciprofloxa-
vée dans la viande de poulet échantillonnée, et la résistance la cina. Esto sugiere una posible transmisión horizontal de genes
plus souvent observée était contre la ciprofloxacine. Cela sug- resistentes a quinolonas mediados por plásmidos, lo que podría
gère la possibilité d’un transfert horizontal des gènes de résis- comprometer el uso clínico de fluoroquinolonas. Por lo tanto,
tance à la quinolone à médiation plasmidique, ce qui pourrait la mejora de la higiene y la provisión de instalaciones adecua-
compromettre l’utilisation clinique des fluoroquinolones. Ainsi, das en los centros de procesamiento de carne podrían ayudar a
l’amélioration de l’hygiène et la mise en place d’installations limitar la contaminación de la carne y la transmisión a través de
adaptées dans les centres de transformation de la viande pour- los alimentos de los serotipos multi resistentes de Salmonella no
raient contribuer à limiter la contamination de la viande et la tifoidea de pollos a humanos.
transmission aux humains des sérotypes multirésistants non
typhiques de Salmonella par la viande de poulets. Palabras clave: Salmonella, resistencia a los antibióticos,
carne de pollo, sacrificio, Nigeria
Mots-clés : Salmonella, résistance aux antibiotiques, viande
de poulet, abattage d’animaux, Nigeria

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