Changes in the Phenotypes of Salmonella spp. in Japanese Broiler Flocks

Abstract Salmonella infections represent a leading cause of foodborne illnesses; resistance to third-generation cephalosporins (TGCs), which are a first-choice antimicrobial for treating human Salmonella enteritis, has become a serious public health concern worldwide. Because the consumption of undercooked chicken meat products is a major cause of foodborne salmonellosis in Japan, we conducted three surveys at different periods between 2017 and 2022, with the cooperation of four abattoirs (two in Eastern and two in Western Japan). The first survey was conducted at abattoir A, which is located in Eastern Japan. Salmonella was detected in 84.4% of broiler flocks tested (27/32); among them, all the TGC-resistant isolates obtained from one farm (farm FA) were identified as S. Infantis. Salmonella was recovered from 62.5% of breast meat samples (20/32), with one case suggesting cross-contamination. The second survey was conducted at three other abattoirs to examine the prevalence of TGC-resistant Salmonella, in both Western (abattoirs B and C) and Eastern (abattoir D) Japan. Salmonella was detected in 90.6% of broiler flocks examined (29/32). TGC-resistant S. Infantis was isolated from 2 flocks until 2018 and not thereafter. Subsequently, isolates were identified as TGC-susceptible S. Schwarzengrund in both regions. The third survey was performed at abattoir A to elucidate whether there were changes in the phenotypes. Of the 11 broiler flocks introduced from farm FA, 10 were positive for Salmonella (90.9%); all the isolates were S. Schwarzengrund susceptible to TGC. This study shows that TGC-susceptible S. Schwarzengrund has replaced the resistant phenotypes among broiler flocks in both Eastern and Western Japan. Although chicken meat products could be cross-contaminated with Salmonella during the slaughtering process, reducing the prevalence of Salmonella in broiler flocks remains important to decrease Salmonella enteritis in humans.


Introduction
Salmonella is an enteric bacterial pathogen that causes foodborne illnesses in humans worldwide.In Japan, Kumagai et al. estimated the national burden of gastroenteritis caused by Salmonella species in 2011 1) and found that the annual incidence was approximately 31.7 cases per 100,000 population.The calculated disability-adjusted life years (DALYs) lost, based on the incidence estimates along with disability weights, were 3,145 DALYs.Foodborne diseases caused by Campylobacter species and enterohemorrhagic Escherichia coli have led to estimated losses of 6,099 and 463 DALYs, respectively.Similarly, Salmonella infections may represent a serious problem leading to social and economic losses.
Although antimicrobial therapy is generally not recommended for acute self-limiting enteritis, the administration of fluoroquinolones and third-generation cephalosporins (TGCs) may be effective for treating patients with severe symptoms and in health risk groups 2,3) .In Japan, TGC administration is not approved in the poultry industry; however, off-label use was performed at a few hatcheries until it was voluntarily ceased in 2012 4,5) .A nationwide survey conducted between 2007 and 2010 showed that 26.3% of Salmonella isolates from broiler flocks were resistant to the TGC, ceftiofur 6) .Duc et al. found that the prevalence of TGC resistance in Salmonella in broiler chickens decreased from 52.7% to 25.8% in Kagoshima Prefecture (Southern Japan) after the withdrawal of TGC administration 7,8) .Other studies reported that the prevalence of TGC resistance in Salmonella had increased in domestic retail chicken meat products until 2012 9,10) and decreased thereafter 10) .This is consistent with findings from Canada, where there was a decrease in the prevalence of ceftiofur resistance in E. coli and S. Heidelberg isolated from retail chicken meat products after the withdrawal of ceftiofur administration 11) .
From the perspective of food safety, it is important to continue monitoring the prevalence and antimicrobial resistance profiles of Salmonella in foods, especially chickens and their products, because most cases of human salmonellosis are foodborne 1) .They are associated in part with the consump-tion of raw/undercooked contaminated poultry products 12,13) .In this study, we conducted three surveys at different periods between 2017 and 2022, with the cooperation of four abattoirs, to determine whether the decreased prevalence of TGC resistance in Salmonella is observed in both Eastern and Western Japan.

Sample Collection
Three surveys were conducted in this study (Table 1).The first survey was conducted between December 2017 and February 2018.Cecal contents and breast meat samples were collected from broiler chickens on 16 different days at abattoir A, which is located in Eastern Japan.On each day of sample collection, the cecal contents were obtained from three broilers each of the first two consecutive flocks.One package of breast meat sample (weighing 2 kg) was also obtained from each flock, which comprised six pieces of half breast meat.The second survey was conducted at three other abattoirs (B and C in Western, and D in Eastern Japan) between November 2018 and November 2020.Cecal contents were collected from three broiler chickens each of 32 flocks, which were transported from different farms to abattoir B, C, or D. The third survey was conducted between October 2021 and October 2022 at abattoir A; cecal contents were collected from three broiler chickens each of 11 flocks, which were transported from farm FA.Farm FA was the largest farm surveyed in this study, containing 29 broiler houses.It shipped one flock approximately once every 2 days to abattoir A. Flocks from farm FA account for 27% of the poultry slaughtered annually at abattoir A.

Isolation of Salmonella spp.
Isolation of Salmonella spp. was carried out according to the Japanese standard method, NIHSJ-01 14,15) , with some modifications.Approximately 1 g of each cecal content and 150 g of each breast meat sample were placed in a buffered peptone water (Eiken Chemical Co., Ltd., Tokyo, Japan) of nine-fold volume to prepare a 1:10 suspension.The ce-cal and breast meat suspension was homogenized using VORTEX-GENIE 2 (Scientific Industries, Inc., Bohemia, NY, USA) and a Masticator (IUL, S.A., Barcelona, Spain), respectively, followed by incubation at 37 °C for 22 h ± 2 h.Aliquots of 0.1 and 1 mL of each culture were transferred to 10 mL of Rappaport-Vassiliadis broth (Oxoid Ltd., Basingstoke, Hampshire, UK) and 9 mL of tetrathionate broth base, Hajna (Eiken Chemical), respectively, and incubated at 42 °C for 22 h ± 2 h.Xylose-lysine-deoxycholate agar (Kanto Chemical Co., Inc., Tokyo, Japan) and CHROMagar Salmonella (CHROMagar, Paris, France) were used for selective isolation.After incubation at 37 °C for 22 h ± 2 h, typical or suspected colonies were purified and identified biochemically 14,15) .Serotyping was performed according to the Kauffmann-White scheme 16) using somatic and flagellar antisera (Denka Co., Ltd., Tokyo, Japan).A flock was considered positive for Salmonella when Salmonella was isolated from at least one sample of cecal contents.One isolate from each serovar was selected per flock and subjected to subsequent antimicrobial susceptibility testing.

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing was performed using the broth microdilution method with 96-well microtiter plates (Eiken Chemical).The following 12 antimicrobial agents were examined: ampicillin, cefazolin, cefotaxime, streptomycin, gentamicin, kanamycin, tetracycline, nalidixic acid, ciprofloxacin, colistin, chloramphenicol, and trimethoprim.The resistance breakpoints were determined in accordance with the guidelines by Clinical and Laboratory Standards Institute 17) and National Veterinary Assay Laboratory of Japan 18) .E. coli ATCC 25922 was used as the quality control strain.

Genetic Analysis of Salmonella Isolates
Cefotaxime-resistant isolates were analyzed to detect plasmid-mediated AmpC β-lactamase genes using multiplex polymerase chain reaction 19) , and sequence analysis was subsequently performed with primer pairs reported by Noda et al. 20) on a 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA).When cross-contamination was suspected in the first survey, the relevant isolates were subjected to whole-genome sequencing (WGS) analysis 21) to determine the antimicrobial resistance genes and sequence types based on multi-locus sequence typing (MLST).Sequencing libraries for each isolate were prepared using the QIAseq FX Library Kit (QIAGEN N.V., Venlo, The Netherlands) to obtain paired-end sequences (300 bp × 2) on the Illumina MiSeq platform (Illumina, Inc., San Diego, CA, USA).The draft genome sequence was assembled using A5-miseq 22) and gene annotation was performed using DFAST version 1.2.3, with the following databases: DFAST default database 23) , ResFinder database 24) , and Bacterial Antimicrobial Resistance Reference Gene database (PRJNA313047).MLST was performed in accordance with the protocols available in the MLST database (https://pubmlst.org/).

Results and Discussion
At the time of the start of this study in 2017, five years had already passed since TGC administration was ceased voluntarily in the poultry industry in Japan.The first survey was conducted at abattoir A in Eastern Japan; 27 out of 32 broiler flocks were positive for Salmonella (84.4%), and 29 isolates were eventually obtained (Table 2).The most frequent serovar was S. Infantis, followed by S. Schwarzengrund.Nine isolates of S. Infantis were resistant to cefotaxime, a TGC, and harbored bla CMY-2 as the resistance gene.They were isolated from 9 flocks introduced from one farm (farm FA).Farm FA did not use the "all-in/all-out" system at the farm level, whereas the other 14 farms included in the first survey used this system.These findings suggest that chicks colonized by TGC-resistant Salmonella were not introduced into the broiler farms at around the time of the study; however, they were introduced before and survived while moving from one broiler house to another in farm FA.
The second survey was conducted to examine the prevalence of TGC-resistant Salmonella, in both Western (abattoirs B and C) and Eastern (abattoir D) Japan, because a regional difference in the serological distribution of Salmonella was observed in broiler chickens in a previous study 6) .In total,  Schwarzengrund was predominant (Table 2).Cefotaximeresistant Salmonella was never isolated during the third survey.The prevalence of TGC resistance in Salmonella isolates was 31.0% in the first, 6.9% in the second, and 0% in the third survey.
To elucidate whether cross-contamination occurs during the slaughtering process, Salmonella was also isolated from the breast meat samples collected in the first survey.Twenty-three isolates were obtained from breast meat samples derived from 19 Salmonella-positive and one Salmonella-negative flocks (Table 3); 18 isolates exhibited the same serovars and antimicrobial resistance profiles as those from the cecal contents of the corresponding flocks.Flocks B21 and B22 were introduced from different farms and slaughtered consecutively on the same day; S. Infantis exhibiting the same antimicrobial resistance profile was isolated from the breast meats from both flocks.The same phenotype was also obtained from the cecal contents of flock B21, whereas Salmonella was not isolated from those of flock B22.To gain more information on these three isolates, they were subjected to WGS analysis.These isolates harbored the same antimicrobial resistance genes and were classified into the same sequence type (Table 4).Although the MLST technique does not thoroughly distinguish genomic heterogeneity among S. Infantis strains 30) , the three strains could not have been differentiated from each other based on the serological and antimicrobial characteristics.We previously reported that chicken meat products derived from a Campylobacter-negative flock could be cross-contaminated by a Campylobacter-positive flock slaughtered right before 25,26) .As both Campylobacter and Salmonella colonize the intestinal tract of broilers, and several broiler flocks from different farms are slaughtered consecutively in a single abattoir, cross-contamination of chicken meat products with Salmonella could occur in a similar manner to Campylobacter contamination.Nevertheless, because of the limited number of breast meat samples included in this study, intensive investigation is needed to determine how frequently cross-contamination occurs during the slaughtering process.
In the first decade of the 2000s, S. Infantis was the most frequent isolate in broiler chickens [27][28][29] , and the resistance rate to ceftiofur was low (1.8%) 29) .The ceftiofur-resistance rate of Salmonella isolated from broiler flocks was 26.3%, especially that of S. Infantis was 38.1%, according to a nationwide survey conducted between 2007 and 2010 6) .Duc et al. showed that the increased prevalence of TGC-susceptible S. Schwarzengrund led to a decrease in the prevalence of TGC-resistant Salmonella in Southern Japan 7,8) .This study has some limitations; for example, the number of samples and survey area were relatively small.However, our results imply the general tendency that TGC-susceptible S. Schwarzengrund has replaced TGC-resistant Salmonella as the predominant phenotype among broiler flocks in both Eastern and Western Japan. In his study, bla CMY-2 was detected as the resistance gene.Extended-spectrum β-lactamaseproducing Salmonella strains carrying bla TEM-52 were reported for the first time from broiler chickens in Japan during 2004-2006 31) .Salmonella strains harboring bla CMY-2 , bla TEM-20 , or bla CTX-M-25 appeared in 2007 and 2008 in Southern Japan 32) .The prevalence of bla TEM-52 remarkably increased 32) and became predominant, followed by bla CMY-2 , in 2010 and 2011 33) .In retail chicken meat products, the first Salmonella spp.harboring bla CMY-2 were isolated in the mid 2000s 34) .The prevalence of bla CMY-2 increased until 2011, and decreased after 2012, partially due to the change in the prevalence of TGC-resistant Salmonella (mainly S. Infantis) harboring bla CMY-2 10,20,35) .Our results are consistent with these previous reports.
In conclusion, TGC-susceptible Salmonella has replaced the resistant phenotypes among broiler flocks in both Eastern and Western Japan, leading to a decreased prevalence of TGC-resistance in Salmonella.Chicken meat products could be cross-contaminated with Salmonella during the slaughtering process; however, reducing the prevalence of Salmonella in broiler flocks remains an important measure to decrease Salmonella infections in humans because the relationship  between a reduction in the prevalence of Salmonellacontaminated chickens and the corresponding reduction in the risk of human illness was reported to be one-to-one, 12) and the prevalence of Salmonella in broiler flocks remains high (>70%) as revealed in this study.From a food safety perspective, continuous nationwide surveillance is needed to determine the transition of the prevalence, serovars, and antimicrobial resistance profiles of Salmonella in humans, chickens, and chicken meat products to identify, implement, and evaluate effective countermeasures for human salmonellosis.

Table 1 .
Summary of the three surveys in this study.
a The values in parentheses refer to the number of flocks positive for Salmonella spp.

Table 2 .
Serovars and antimicrobial resistance profiles of Salmonella isolated from the cecal contents of broilers in the first, second, and third surveys.
The values in parentheses refer to the number of isolates from farm FA out of the total.
a ABPC; ampicillin, CEZ; cefazolin, CTX; cefotaxime, SM; streptomycin, KM; kanamycin, TC; tetracycline, NA; nalidixic acid, TMP; trimethoprim.b The values refer to the number of isolates.c ing to the results of the second survey, TGC-susceptible S. Schwarzengrund was considered to have recently replaced TGC-resistant S. Infantis in Japanese broiler flocks.Based on this hypothesis, we investigated the presence of TGCresistant S. Infantis once again in broiler flocks introduced from farm FA at abattoir A in the third survey.Of the 11 broiler flocks tested from 2021 to 2022, 10 were positive for Salmonella (90.9%), and 10 isolates were eventually obtained; cefotaxime-susceptible and kanamycin-resistant S.

Table 3 .
Comparison of Salmonella isolates obtained from cecal contents and breast meat of broilers in the first survey.

Table 4 .
Genetic characteristics of three strains of S. Infantis isolated from flocks B21 and B22.Nucleotide sequence data reported are available in the DDBJ Sequence Read Archive under the accession numbers DRR524581 (the isolate from the cecal contents, flock B21), DRR524582 (the isolate from the breast meat, flock B21), and DRR524583 (the isolate from the breast meat, flock B22). a ABPC; ampicillin, CEZ; cefazolin, CTX; cefotaxime, SM; streptomycin, TC; tetracycline, TMP; trimethoprim.