Increased kanamycin-resistant Salmonella Schwarzengrund, serovar changing and antimicrobial susceptibility of Salmonella enterica from broiler chickens in Kagoshima, Japan between 2019 and 2023

Abstract

Background

This study investigated the prevalence of Salmonella, determined the minimum inhibitory concentrations (MICs) of antimicrobials, and examined antimicrobial resistance genes and plasmids in Salmonella strains from broiler chickens in Kagoshima Prefecture, Japan.

Results

A total of 3,774 cecal samples were collected from 236 broiler flocks between 2019 and 2023. Salmonella was prevalent in 202 (85.6%) and 753 (19.9%) broiler flocks and cecal content samples, respectively. Salmonella Manhattan, S. Schwarzengrund, and S. Infantis were the only serovars found in broiler chickens in Kagoshima Prefecture. Since 2021, the isolation rate of S. Schwarzengrund has been higher than that of other serovars. A total of 278/753 (36.9%) Salmonella strains were resistant to kanamycin, 99.3% of which were S. Schwarzengrund strains (MIC value ≥ 512 µg/mL). Furthermore, of the 268 Salmonella isolates that were tested for susceptibility to all antimicrobials used in this study, all were susceptible to cefoxitin and ofloxacin, and the majority were resistant to streptomycin-S (98.5%), sulfamethoxazole-Su (90.3%), tetracycline-T (79.5%), and kanamycin-Km (28.7%). In addition, three plasmid replicons (IncFIB, IncP1, and IncX4) were identified in Salmonella. IncFIB was present in all the Salmonella isolates examined and contained antimicrobial resistance genes. IncX4 and IncP1 were identified in streptomycin, sulfamethoxazole, and tetracycline (SSuT)-resistant S. Manhattan and streptomycin, sulfamethoxazole, tetracycline, and kanamycin (SSuTKm)-resistant S. Infantis, respectively, and contained none of the resistance genes.

Conclusion

This study revealed an increased incidence of S. Schwarzengrund in broiler chickens, which exhibit high kanamycin resistance. Additionally, a significant percentage of the Salmonella isolates in this study were resistant to streptomycin, tetracycline, and sulfamethoxazole. This finding suggests that these antimicrobials are ineffective at controlling infections in broilers. In addition, we speculate that the spread of antibiotic resistance in Salmonella is enhanced by plasmids.

Background

Salmonella enterica is an important foodborne pathogen that has significant economic and health impacts on humans and animals worldwide. It causes an estimated 90 million cases of gastroenteritis worldwide every year, with approximately 155,000 mortalities [1]. Salmonella is classified into more than 2,600 serotypes on the basis of the presence of somatic (O), flagellar, and capsular surface antigens [2]. Chickens are well-known reservoirs of various Salmonella serovars that may spread to humans after they consume contaminated raw or undercooked chicken products [3].

Salmonella serovars Schwarzengrund, Manhattan, and Infantis are mostly detected in chicken meat and ceca in Japan [4–6], and these serovars have also been reported to contaminate humans [7]. However, the isolation rate of S. Schwarzengrund from food animals and humans has recently increased, and countries, including Thailand, Slovakia, New Zealand, Venezuela, Brazil, the U.S., and Denmark, have reported a significant increase in this serovar [8–10].

In Japan, this serovar ranked among the top five serotypes after 2015, and it became the most common serovar in 2018 [11]. Additionally, the isolation rate of S. Schwarzengrund in food increased from 13.3% (2012–2014) to 51.3% (2018–2020) in Tokyo, Japan [12]. Moreover, several studies have reported an increased rate of isolation of S. Schwarzengrund in both broiler chickens and their meat [4, 6, 13, 14]. The increase in the isolation rate of S. Schwarzengrund is not only noteworthy; there is also evidence of a higher frequency of antimicrobial resistance (AMR) within the strains of this serovar that are spreading globally [15].

Antimicrobials, including penicillins, tetracyclines, aminoglycosides, fluoroquinolones, sulfonamides, and macrolides, are used to treat bacterial infections in broilers in Japan [16]. In addition, a number of studies have reported multidrug-resistant Salmonella strains isolated from broilers in Japan [4, 7, 13, 14, 17]. These studies revealed that AMR in Salmonella is caused by the overuse of antimicrobial agents on broiler farms and that antimicrobial resistance has spread widely among Salmonella strains. In addition, Duc et al. [5, 18] reported that the switching of Salmonella serovars in broiler chickens in Kagoshima influenced the pattern of antimicrobial resistance.

Conjugative plasmids are key agents of horizontal gene transfer that promote the dissemination of antimicrobial resistance genes (ARGs) and virulence genes (VGs) among bacteria [19]. Furthermore, the transfer of plasmids in pathogens has led to the worldwide spread of numerous ARGs encoding resistance to β-lactams, quinolones, aminoglycosides, tetracyclines, sulfonamides, and many other drug classes [20].

The multidrug resistance of Salmonella, the diverse mechanisms of AMR spread among Salmonella, and the periodic switching of the Salmonella serovar distribution in broiler chickens make the control of salmonellosis challenging. Consequently, ongoing monitoring and periodic assessment of the distribution, AMR profile and mechanisms involved in the emergence and spread of AMR in Salmonella from broiler chickens are necessary. The aim of this study was to investigate the prevalence, determine the minimal inhibitory concentration (MIC) of antimicrobials, and examine the resistance genes and plasmids associated with the antimicrobial resistance of Salmonella serovars isolated from broiler ceca in Kagoshima, Japan, from 2019 to 2023.

Results

Prevalence and serovar changes

The prevalence and distribution of Salmonella in broiler chickens from 2019 to 2023 in Kagoshima Prefecture, Japan, are presented in Table 1. Overall, the percentages of Salmonella-positive flocks and Salmonella-positive samples were 202/236 (85.6%) and 753/3774 (19.9%), respectively. The prevalence of Salmonella-positive flocks significantly (p < 0.05) increased in the first three years of this study, increasing from 72.9% in 2019 to 89.6% in 2020 and 93.8% in 2021. However, the prevalence decreased significantly (p < 0.05) to 79.2% in 2022 before increasing again to 93.2% in 2023. Similarly, the prevalence of Salmonella-positive samples increased to 21.7% in 2020, from 13.2% in 2019 (p < 0.05) and subsequently to 23.6% in 2021. The prevalence then decreased dramatically (p < 0.05) to 17.7% in 2022 before significantly increasing (p < 0.05) to 23.9% in 2023.

Table 1.

The prevalence and distribution of Salmonella serovars isolated from broiler chickens from 2019–2023

Year	No. of flocks	No. of positive flocks (%)	No. of samples	No. of positive samples (%)	Salmonella serovars
					Schwarzengrund isolates	Manhattan isolates	Infantis isolates
2019	48	35(72.9)	768	101(13.2)	36	59	6
2020	48	43(89.6)#↑	768	167(21.7)#↑	55	105	7
2021	48	45(93.8)#↑	768	181(23.6)	111#↑	70#↓	0
2022	48	38(79.2)#↓	768	136(17.7)#↓	83	52	1
2023	44	41(93.2)*↑	702	168(23.9)#↑	126#↑	42#↓	0
Total	236	202(85.6)	3,774	753(19.9)	411(54.6)#	328 (43.6)	14(1.9)

# significantly higher proportion (p < 0.05)

#↓ significant decrease from the previous year (p < 0.05)

#↑ significantly increased from the previous year (p < 0.05)

Among the 753 isolates of Salmonella obtained in this study, S. Schwarzengrund was the most prevalent serovar (p < 0.05) at 411/753 (54.6%), followed by S. Manhattan at 328/753 (43.6%) and S. Infantis at 14/753 (1.9%). Additionally, serovar switching occurred, especially between S. Manhattan and S. Schwarzengrund, whereby S. Manhattan was the dominant serovar in 2019 (59/101) and 2020 (105/167); however, beginning in 2021, S. Schwarzengrund surpassed S. Manhattan and became the dominant serovar. In addition, the prevalence of Salmonella infantis was low throughout the study period.

Antimicrobial resistance of Salmonella isolates

The comparison of the antimicrobial resistance profiles of Salmonella isolates from 2019 to 2020 from this study with those from 2017 to 2018 cited from earlier study [18] is presented in Table 2. We included the antimicrobial results of Salmonella isolates for the first two years of this study, allowing comparison with earlier studies. All 268 (100%) Salmonella isolates were susceptible to cefoxitin and onfloxacin. High resistance rates were detected for streptomycin (98.5%), sulfamethoxazole (90.3%), tetracycline (79.5%) and kanamycin (28.7%), whereas low resistance rates were detected for ampicillin (5.2%), ceftiofur (5.2%), cefotaxime (4.8%), and chloramphenicol (0.4%).

Table 2.

Comparison of the antimicrobial resistance profiles of Salmonella isolates from 2019–2020 with those from 2017–2018

Antimicrobial agent	MIC-Break-point (µg/ml)	No. of resistant isolates (%)
		Previous study a)	Current study b)
		2017–2018	2019–2020
		n = 264 a)	n = 268 b)
AMP	≥ 32	20 (7.6)	14 (5.2)
CTX	≥ 4	15 (5.7)	13 (4.8)
CFX	≥ 32	2 (0.8)	0 (0.0)
CTF	≥ 8	20 (7.6)	14 (5.2)
OFLX	≥ 2	5 (1.9)	0 (0.0) #↓
CP	≥ 32	0 (0.0)	1 (0.4)
SM	≥ 16	249 (94.3)	264 (98.5)#↑
SUL	≥ 512	213 (80.7)	242 (90.3)#↑
OTC	≥ 16	217 (82.2)	213 (79.5)
KM	≥ 64	77 (29.2)	77 (28.7)

AMP ampicillin, CTX cefotaxime, CFX cefoxitin, CTF ceftiofur, OFLX ofloxacin, CP chloramphenicol, SM streptomycin, SUL sulfamethoxazole, OTC oxytetracycline, KM kanamycin

^(a)Salmonella isolates cited from Duc et al., 2022 [18] 

^(b)Salmonella isolates of this study (the n = 268 includes isolates from 2019 to 2020) 

#↓ significantly decreased from the previous year (p < 0.05)

#↑ significantly increased from the previous year (p < 0.05)

In this study, we observed a significant (p < 0.05) increase in the rate of Salmonella resistance to streptomycin (98.5%) and sulfamethoxazole (90.3%) compared with 94.3% and 80.7% in the 2017 and 2018 survey periods (Duc et al., 2022), respectively. Furthermore, there was an increase in the resistance rate to 0.4% chloramphenicol from 0.0% in a previous study; however, the increase was not significant. On the other hand, Salmonella strains showed a decreased rate of resistance to oxytetracycline (79.5%), kanamycin (28.7%), ampicillin (5.2%), ceftiofur (5.2%), cefotaxime (4.8%), cefoxitin (0%), and onfloxacin (0%) in this study compared with 82.2%, 29.2%, 7.6%7.6%, 5.7%, 0.8%, and 1.9%, respectively, in the 2017–2018 survey period. The decrease in the rate of resistance of Salmonella to antimicrobials other than ofloxacin was not significant.

Table 3 shows the comparison of antimicrobial resistance profiles for each Salmonella serovar from 2019 to 2020. The serovars S. Schwarzengrund and S. Infantis were all (100%) susceptible to ampicillin, cefotaxime, ceftiofur, and chloramphenicol. Like S. Manhattan, which were all susceptible to kanamycin, all three serovars were susceptible to cefoxitin and ofloxacin. In addition, high resistance rates to streptomycin, sulfamethoxazole, and oxytetracycline were observed for all the Salmonella Schwarzengrund and S. Infantis strains, and resistance rates of 96.9%, 84.1%, and 65.9%, respectively, were exhibited by S. Manhattan to the aforementioned antimicrobial agents.

Table 3.

Comparison of the antimicrobial resistance of Salmonella serovars from 2019–2020

Antimicrobial agent	Number of resistant isolates (%)
	S. Schwarzengrund n = 91	S. Manhattan n = 164	S. Infantis n = 13
AMP	0 (0.0)	14 (8.5)	0 (0.0)
CTX	0 (0.0)	13 (7.9)	0 (0.0)
CFX	0 (0.0)	0 (0.0)	0 (0.0)
CTF	0 (0.0)	14 (8.5)	0 (0.0)
OFLX	0 (0.0)	0 (0.0)	0 (0.0)
CP	0 (0.0)	1 (0.6)	0 (0.0)
SM	91 (100)	159 (96.9)	13(100)
SUL	91 (100)	138 (84.1)	13(100)
OTC	91 (100)	108 (65.9)	13(100)
KM	75 (82.4) #	0 (0)	1 (7.7)

AMP ampicillin, CTX cefotaxime, CFX cefoxitin, CTF ceftiofur, OFLX ofloxacin, CP chloramphenicol, SM streptomycin, SUL sulfamethoxazole, OTC oxytetracycline, KM kanamycin

# significantly greater proportion of kanamycin-resistant strains than other serovars (p < 0.05)

In contrast, low resistance to ampicillin (8.5%), ceftiofur (8.5%), cefotaxime (7.9%), and chloramphenicol (0.6%) was observed in S. Manhattan. In particular, S. Schwarzengrund presented a significantly (p < 0.05) high rate of kanamycin resistance (82.4%), as opposed to other serovars whose majority were susceptible.

The incidence of kanamycin resistance in Salmonella serovars

As shown in Table 4, the overall incidence of kanamycin-resistant Salmonella obtained in this study was 278/753 (36.9%), which increased significantly (p < 0.05) each year during the present survey period, except in 2020, when the proportion decreased from 28.7% in 2019 to 28.1%. Notably, S. Schwarzengrund presented a significantly (p < 0.05) greater percentage (276/411 (67.2%)) of kanamycin resistance than did S. Manhattan (1/328 (0.3%) and S. Infantis (1/14 (7.1%) strains. The proportion of kanamycin resistance within S. Schwarzengrund varied each year during the study period. It increased from 77.8% in 2019 to 85.5% in 2020. However, in 2021, the proportion significantly decreased (p < 0.05) to 55.9%. In 2022, the proportion rose again to 69.9% before dropping to 64.3% in 2023.

Table 4.

Incidence of Kanamycin resistance in Salmonella serovars from broilers from 2019–2023

Year	No. of KmR S. S/ No. of S. S (%)	No. of KmR S. M/ No. of S. M (%)	No. of KmR S. I/ No. of S. I (%)	KmR total/Total No. of isolates (%)
2019	28/36 (77.8)	0/59 (0.0)	1/6 (16.7)	29/101(28.7)
2020	47/55 (85.5)	0/105 (0.0)	0/7 (0.0)	47/167(28.1)
2021	62/111 (55.9)#↓	0/70 (0.0)	0/0 (0.0)	62/181(34.3) #↑
2022	58/83 (69.9) #↑	1/52 (1.9)	0/1 (0.0)	59/136(43.4) #↑
2023	81/126 (64.3)	0/42 (0.0)	0/0 (0.0)	81/168 (48.2) #↑
Total	276/411 (67.2) #	1/328 (0.3)	1/14 (7.1)	278/753 (36.9)

No. of KmR-number of kanamycin-resistant strains; S. S., S. M. and S.I. are Salmonella serovars Schwarzengrund, Manhattan, and Infantis, respectively

#↓ significantly decreased from the previous year (p < 0.05)

#↑ significantly increased from the previous year (p < 0.05)

The minimum inhibitory concentration (MIC) and distribution of genes conferring kanamycin resistance (aphA1) in kanamycin-resistant Salmonella isolates are shown in Table 5. All kanamycin-resistant S. Schwarzengrund 276/276 (100%) and S. Infantis 1/1 (100%) strains had a kanamycin MIC value of ≥ 512 µg/ml, and in addition, they all carried the aphA1 gene, contrary to kanamycin-resistant S. Manhattan 1/1 (100%), which not only had a kanamycin MIC value of 256 µg/ml but also lacked the aphA1 gene.

Table 5.

MIC values and aphA1 gene distribution in kanamycin-resistant Salmonella serovars from broilers from 2019–2023

Serovar (No. of isolates)	MIC of kanamycin (µg/ml)	No. of KmR isolates	No. of isolates positive for aphA1 gene (%)
S. Schwarzengrund (411)	512	276	276(100)
	256	0	0
	128	0	0
S. Manhattan (328)	512	0	0
	256	1	0(0)
	128	0	0
S. Infantis (14)	512	1	1(100)
	265	0	0
	128	0	0
Total		278	277(99.6)

The aphA1 (another name, aph (3")-Ia) gene was detected by PCR

Whole genome sequencing (WGS) and plasmid analysis

Table 6 shows the ARGs and plasmid profiles detected in the Salmonella isolates via WGS. ResFinder identified five genes—ant (3”)-Ia, sul1, tet(A), aph(3’)-Ia, and dfrA14—that confer resistance to streptomycin, sulfamethoxazole, tetracycline, kanamycin, and trimethoprim, respectively. However, S. Manhattan lacked the aph(3’)-Ia gene despite being phenotypically resistant.

Table 6.

Antimicrobial resistance genes and plasmid sequence analyses of Salmonella serovars from broilers

Serovar	Phenotype	Resistance genes on plasmids	No. of plasmids	Plasmid replicons	Plasmid size(bp)
S. Schwarzengrund	SSuT	ant(3”)-Ia, sul1, tet(A), dfrA14	1	IncFIB	284,635
S. Schwarzengrund	SSuTKm	ant(3”)-Ia, sul1, tet(A), dfrA14, aph(3”)-Ia	1	IncFIB	284,630
S. Manhattan	SSuT	ant(3”)-Ia, sul1, tet(A) none	2	IncFIB IncX4	278,655 29,617
S. Manhattan	SSuTKm	ant(3”)-Ia, sul1, tet(A)	1	IncFIB	276,478
S. Infantis	SSuT	ant(3”)-Ia, sul1, tet(A)	1	IncFIB	270,117
S. Infantis	SSuTKm	ant(3”)-Ia, sul1, tet(A), dfrA14, aph(3”)-Ia none	2	IncFIB IncP1	291,123 41,696

S- Streptomycin, Su- sulfamethoxazole, T-tetracycline and Km- kanamycin resistant; ant (3”)-Ia (aadA1), sul1, tet (A), aph(3’’)-Ia (aphA1) and dfrA14 are genes involved in resistance to streptomycin, sulfamethoxazole, tetracycline, kanamycin and trimethoprim, respectively

“None” - no resistance gene detected on the plasmid.

Additionally, three plasmid replicons (IncFIB, IncP1, and IncX4) were identified via PlasmidFinder. The IncFIB plasmid was identified in all six isolates and contained resistance genes, whereas IncX4 and IncP1 were identified in SSuT-resistant S. Manhattan and SSuTKm-resistant S. Infantis, respectively, but contained none of the resistance genes. In addition, the size of the IncFIB plasmid was between 270 and 291 kb, whereas the IncP1 and IncX4 plasmid sizes were 4.1 kb and 2.9 kb, respectively.

Moreover, the replicon groups IncFIB, IncP1, and IncX4 identified in the Salmonella isolates from this study were 100% identical to the Salmonella enterica subsp. enterica serovar Infantis strain N55391 plasmid pN55391, the Escherichia coli strain CH613_eco plasmid, and the Escherichia coli UMNF18 plasmid pUMNF18_32 strains with accession numbers CP016411, CM007914, and CP002895, respectively, in the National Institutes of Health (NIH) genetic sequence database (GenBank).

Discussion

In this study, the overall prevalence rates of Salmonella-positive flocks and samples were 85.6% and 19.9%, respectively, which were higher than the 81.3% and 17.9% prevalence rates of Salmonella reported in a similar earlier study [18]. However, our findings are consistent with those of a study in eastern Japan [13], which reported an 84.4% prevalence of Salmonella in broiler flocks. Additionally, our results contrast with those of a study in Poland, where the prevalence of Salmonella in broilers was 1.57% [21].

Three Salmonella serovars, S. Schwarzengrund, S. Manhattan, and S. Infantis, were the only serovars detected in broiler chickens in Kagoshima, Japan, in the present study, which is in agreement with earlier studies [5, 14, 18]. However, the frequency of isolation varied among these serovars in each survey period, and in the present survey period, S. Schwarzengrund was significantly (p < 0.05) the most frequent serovar (411/753, 54.6%), followed by S. Manhattan (328/753, 43.6%) and S. Infantis (14/753, 1.9%). This serovar has also been reported to emerge and cause multiple infections in other countries, including the United States of America, Thailand, Slovakia, New Zealand, and Venezuela [10, 22]. The introduction of the S. Schwarzengrund ST241 clone in chickens has been linked to the current increase in S. Schwarzengrund in broilers [23].

In addition, we noted a periodic change in predominance among the three serovars; in 2019 and 2020, serovar S. Manhattan was more frequently isolated than S. Schwarzengrund was, and the isolation rate of the latter serovar increased starting in 2021 and recently became a dominant serovar, whereas that of S. Manhattan progressively declined. Similarly, Duc et al. [5] reported serovar switching for the first time in 2019, when the isolation rate of S. Manhattan increased over that of S. Infantis. Salmonella serovar switching in broiler chickens has been associated with changes in the patterns of antimicrobial resistance of Salmonella strains from broilers in Kagoshima, Japan [18].

Salmonella is highly resistant to streptomycin (98.5%), sulfamethoxazole (90.3%), oxytetracycline (79.5%) and kanamycin (28.7%). A similar finding was reported in earlier studies of Salmonella in chickens in Japan [6, 7]. The extensive use of these antimicrobials for bacterial control in poultry hatcheries and breeder farms in Japan may account for the observed increased resistance [4]. In contrast, all the isolates were susceptible to cefoxitin and ofloxacin, and only a small proportion of the isolates were resistant to ampicillin, ceftiofur, cefotaxime, and chloramphenicol. The decreasing trend of Salmonella resistance to β-lactam and extended-spectrum cephalosporin antimicrobials has been associated with the cessation of ceftiofur in ovo administration by the Japanese poultry industry [17, 18].

The increased resistance of Salmonella isolates to streptomycin and sulfamethoxazole in the present study was significantly greater than that in the earlier survey period (Table 2). This may be due to the increased isolation rate of S. Schwarzengrund isolates, the majority of which are resistant to the aforementioned antimicrobials. On the other hand, we noted decreased resistance rates of Salmonella against oxytetracycline, kanamycin, ampicillin, cefotaxime, cefoxitin, ceftiofur, and ofloxacin in the present study compared with the previous survey period; however, the decrease was not statistically significant except for that of ofloxacin.

This study revealed interesting findings about kanamycin resistance among Salmonella serovars: while all S. Manhattan isolates were susceptible to kanamycin and only one isolate of S. Infantis was resistant to the drug, a high proportion, 75/91 (82.4%, p < 0.05), of S. Schwarzengrund were resistant to the drug during the 2019–2020 survey period (Table 3).

Similarly, S. Schwarzengrund contributed 276/278 (99%) to the overall incidence of 278/753 (36.9%) kanamycin-resistant Salmonella strains during the entire 2019–2023 survey period (Table 4). According to earlier comparable investigations conducted in Japan [4, 13, 23], S. Schwarzengrund isolated from chicken meat, chicken meat products, and chicken feces showed increased kanamycin resistance, indicating the extensive use of this antimicrobial in Japanese poultry farming. Additionally, a study examining the distribution, virulence, and antimicrobial resistance of Salmonella enterica serovar Schwarzengrund strains, which were isolated from food-animal-associated sources and human patients in the United States, the United Kingdom, Canada, Germany, France, Brazil, India, Thailand, Taiwan, and Vietnam, revealed a high prevalence of the aminoglycoside (aph(3'')-Ib) gene of this serovar [22]. In their study, they did not find a significant prevalence of kanamycin resistance in S. Schwarzengrund, possibly due to differences in the drug of choice from the aminoglycoside class and sequence types between nations. For instance, Japan’s S. Schwarzengrund strains from chickens belong to ST241, which shares a common ancestor regardless of antimicrobial resistance pattern, product district, or isolation year. This ST241 isolate differs from the globally distributed S. Schwarzengrund [23]. However, aminoglycoside resistance in the Salmonella enterica serovar Schwarzengrund is a growing global concern. In the present study, we were unable to identify the primary reason for the significant kanamycin resistance observed in this serovar. However, we speculate that plasmids may have played a role in facilitating the spread of this resistance.

Added to that, the incidence of kanamycin resistance decreased considerably (p < 0.05) from 85.5% in 2020 to 55.9% in 2021 before increasing to 69.9% in 2022, even though the number of S. Schwarzengrund isolates doubled from 55 in 2020 to 111 in 2021 (Table 4). In 2021, nearly half of S. Schwarzengrund, at 44%, were not resistant to kanamycin. This could be the result of either less kanamycin use on chicken farms or the fact that these strains simply did not have the genes for kanamycin resistance.

Besides, all the kanamycin-resistant S. Schwarzengrund and S. Infantis isolates in this study presented a kanamycin MIC value of ≥ 512 µg/ml and carried the aphA1 gene. In contrast, kanamycin-resistant S. Manhattan, which had an MIC value of 256 µg/ml, lacked the aphA1 gene. Our findings are consistent with those of a previous study [24], which reported that kanamycin-resistant Salmonella isolates with an MIC value of ≥ 512 µg/ml carried the aphA1 gene. This finding suggests that the serovars S. Schwarzengrund and S. Infantis are likely to have a relatively high affinity for kanamycin resistance and a comparable mechanism mediating resistance, unlike S. Manhattan.

Furthermore, the plasmid replicons IncFIB, IncP1, and IncX4 were identified in the representative Salmonella isolates in this study. The IncFIB gene was the predominant replicon identified in all the isolates, and this replicon contained the ant(3”)-Ia, sul1, tet(A), aph(3’)-Ia, and dfrA14 resistance genes. This finding is in agreement with an earlier study [25] on Salmonella in chicken meat in Japan, which revealed similar antimicrobial resistance gene combinations contained in the IncFIB plasmid. This finding, however, contrasts with previous research [26], which did not identify the IncFIB replicon in the Salmonella serovars investigated in this study. Instead, the replicon was found to contribute to the acquisition of antibiotic resistance in the Salmonella serovars Heidelberg, Typhimurium, and Kentucky. In addition, studies in the United States by Felix et al. [22], Sopovski et al. [27], and Felix et al. [15] reported that the predominance of the IncFIB replicon was linked to aminoglycoside resistance in S. Schwarzengrund strains isolated from humans, food, and animals.

The IncP1 and IncX4 replicons did not contain a resistance gene, even though they were identified in SSuT-resistant S. Manhattan and SSuTKm-resistant S. Infantis, respectively. The lack of resistance genes in these replicons may explain their small sizes of 4.1 kb and 2.9 kb, as observed in this study. This finding corresponds with an earlier study [25], which revealed the coexistence of the IncFIB replicon with Inc1-I in S. Schwarzengrund and with IncX4 in S. Infantis, which did not carry the resistance gene.

Conclusion

The results of this study indicate that S. Schwarzengrund is currently the most prevalent strain in broiler chickens from Kagoshima and has a strong affinity for kanamycin resistance. Streptomycin, tetracycline, and sulfamethoxazole are no longer effective antimicrobials for controlling Salmonella in broiler chickens. This ineffectiveness is due to the high resistance rates observed in this study as well as in similar previous studies. In addition, we identified three plasmid replicons from Salmonella isolates, but in particular, the IncFIB plasmid has been found to carry multiple resistance genes, including the gene mediating kanamycin resistance. While further research on the causes of high kanamycin resistance, particularly in the S. Schwarzengrund serovar, is recommended, the findings from this study indicate the need for ongoing surveillance of the prevalence and antimicrobial susceptibility of Salmonella in broiler chickens. Additionally, the high resistance of Salmonella to streptomycin, sulfamethoxazole, oxytetracycline, and kanamycin encouraged broiler companies to seek alternative antimicrobials.

Methods

Sampling

A total of 3,774 cecal specimens derived from 236 broiler flocks (approximately 10,000 birds per flock) were collected from poultry processing plants in Kagoshima Prefecture, Japan, from 2019 to 2023. Briefly, cecum samples (n = 32), representative of two broiler flocks (16 randomly selected samples per flock), were collected every two weeks by prefectural officials from January through December every year throughout the survey period. The samples were collected in sterile bags and transported in a cold chain until arrival at the laboratory for further processing on the same day.

Salmonella isolation

The isolation, identification and serotyping of Salmonella were performed as previously described [28]. Briefly, approximately 1 g of cecal contents was aseptically collected into 5 mL of sterilized distilled water and homogenized by vortexing. Then, 1 mL of the suspension was preenriched in 5 mL of Hajna tetrathionate broth (Eiken Chemical Co. Ltd., Tokyo, Japan) and incubated in a water bath at 42 °C. After 24 h of incubation, a loopful from each of the enriched broths was streaked onto plates of selective Rambach (Rambach, 1990) agar and incubated at 37 °C for 24 h. Pink colonies were selected from each plate and streaked on nutrient agar slants. Presumptive Salmonella isolates were identified via biochemical tests, including fermentation of glucose, lactose and sucrose; hydrogen sulfide production; citrate; lysine decarboxylation; and methyl red and indole tests. The isolates were serotyped via slide agglutination with Salmonella group O antisera (Denka Co., Tokyo, Japan) for somatic antigens and tube agglutination with H-antisera (Denka Co., Tokyo, Japan) for flagellar (H) antigens. The serovars were determined on the basis of their reactivity with O and H group antigens according to the Kauffmann–White scheme [29].

Antimicrobial susceptibility tests

The minimum inhibitory concentrations (MICs) of ten antimicrobial agents (ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, oxytetracycline, kanamycin, onfloxacin, cefotaxime, cefoxitin, and ceftiofur) against Salmonella isolates were determined via the agar dilution method on Mueller‒Hinton agar (Oxoid Ltd., Basingstoke, UK) plates according to the guidelines of the Clinical Laboratory Standards [30]. However, while all 753 isolates were tested for kanamycin susceptibility, only 268 Salmonella isolates from 2019 to 2020 were tested for all ten antimicrobials for comparison with earlier studies. The MIC range for the tested antimicrobials was 0.25–512 µg/mL. The MIC breakpoints were interpreted according to the Clinical and Laboratory Standards Institute [30] guidelines. Escherichia coli (E. coli) ATCC 25,922 and Staphylococcus aureus ATCC29213 were used as quality control strains.

Antimicrobial resistance gene detection

The genes conferring resistance to streptomycin [aadA1 (another name, ant(3”)-Ia)], sulfamethoxazole (sul1), tetracycline [tet A (tet(A))], and kanamycin [aphA1 (aph(3’)-Ia)] were detected via simplex PCR as described previously [31, 34] with minor modifications.

Whole-genome sequencing (WGS) and plasmid analysis

A total of six representative Salmonella isolates (two from each serovar) were selected on the basis of their resistance phenotypes (SSuT and SSuTKm, one strain each) for plasmid whole-genome sequencing. The genes that confer resistance to SSuTKm antimicrobials are carried by the plasmid. Therefore, we analyzed plasmids of the representative strains, interested in any alterations that may have led to the spread of kanamycin resistance, especially in S. Schwarzengrund. The genomic DNA were extracted via the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturers’ instructions for whole-genome sequencing (WGS). For short-read sequencing, libraries were prepared via the QIAseq FX DNA Library Kit (QIAGEN) and sequenced on the MiSeq platform (Illumina, San Diego, CA, USA) via the MiSeq Reagent Kit v3 (600 cycles). For long-read sequencing, libraries were constructed via the Rapid Barcoding Kit (Oxford Nanopore Technologies, Oxford, UK), loaded onto an R10.4.1 flow cell, and sequenced with the MinION Mk1B device (Oxford Nanopore Technologies) via MinKNOW software.

Hybrid assembly of the short- and long-read data was performed to obtain complete plasmid sequences. The raw short reads were trimmed for adapter sequences and low-quality bases via Fastp v0.23.4 [32], whereas the long reads were preprocessed via Porechop v0.2.4 (https://github.com/rrwick/Porechop) and NanoFilt v2.8.0 [33]. Assembly was conducted with Unicycler v0.5.1 [34] or Hybracter v0.11.2 [35] to generate complete plasmid sequences. The assembled genomes were subsequently polished with Pilon v1.24 [36]. Plasmid replicon types and antimicrobial resistance genes were identified via the PlasmidFinder v2.0.1 [37] and ResFinder v4.6.0 [38] databases via the staramr v0.11.0 tool [39], respectively. Briefly, the input files were assembled into contigs in FASTA format; the minimum percent identity was set at 95% for PlasmidFinder and 90% for ResFinder, both with the minimum length coverage set at 60%.

Statistical analysis

Using the chi-square test in the Microsoft Excel spreadsheet, the prevalence and distribution of Salmonella isolates, changes in the proportion of resistant isolates in each antimicrobial agent, and the incidence of kanamycin resistance in Salmonella isolates in each year during the study period were compared for significant differences. The result was considered significant when the computed p value was < 0.05.

Abbreviations

S. S

Salmonella Schwazerngrund

S. I

Salmonella Infantis

S. M

Salmonella Manhattan

AMR

antimicrobial resistance

MIC

minimum inhibitory concentration

AMP

ampicillin

CTX

cefotaxime

CFX

cefoxitin

CTF

ceftiofur

OFLX

ofloxacin

CP

chloramphenicol

SM

streptomycin

SUL

sulfamethoxazole

OTC

oxytetracycline

KM

kanamycin

No

number

KmR

kanamycin resistant

SSuTKm

S-streptomycin, Su-sulfamethoxazole, T-tetracycline and Km-kanamycin

IncFIB

incompatibility group FIB

IncP1

incompatibility group P1

IncX4

incompatibility group X4

Authors’ contributions

GJS contributed conceptualization, data analysis, manuscript preparation, writing and editing. GJS, SH, RM and VMD performed the isolation, identification, and serotyping of bacteria and antimicrobial susceptibility tests. TO, HS and TC conceptualization; data analysis; methodology; software; resources; and manuscript writing, review and editing. TC supervised and planned the research. All the authors have approved the final manuscript.

Funding

This study was funded by the Graduate School of Veterinary Medicine, Kagoshima University. However, the funder did not influence or be involved in the design, analysis or reporting of the study.

Data availability

The data supporting the results of this study are contained within the article.

Declarations

Ethics approval and consent to participate

Specimen collection and carcass handling were conducted in accordance with Japanese regulations on poultry carcass inspection. The samples were released from the accredited poultry processing plant to the analysis laboratory under Japanese regulations on poultry processing. This study was approved by the Institutional Animal Care and Ethics Committee of the Joint Faculty of Veterinary Medicine, Kagoshima University.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.