Increased Salmonella Schwarzengrund prevalence and antimicrobial susceptibility of Salmonella enterica isolated from broiler chickens in Kagoshima Prefecture in Japan between 2013 and 2016.

This study aimed to analyze the Salmonella serovars, measure the minimum inhibitory concentration of antimicrobials, and examine the antimicrobial resistance genes of Salmonella isolated from 192 broiler flocks in Kagoshima Prefecture in Japan, from 2013 to 2016. We found that all Salmonella isolates belonged to three serovars: Salmonella Manhattan, S. Infantis, and S. Schwarzengrund. Among them, S. Schwarzengrund prevalence has recently increased annually making the main serovar. Most recovered isolates were highly resistant to streptomycin, sulfamethoxazole, and oxytetracycline. We saw the reduction of third-generation cephalosporin resistance and identified the reason of increased kanamycin resistance to be the increased number of S. Schwazengrund isolates. Among the kanamycin-resistant Salmonella isolates, aphA1 constituted the main resistance gene detected.

Salmonellosis, one of the most important diseases in both humans and animals, has been described as the second most commonly caused foodborne bacterial disease worldwide [12]. Salmonella is one of the four key global causes of diarrheal diseases, with 2579 serovars identified till date [17]. Antimicrobial agents are widely used during poultry production for growth promotion, or treatment purposes [14]. Resistance to antimicrobial agents in bacteria is mediated by several mechanisms including changes in bacterial cell wall permeability, energy-dependent removal of antimicrobials via membrane-bound efflux pumps, modification of the site of drug action, and destruction or inactivation of the drug [3, 19]. Bacteria can acquire resistance genes through mobile elements, such as plasmids, which provide flexibility to the host bacteria and promote the spread and distribution of these genes across the diverse bacterial population [4].

Notably, we recently reported an increase in the prevalence of Salmonella in broiler chickens in Japan including the first report of Salmonella Schwarzengrund detection in 2012, which is the main serovar detected in Kagoshima Prefecture, Japan presently [9]. S. Schwarzengrund has been reported as an emerging pathogen in Asia, Denmark, the United States of America and Brazil [1, 2, 15]. In this study, we analyze the Salmonella serovars, measure the minimal inhibitory concentration (MIC) of antimicrobials, and examine the resistance genes in order to describe the recent fluctuations of antimicrobial susceptibility of Salmonella in broiler chickens and investigate its mechanism.

During 2013 to 2016, we analyzed 3069 cecal specimens from 192 broiler flocks (approximately 10,000 birds per flock) collected by the prefectural officials at an accredited poultry processing plant in Kagoshima Prefecture, Japan. Samples were delivered to the Laboratory of Veterinary Public Health, Kagoshima University, and cultured on the day of arrival. [8, 9]. The antimicrobial susceptibility of the Salmonella isolates was ascertained by the agar dilution method using Mueller Hinton agar (Oxoid Ltd., Basingstoke, UK) [20, 21, 25]. Two kanamycin resistance genes aphA1, and kn were detected by using PCR [7, 11, 13].

Salmonella prevalence in broiler chickens from 2013 to 2016 in Kagoshima Prefecture, Japan is shown in Table 1. Overall, the prevalence of Salmonella- positive flocks exhibited a dramatic increase during the last three years in the study period compared to that during the first year. In general, the incidence of Salmonella in the flocks was 78.6% (151/192; 48 flocks per year for four years) and the proportion of Salmonella-positive samples in the total number of samples from broiler chickens was 17.8% (546/3069). As shown in Table 1, Salmonella prevalence at both the flock and individual broiler chicken levels in the present study (78.6%) is much higher than that in our previous study (49.0%) [9]. Our report was similar to the Salmonella prevalence in Japan reported by Yamazaki et al. [24], and Sasaki et al. [18]. Alternatively, Salmonella prevalence was reported to vary considerably across different geographic regions worldwide. In Sweden, a study from 2007 to 2015 on housed broilers and laying hens reported that the percentage of Salmonella-positive broiler flocks was 2.0% [23]. A study in Egypt reported that 41.0% of tested broiler flocks were positive for Salmonella along with 1.09% of tested samples [10].

Table 1.

Prevalence of Salmonella in broiler chickens during 2013–2016 in Kagoshima, Japan

Year	No. of flocks	No. of positive flocks (%)	No. of samples	No. of positive samples (%)
2013	48	31 (64.6)	767	82 (10.7)
2014	48	41 (85.4)	767	153 (19.9)
2015	48	40 (83.3)	768	157 (20.4)
2016	48	39 (81.3)	767	154 (20.1)
Total	192	151 (78.6)	3,069	546 (17.8)

The Salmonella isolates from broiler chickens in Kagoshima Prefecture, Japan belongs to three serovars: Infantis, Manhattan, and Schwarzengrund across the four years of the present study, as also reported in our previous study [9], although the relative proportions differed as shown in Table 2. The largest differences were observed in Infantis and Schwarzengrund serovars. Across both studies, S. Infantis proportion exhibited a dramatic decrease. In contrast, S. Schwarzengrund and S. Manhattan percentage steadily increased from 2.1 and 40.3%, respectively, in 2009–2012 to 21.3 and 51.8%, respectively, in 2013–2016.

Table 2.

Incidence of Salmonella serovars in broiler chickens in Kagoshima, Japan during two periods (2009–2012 and 2013–2016)

Serovar	Survey period
	2009–2012a) (%)	2013–2016b) (%)
No. of S. Infantis	140 (57.6)	147 (26.9)
No. of S. Manhattan	98 (40.3)	283 (51.8)
No. of S. Schwarzengrund	5 (2.1)	116 (21.3)
Total	243	546

a) Cited from our previous study [9]. b) This study.

In Japan, S. Schwarzengrund proportion of broiler chicken origin increased from 0% in 2000–2003 to 28.1% in 2005–2007 and was resistant to streptomycin, oxytetracyclin and kanamycin [2]; a high incidence of S. Schwarzengrund was also detected in Kyushu region, Japan with 123 positive samples from 184 Salmonella strains (66.8%) isolated from broiler chickens [24]. Moreover, a study conducted in Taiwan from 2004 to 2006 indicated S. Schwarzengrund contamination prevalence in raw chicken meat samples as 30.5% [6]. In our present study, the number of S. Schwarzengrund strains isolated increased dramatically from 5 to 116 (Table 2). Together, these studies support that S. Schwarzengrund has become one of the most prevalent serovars in broiler chickens in East Asia.

Table 3 describes that the proportion of Salmonella antimicrobial resistance slightly changed across the previous (2009–2012) [9] and current (2013–2016) study periods. Ampicillin, cefotaxime, and ceftiofur resistance was concurrently and markedly decreases. Conversely, kanamycin-resistant Salmonella proportion increased from 6.6% in 2009–2012 to 13.7% in 2013–3016. The majority of S. Schwarzengrund were sensitive to ampicillin, cefotaxime, cefoxitin, and ceftiofur (zero percent resistance).

Table 3.

Antimicrobial susceptibility profiles from the current study and our previous study [9] of Salmonella isolates from broiler chickens in Kagoshima, Japan

Antimicrobial agent	MIC Break-point (µg/ml)	No. of resistant isolates (%)
		Previous studya)	Current studyb)
		2009–2012	2013–2016
		n=243a)	n=511*b)
AMP	≥32	134 (55.1)	148 (29.0)
CTX	≥4	128 (52.7)	132 (25.8)
CFX	≥32	15 (6.2)	42 (8.2)
CP	≥32	0 (0.0)	0 (0.0)
SM	≥16	231 (95.1)	484 (94.7)
SUL	≥512	221 (91.0)	463 (90.6)
OTC	≥16	222 (91.4)	451 (88.3)
KM	≥64	16 (6.6)	70 (13.7)
OFLX	≥2	4 (1.6)	3 (0.59)
CTF	≥8	124 (51.0)b)	112 (22.0)

a) Cited from our previous study [9]. b) This study. *The number of strains (511) differs from the total given in Table 2 (546) because at the time of MIC testing, some strains were dried and not suitable for use. AMP, ampicillin; CTX, cefotaxime; CFX, cefoxitin; CP, chloramphenicol; SM, streptomycin; SUL, sulfamethoxazole; OTC, oxytetracycline; KM, kanamycin; OFLX, ofloxacin; CTF, ceftiofur.

As shown in Table 4, almost all Salmonella strains of the three serovars were sensitive to chloramphenicol and ofloxacin, whereas over 80% of each serovar exhibited resistance to streptomycin, sulfamethoxazole, and oxytetracycline. In our survey from 2009 to 2012, the increased proportion of the S. Manhattan serovar led to an annual increase in resistance to ampicillin, cefotaxime, and ceftiofur [9]. In the present study, although S. Manhattan percentage was 51.8% (Table 2) the resistance rate of all Salmonella serovars decreased compared to that from 2009 to 2012 [9]. This may be due to the decrease in the number of S. Infantis and increase in S. Schwarzengrund from 2013 to 2016, as all isolated S. Schwarzengrund (109 isolates) were sensitive to ampicillin, cefotaxime, and ceftiofur (Table 4). The β-lactam antimicrobial resistance rate of S. Manhattan was higher than those of S. Infantis and S. Schwarzengrund. In addition, considerable differences in kanamycin resistance were detected among the three serovars. While the majority of S. Manhattan was susceptible to kanamycin, S. Infantis exhibited a resistance rate at 10.8% and S. Schwarzengrund showed the maximum rate, with 47.7% resistance to kanamycin. The reduction of β-lactam resistance proportion in our study may be the same as reported by Mauro et al. [16], where the authors indicated that the off-label use of ceftiofur with Marek’s vaccine is associated with the short-term increase in ESBL-producing Escherichia coli in the gut of broiler chickens. In Japan, the same situation appeared following the cessation of ceftiofur use by the Japanese poultry industry [22].

Table 4.

Comparison of antimicrobial resistance of Salmonella Schwarzengrund, S. Manhattan and S. Infantis during the 2013–2016 study period

Antimicrobial agent	No. of resistant isolates (%)
	S. Schwarzengrund	S. Manhattan	S. Infantis
	n=109	n=263	n=139
AMP	0 (0.0)	119 (45.2)	29 (20.9)
CTX	0 (0.0)	109 (41.4)	23 (16.5)
CFX	0 (0.0)	27 (10.3)	15 (10.8)
CP	0 (0.0)	0 (0.0)	0 (0.0)
SM	109 (100)	257 (97.7)	118 (84.9)
SUL	102 (93.6)	244 (92.8)	117 (84.2)
OTC	101 (92.7)	238 (90.5)	112 (80.6)
KM	52 (47.7)	3 (1.1)	15 (10.8)
OFLX	0 (0.0)	0 (0.0)	3 (2.2)
CTF	0 (0.0)	90 (34.2)	22 (15.8)

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

Figure 1 shows a comparison of the specific antimicrobial resistance rates for S. Infantis (Fig. 1a) and S. Manhattan (Fig. 1b) between the current (2013–2016) and previous (2009–2012) [9] study periods. However, as only five strains of S. Schwarzengrund were isolated in the previous period [9], we did not perform the comparison for this serovar. S. Infantis proportion exhibiting antimicrobial resistance to ampicillin, cefotaxime, streptomycin, sulfamethoxazole, and oxytetracycline slightly decreased in the current study period compared to that in the previous study period (Fig. 1a). No change was observed in cefoxitin, chloramphenicol, and ofloxacin resistance, whereas kanamycin and ceftiofur resistance was slightly increased. In comparison, the resistance rate of S. Manhattan to streptomycin, sulfamethoxazole, oxytetracycline, chloramphenicol, kanamycin, and ofloxacin minimally fluctuated between the two periods. The percentage of resistance to three antimicrobials decreased in the present period: ampicillin (from 94.9% to 45.2%), cefotaxime (from 93.9% to 41.4%), and ceftiofur (from 74.5% to 30.0%). In contrast, cefoxitin-resistant S. Manhattan resistance increased from 0 to 10.3% between the previous and current study periods.

Fig. 1.

Change of antimicrobial resistance from 2009–2012 to 2013–2016 among (a) Salmonella Infantis and (b) S. Manhattan. AMP, ampicillin; CTX, cefotaxime; CFX, cefoxitin; CP, chloramphenicol; SM, streptomycin; SUL, sulfamethoxazole; OTC, oxytetracycline; KM, kanamycin; OFLX, ofloxacin; CTF, ceftiofur.

We further evaluated 68 kanamycin-resistant S. enterica isolates from Kagoshima Prefecture, Japan during the present study period (2013–2016) (13 S. Infantis, 3 S. Manhattan, and 52 S. Schwarzengrund) for kanamycin resistance genes (kn and aphA1) by PCR. None of the 68 isolates carried kn, whereas 65/68 (95.6%) carried aphA1 (Table 5). All the 13 S. Infantis isolates (MIC: 512 µg/ml) carried aphA1. Of the three S. Manhattan isolates, one (MIC: 512 µg/ml) carried aphA1 whereas two others (MIC: 256 and 128 µg/ml) did not. The 51 S. Schwarzengrund isolates with MIC of 512 µg/ml carried aphA1; that with MIC of 256 µg/ml did not.

Table 5.

Distribution of the aphA1 kanamycin resistance gene from Salmonella serovars isolated from broiler chickens during the 2013–2016 study period

Serovar (no. of isolates)	MIC of kanamycin (µg/ml)	No. of isolates tested	No. of isolates positive for aphA1 resistant gene (%)
S. Infantis (13)	512	13	13 (100)
	256	-	-
	128	-	-
S. Manhattan (3)	512	1	1 (100)
	256	1	0 (0.0)
	128	1	0 (0.0)
S. Schwazengrund (52)	512	51	51 (100)
	256	1	0 (0.0)
	128	-	-
Total		68	65 (95.6)

MIC, minimal inhibitory concentration.

aph gene was found in almost kanamycin-resistant of S. enterica serovars isolated in some regions of the United States of America in 2005 [5]. A study in the United States of America and China [7] found aph in S. Enteritidis, S. Haardt, and an unidentified serovar from chicken meat and S. Derby from pork. In comparison, we found the aph gene (but not kn) in three serovars: S. Infantis, S. Manhattan, and S. Schwarzengrund. This may suggest that this gene commonly serves to provide kanamycin resistance in numerous Salmonella serovars.

Together, our findings revealed that there has been a recent increase in the population of the S. Schwarzengrund-strain, making it the main serovar of Salmonella isolated from broiler chickens in Kagoshima Prefecture in Japan, followed by S. Manhattan. In turn, the increase of S. Schwarzengrund, which exhibited a high level of kanamycin resistance, led to a decrease in the rate of antimicrobial resistance to ampicillin, cefotaxime, and ceftiofur among all Salmonella isolates and affected the increase in the percentage of kanamycin-resistant isolates. In addition, the resistance rate of S. Manhattan to β-lactams in this study slightly decreased compared to that in our previous study [9], which also affected the overall rate of resistance to β-lactams. Moreover, we demonstrated that aphA1 is the main antimicrobial resistance gene in Salmonella isolates. These changing profiles indicate the need for continual evaluation and research regarding the molecular characteristics of Salmonella in broiler chickens.

CONFLICT OF INTEREST

The authors declare that this research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.