Chronological Change of Resistance to β-Lactams in Salmonella enterica serovar Infantis Isolated from Broilers in Japan

Epidemiologic surveillance study was conducted in southern Japan to determine the antimicrobial resistance phenotypes and characterize the β-lactamase genes and the plasmids harboring these genes in Salmonella enterica serovar Infantis (S. Infantis) isolates from broilers. Between January, 2007 and December, 2008, a total of 1,472 fecal samples were collected and examined at the Laboratory of Veterinary Public Health, Kagoshima University, Japan. In 93 (6.3%) isolates recovered, 33 (35.5%) isolates showed resistance to cefotaxime, an extended-spectrum cephalosporin (ESC), conferred by TEM-20, TEM-52 and CTX-M-25 extended-spectrum β-lactamases (ESBLs). In addition to ESC-resistance, eight (8.6%) isolates exhibited resistance to cefoxitin mediated by CMY-2 AmpC β-lactamase. Plasmid analysis and polymerase chain reaction replicon typing revealed the blaTEM-20 and blaCMY-2 genes were associated with IncP plasmids, blaTEM-52 was linked with a non-typable plasmid and blaCTX-M-25 was carried by an IncA/C plasmid. Non-β-lactam resistance to streptomycin, sulfamethoxazole, and oxytetracycline encoded by the aadA1, sul1, and tet(A) genes, respectively, was found in 86 (92.5%) isolates. Resistance to kanamycin and ofloxacin was exhibited in 12 (12.9%) and 11 (11.8%) isolates, respectively, the former was mediated by aphA1-Iab. These data indicate that S. Infantis isolates producing ESBLs and AmpC β-lactamase have spread among broiler farms in Japan. These data demonstrated that the incidence of ESC-resistant S. Infantis carrying blaTEM-52 remarkably increased and S. Infantis strains harboring blaCMY-2, blaTEM-20, or blaCTX-M-25 genes emerged from broilers in Japan for the first time in 2007 and 2008.


INTRODUCTION
Non-typhoidal Salmonella enterica serovars (Salmonella) are a major cause of bacterial food-borne diseases world-wide (Majowicz et al., 2010). The food poisoning statistics in Japan show that bacterial food poisoning patients during the year 2008 numbered at 10,331 and Salmonella were the leading etiological agents accounting for 24.7% of the cases [http://idsc.nih.go.jp/ iasr/29/342/graph/t3421.gif]. Poultry products are important vehicles in the transmission, and have been incriminated in several Salmonella outbreaks (Kimura et al., 2004;Chittick et al., 2006). Since the late 1990s, Salmonella enterica serovar Infantis (S. Infantis) has been the commonest serovar of Salmonella isolated from both broiler flocks and retail chicken meat in Japan (Asai et al., 2007b;Iwabuchi et al., 2011).
The emergence of multidrug-resistant Salmonella has become a serious global health problem because antimicrobial treatment is lifesaving for invasive infections, particularly in neonates of <1 year of age. Preventive antimicrobial treatment is also generally given to patients suffering from immunosuppressive or other predisposing conditions (Hohmann, 2001). The options of first-line therapy for Salmonella infection include ampicillin (AMP), sulfamethoxazole-trimethoprim, fluoroquinolones, and extended-spectrum cephalosporins (ESCs). ESCs are among the preferred drugs because of resistance to the other aforementioned drugs is relatively frequent in Salmonella isolates (Hohmann, 2001). At present, ESC-resistance in Salmonella is mainly attributed to the acquisition of plasmid-mediated extendedspectrum β-lactamases (ESBLs) and AmpC β-lactamases (Bonnet, 2004;Arlet et al., 2006).
Salmonella carrying the bla CMY−2 gene were recently recovered from bovine and porcine salmonellosis cases (Dahshan et al., 2010;Sugawara et al., 2011). Besides, S. Infantis isolates harboring the bla TEM−52 gene were reported from broilers for the first time in the year 2004 (Shahada et al., 2010a). Thus, a major concern has been increased prevalence of resistance to ESCs noted in S. Infantis isolates. This study was conducted to determine the antimicrobial resistance phenotypes and characterize the β-lactamase genes and the plasmids harboring these genes in S. Infantis isolated from broiler flocks.

BACTERIAL ISOLATES
Between January, 2007 and December, 2008, a total of 1,472 cecal specimens derived from 92 broiler flocks (ca. 10,000 birds per flock) were collected from a poultry processing plant located in the southern part of Japan. Usually, 16 samples per flock were randomly selected fortnightly. The isolation, identification and serotyping of S. Infantis isolates were performed at the Laboratory of Veterinary Public Health, Kagoshima University, Japan as previously described (Shahada et al., 2008).

DETERMINATION OF MINIMUM INHIBITORY CONCENTRATIONS
Antimicrobial susceptibility testing was assayed by the agar dilution method on Mueller-Hinton (MH) agar (Oxoid Ltd., Basingstoke, Hampshire, England) plates according to the National Committee for Clinical and Laboratory Standards guidelines (National Committee for Clinical Laboratory Standards, 2001).

DOUBLE-DISK SYNERGY ASSAY
The double-disk synergy testing was conducted to screen for ESBLs and AmpC β-lactamases as previously described (Shahada et al., 2010a). This test was performed as a standard Kirby-Bauer disk diffusion assay on MH agar (Oxoid) plates.

DETECTION OF RESISTANCE DETERMINANTS
All DNA templates were prepared using the InstaGene Matrix kit (Bio-Rad Laboratories, Hercules, CA, USA). Detection of resistance genes was performed by polymerase chain reaction (PCR). The amplification reactions were carried out using primers and conditions as previously described (Dahshan et al., 2010;Shahada et al., 2010b). Briefly, the targets were as follows: bla TEM , bla OXA , bla PSE, bla SHV , bla CTX−M−1 group, bla CTX−M−2 group, bla CTX−M−25 , and Toho-1 encoding for penicillinases; bla CIT , bla CMY−2 , bla DHA , bla FOX , bla MOX , bla ACC , and bla EBC encoding for cephalosporinases; tet(A), tet(B), and tet(G) mediating tetracycline efflux proteins; aadA1 and aadA2 encoding for resistance to STR and spectinomycin; sul1 conferring resistance to SUL; and aphA1-Iab encoding for resistance to KAN.
When β-lactamase-encoding genes were positive following PCR amplification, obtained products were directly sequenced using a BioDye Terminator version 3.1 Ready Reaction sequencing kit and ABI 3100 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). The DNA alignments and deduced amino acid sequences were examined using the BLAST program (National Center for Biotechnology Information, USA).

PLASMID ANALYSIS
All S. Infantis isolates were examined for the carriage of plasmids by employing the alkaline lysis method as previously described (Kado and Liu, 1981). The molecular size of plasmids was determined by using the standard Salmonella enterica serovar Choleraesuis ATCC 7001 (50 kbp) and S. Typhimurium DT104 strain 300-98 (90 kbp). The plasmid size was estimated by graphing the molecular size of standard strains versus the distance traveled from the wells using logistic graph paper. Conjugation experiments were performed as described previously (Shahada et al., 2010a). In brief, S. Infantis donor isolates and rifampicin-resistant E. coli DH5α recipient derivatives were used for conjugal mating. Conjugants were selected onto deoxycholate hydrogen sulfide lactose agar containing 128 μg/ml rifampicin and 128 μg/ml AMP, followed by antimicrobial testing and the detection of transferred resistance genes as above. Plasmids were characterized by the PCRbased replicon typing method as described previously (Carattoli et al., 2005) to detect the plasmid types: IncI1, IncA/C, IncHI1, IncHI2, IncN, IncX, IncW, IncY, IncP, IncT, IncFIIs, IncL/M, IncFIA, IncFIB, IncFIC, IncFrepB, IncK/B, and IncB/O.

PLASMID CHARACTERIZATION
Three plasmid profiles were defined among S. Infantis isolates. Seventy isolates harbored ca. 180-kbp plasmids (profile I), 22 isolates carried two plasmids of ca. 50 and 180 kbp (profile II), and one isolate possessed three plasmids of ca. 50, 125, and 180 kbp (profile III; Figure 1). Isolates exhibiting resistance to ESCs displayed diverse profiles: Of 34 ESC-resistant S. Infantis isolates, 22 belonged to profile II, 11 were classified into profile I and one isolate was categorized into profile III (

DISCUSSION
The results obtained in this study show that resistance to ESCs has increased to 35.5% from the previous 9.2% reported during an earlier investigation (Shahada et al., 2010b). Other new findings include the detection of S. Infantis isolates harboring the bla TEM−20 , bla CTX−M−25, and bla CMY−2 genes. Demonstration of these resistance traits in S. Infantis serovar is a rare phenomenon. This is the first report describing these mechanisms of ESCresistance exhibited by S. Infantis isolates derived from broilers.
In the present study, we identified one S. Infantis isolate harboring the bla TEM−20 gene while other isolates had bla TEM−52 . It's worth noting that the wild-type bla TEM−1 gene was not detected during this study suggesting the likelihood of occurrence of point mutations which led to the emergence of observed variants, bla TEM−20 and bla TEM−52 . This hypothesis is supported by deduced amino acid sequence analysis which revealed that the bla TEM−20 gene differs from bla TEM−1 by two substitutions, Met182→Thr and Gly238→Ser; whereas the bla TEM−52 gene differs from bla TEM−1 by three substitutions, Glu104 Lys, Met182→Thr, and Gly238→Ser (Arlet et al., 1999;Weill et al., 2004). This phenomenon has the implications for bacterial adaptation mechanism because bla TEM−1 seems to have lost its fitness as a potential resistance trait necessary for survival of Salmonella. Thus, point mutations have taken place as the evolutionary adaptation mechanism crucial for successful colonization of the broiler chicken intestinal tract.
In Japan, CTX-M-type ESBL-producing Enterobacteriaceae are important nosocomial infectious agents raising considerable concern in the public health community. Similarly, the detection of CTX-M-2 and CTX-M-25 ESBL-producing E. coli isolates from chickens affected with colibacillosis has raised concerns in the veterinary public health community (Kojima et al., 2005;Asai et al., 2011). We report for the first time one S. Infantis isolate harboring bla CTX−M−25 on IncA/C plasmid and bla TEM−52 on non-typable plasmid. Because bla CTX−M−25 was initially demonstrated in E. coli (Asai et al., 2011) a comprehensive molecular study is required to determine the likely source and elucidate mechanisms involved in collecting antimicrobial resistance traits and mobilizing them across taxonomical borders.
To date, several reports have described the emergence of AmpC β-lactamase-producing Salmonella derived from farm animals affected with salmonellosis. One of the studies involving Salmonella enterica serovar Typhimurium (S. Typhimurium) identified bla CMY−2 associated with self-transmissible IncI1-Iγ and A/C plasmids (Sugawara et al., 2011). Another study revealed a novel chromosomally integrated multi-drug resistance genomic island harboring bla CMY−2 among clonally related S. Typhimurium isolates (Shahada et al., 2011). Consequently, the detection of bla CMY−2 in several S. Infantis isolates from broilers poses another challenge in the veterinary public health community. Carriers of bla CMY−2 harbored IncP plasmids initially demonstrated in Pseudomonas bacteria (Shintani et al., 2011).
Antimicrobial susceptibility data indicate a gradual decrease of OFX resistance to 11.8 from 20.8% previously reported (Shahada et al., 2010b). Fluoroquinolones (e.g., OFX) are broad-spectrum antimicrobial agents widely used in clinical medicine. Emergence of fluoroquinolone resistance was attributed to overuse of this group of drugs in domestic animals either therapeutically or for the purpose of growth promotion (Asai et al., 2007a). Since 1991, the Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF) approved this class of antimicrobials in veterinary medicine for therapeutic purposes and prohibited its use as feed additives. Fluoroquinolones might have been prescribed prudently among antimicrobial agents used in broiler farms in Japan. This likely has contributed to the decline of resistance to OFX observed in the present study.
Taken together, these results show increased resistance against ESCs mediated by both ESBLs and AmpC β-lactamases. It seems these resistance traits have spread among farm animals in Japan while their likely source remains largely unknown. The probability that resistance to ESCs may continue to spread among members of the Enterobacteriaceae family poses another public health challenge because ESBLs and AmpC β-lactamases limit the effectiveness of cephalosporin therapy.

ACKNOWLEDGMENT
This study was partly supported by a Grants-in-Aid for Scientific Research (No.23580426) from the Japan Society for the Promotion of Science.