| Salmonella spp., Staphylococcus aureus, and Campylobacter jejuni are important foodborne zoonotic pathogenic bacteria. They not only cause illnesses in livestock and poultry, but can also infect humans through the food chain or contact with diseased animals. Antibiotics are the first choice for treating infected animals, but their use promotes the evolution of drug-resistant bacteria that can also infect humans through the food chain or direct contact. Three provinces in central China(Henan, Hubei, and Hunan) comprise a particularly concentrated animal breeding area. This research investigates the isolation, characterization, and identification of bacteria from animal sources(i.e., chickens, swine, and dairy cows), including the determination of serotypes, minimum inhibitory concentration(MIC) drug sensitivity testing, and the identification of drug-resistant genes, virulence factors, and epidemic genotypes for the three bacterial genera. This study offers important observational data and provides suggestions for the clinical application of antibiotics to protect animal health and food safety. Furthermore, it establishes a firm foundation for further scientific research. 1. Collection SamplesFrom March 2010 through July 2011, 2,185 cloaca and anal swab samples were taken from chickens, swine, and dairy cows on farms in Henan, Hubei, and Hunan provinces. In total, 837 chicken samples, 930 swine samples, and 418 dairy cow samples were obtained. Each sample was cultured and Salmonella spp., Staphylococcus aureus, and Campylobacter jejuni bacteria were isolated and identified. 2. Prevalence and Molecule Character of Resistance and Virulence of Salmonella isolates from livestock in Central ChinaThrough biochemical reactions and polymerase chain reaction(PCR)-based identification using a Salmonella-specific gene(inv A), 248(11.35%, 248/2185) strains of Salmonella were identified. The rates of Salmonella isolation from chicken, swine, and dairy cow sources were 12.54%(105/837), 4.09%(38/930), and 25.12%(105/418), respectively. In addition, 209 Salmonella isolates were acquired from the Veterinary Hospital of Huazhong Agricultural University. In total, this study included 457 Salmonella isolates that were characterized by serotyping, antibiotic sensitivity testing, resistance genes, virulence genes, and MLST.The serogroup of 62 Salmonella isolates could not be identified by slide agglutination tests using O and H antigens. For the remaining 395 isolates, 8 serogroups and 41 serotypes were identified. The group B(n = 205, 44.86%) and group C1(n = 141, 30.85%) were the dominant serogroups; Typhimurium 12.47%(n = 57) and IIIb 4.81%(n = 22) were the dominant serotypes. For Salmonella derived from chickens, the dominant serotypes were Typhimurium(n = 20, 4.38%) and Enteritidis(n = 9, 1.97%). The dominant serotypes for Salmonella derived from swine were IIIb(n = 22, 4.81%) and Typhimurium(n = 11, 2.41%). The dominant serotypes for Salmonella derived from dairy cow sources were Typhimurium(n = 26, 5.69%) and Agona(n = 12, 2.63%).The agar dilution method was used to gauge the sensitivity of the Salmonella isolates to 24 different antibiotics. A total of 188 isolates(41.14%) were sensitive to all tested antibiotics and 220 isolates(48.14%) demonstrated resistance to at least one type of antibiotic during MIC testing. All isolates were at least moderately sensitive to imipenem. The highest resistances were observed for nalidixic acid, trimethoprim-sulfamethoxazole, doxycycline, and tetracycline(39.17%, 39.61%, 28.22%, and 27.58%, respectively). The tested isolates demonstrated resistance to all animal-specific antibiotics: enrofloxacin(24.51%), florfenicol(20.13%), ceftiofur(7.44%), and cefquinome(5.25%). They also demonstrated resistance to antibiotics used in both animals and humans: ciprofloxacin(24.07%), gentamicin(19.69%), ceftriaxone(6.34%), and amoxicillin/clavulanate(4.38%). Azithromycin is a macrolide antibiotic and had a resistance rate of 16.19%. Resistance to infrequently used animal antibiotics was also demonstrated. For example, polymyxin B and fosfomycin had resistance rates of 18.82% and 12.04%, respectively. Salmonella from chicken and swine demonstrated resistance to 23 of the 24 antibiotics tested. Their resistance rates were 1.90% ~ 49.52% and 0.40% ~ 48.18%, respectively. Salmonella derived from dairy cows only demonstrated resistance to 12 antibiotics. Their resistance rates were 0.95% ~ 10.48%. Resistance to three or more types of antibiotics was observed for 159 isolates(34.72%) of Salmonella, including 51 isolates from various healthy animals from different provinces and 89 isolates from diseased swine at the Veterinary Hospital of Huazhong Agricultural University.Based on MIC testing and the detection of extended-spectrum β-lactamase(ESBLs), 25 Salmonella isolates(5.47%, n = 457) were ESBL producers. Three isolates derived from swine at the Veterinary Hospital of Huazhong Agricultural University were suspected ESBL producers. The sources of the 25 Salmonella isolates that produced ESBLs were as follows: 2 isolates from healthy chickens in Hubei, 15 isolates from healthy chickens in Henan, and 8 isolates from diseased swine at the Veterinary Hospital of Huazhong Agricultural University.Among the Salmonella isolates that were sensitive to all antibiotics(n = 188), 80 strains and 17 specific serotypes were identified. Typhimurium(n = 28) was the dominant serotype. Among the multidrug-resistant Salmonella isolates, 26 specific serotypes were identified. Enteritidis(n = 10) and IIIb(n = 10) were the dominant serotypes. Enteritidis mainly came from chicken sources, and IIIb mainly came from diseased swine sources. All serotypes were 100% resistant to nalidixic acid. Among the multidrug-resistant Salmonella, bacterial strains with different serotypes demonstrated slightly different drug resistances. The 10 isolates of the Enteritidis serotype were resistant to nalidixic acid, cotrimoxazole, olaquindox, and maquindox. The 10 isolates of the IIIb serotype were resistant to ampicillin, amoxicillin, nalidixic acid, and cotrimoxazole.PCR methods were used to detect 140 drug-resistant genes in the 187 isolates of Salmonella that demonstrated resistance to at least two antibiotics during MIC testing. A total of 58 genes(41.43%) were not detected and 82(58.57%) were detected. There were 27 drug-resistant genes that were detected in 90% or more of the strains, including mur A(n = 173, 92.51%), ISVsa3(n = 177, 94.65%), aad A2(n = 179, 95.72%), aad A1(n = 180, 96.26%), mer R and tol C each in 181 strains(96.79%), pare and class 1 integron each in 182 strains(97.33%), emr B and mdl B each in 184 strains(98.40%), qac EΔ1(n = 185, 98.93%), and gyr A, gyr B, acr A, and mdf A each in 186 strains(99.47%). The following drug-resistant genes were detected in all 187 isolates: par C, tet B, omp C, omp F, ram A, acr B, acr E, acr F, emr A, ydh E, mdt B, and mdt C.There were similarities and differences in the distributions of drug-resistant genes for the three animal sources. One similarity was that 63 of the 81 drug-resistant Salmonella genes that were detected could be found in chickens, swine, and dairy cows alike, including genes encoding ESBL, efflux pumps, and integrons. The following differences were observed: ere B was only detected in isolates from dairy cow sources; mar R, mcp/Tn1721, and rmt B were only detected in isolates from chicken sources; dfr Ia, DHA, tet C, Tn1, Tn2, Tn4, mer A, and rmt A were only detected in isolates from swine sources; dfr VII, CMY-2, tnp/Tn10, tnp R/Tn1721, erm B, and fos A3 were present in isolates from chicken and swine sources.One-hundred and forty Salmonella isolates were producers of ESBLs and Amp C. Ninety-two strains had a single drug-resistant gene subtype: OXA-1(n = 59), TEM-1(n = 23), DHA(n = 5), CTX-M-3(n = 4), and CMY-2(n = 1). Salmonella from chicken and dairy cow sources carried TEM-1, CTX-M-3, and OXA-1. Swine-derived Salmonella carried TEM-1, CMY-2, DHA, and OXA-1. Salmonella isolates from animal sources in Henan and Hunan did not have CMY-2 or DHA. Salmonella isolates from Hubei did not have DHA. Salmonella from animals at the Veterinary Hospital of Huazhong Agricultural University did not have CTX-M-3. Forty-three strains of Salmonella simultaneously carried two kinds of drug-resistant gene subtypes in 7 distinct combinations, TEM-1 + CMY-2, TEM-1 + DAH, TEM-1 + OXA-1, CTX-M-3 + DHA, CTX-M-3 + OXA-1, OXA-1 + CMY-2, and OXA-1 + DHA. These drug-resistant gene combinations were only found in Salmonella from chicken and swine sources. The remaining 5 strains of Salmonella exhibited three different drug-resistant gene subtypes simultaneously. These combinations were TEM-1 + OXA-1 + CMY-2, which was only detected in chicken-derived Salmonella, and TEM-1 + OXA-1 + DHA and CTX-M-3 + OXA-1 + DHA, which were only detected in swine-derived Salmonella.Of the 140 ESBL and Amp C producers, there were 23 definitive serotypes. IIIb(group C1), Enteritidis, and Typhimurium were the dominant serotypes. All 140 strains were resistant to at least 2 antibiotics. Most commonly, the bacteria were resistant to 8 drugs(24 strains), 11 drugs(23 strains), or 10 drugs(20 strains). These 140 strains had 75 of the 81 drug-resistant genes detected in addition to ere B, ESBLs and Amp C(92.59%, 75/81). A total of 81.43% of the isolates(114/140) were carriers of aac(6′)-Ib-cr, 44.29%(62/140) carried oqx A, 42.14%(59/140) carried oqx B, and 41.43(58/140) carried all three of these genes. A total of 97.86%(137/140) and 9.29%(13/140) of isolates carried class 1 and class 1-int-vari integron respectively. Furthermore, 7.86%(11/140) carried all five of these genes simultaneously(i.e., aac(6′)-Ib-cr + oqx A + oqx B + class 1 + class 1-int-vair). Carriers of transposons and insertion elements represented 0.71 ~ 95.00% of isolates.PCR methods were used to detect 26 virulence factors in the 187 Salmonella isolates. Other than gip A, flg K, and sii D, 23(88.46%, 23/26) virulence factors were detected. Among the virulence factors detected, stn, hil A, prg H, sop B, bcf C, fim A, and rpo S were detected in 100% of the isolates. Detection rates for the remaining virulence factors ranged from 11.76% ~ 99.47%. The virulence factors avr A and ssa Q, which are related to T3SS-1 and T3SS-2, were detected in 70.59% and 83.42% of the isolates, respectively.In a multi-locus sequence typing(MLST) analysis, the Salmonella isolates exhibited five sequence types(STs). ST 1(36.90%), ST 6(28.88%), and ST 4(13.90%) were the most prevalent. Among swine-derived Salmonella from the Veterinary Hospital of Huazhong Agricultural University, ST 6(n = 51) and ST 1(n = 39) were the most prevalent. Among Salmonella isolates from healthy animals in the three provinces included in this study, ST 1(n = 30) and ST 4(n = 23) were the most prevalent. In chicken-derived Salmonella strains, ST 4(n = 20) and ST 1(n = 12) were the most prevalent. In swine-derived Salmonella strains, the most prevalent type was ST 1(n = 13). Among Salmonella from dairy cow sources, ST 1(n = 5) was the most prevalent. Among ESBL- and Amp C-producing Salmonella, ST 1(n = 55, 39.29%) was the most prevalent.Based on an analysis of the MLST results and the serotypes of all 187 Salmonella isolates, 66 strains(35.29%, 66/187) had a definitive serotype and ST. In the group B, 21 strains demonstrated 10 different serotypes and 3 STs(ST 1, ST 6, and ST 57). Of those, ST 1(n = 18) was most prevalent, and was found in all serotypes. In the group C1, 25 strains demonstrated 9 different serotypes and 2 STs(ST 1 and ST6). ST 1(n = 8) was detected in isolates with five different serotypes. ST 6(n = 20) was detected in isolates with 7 different serotypes. All 9 strains in the group D1 were ST 4; however, they demonstrated different serotypes(Enteritidis and II). The 3 strains that made up the group E1 had three serotypes and all were ST 65. In the group E4, 8 strains contains 4 different serotypes and 3 STs(ST1, ST4 and ST65). 3. Prevalence and Molecule Character of Resistance and Virulence of Staphylococcus aureus isolates from livestock in Central ChinaFrom the 2,185 samples collected from healthy animals, 85 strains of Staphylococcus aureus were isolated(3.89% isolation rate; 85/2185). The rates for bacteria isolated from chicken, swine, and dairy cow sources were 3.11%(26/837), 3.44%(32/930), and 6.46%(27/418), respectively. Twenty-four isolates(28.24%, 24/85) of S. aureus were coagulase-negative. Chicken-derived isolates accounted for most of those(54.17%, 13/24). PCR-based methods were used to determine S. aureus serotypes. Fifty-six strains(65.88%, 56/85) were cap 5 type, 15 strains(17.65%, 15/85) were cap 8 type, and the remaining 14 strains(16.47%, 14/85) were non-cap 5 and non-cap 8. cap 5 was the dominant serotype observed.The agar dilution method was used to detect the sensitivity of S. aureus isolates to 30 antibiotics. All 85 strains were sensitive to vancomycin, amoxicillin/clavulanate, cefquinome, imipenem, and amikacin. Based on MIC testing, over 50% of the isolates were resistant to the following drugs: penicillin(67.06%), ampicillin(64.71%), amoxicillin(94.12%), tetracycline(60.00%), nalidixic acid(97.65%), cotrimoxazole(77.65%), lincomycin(61.18 %), erythromycin(94.12%), azithromycin(94.12%), and olaquindox(96.47%). All S. aureus isolates demonstrated resistance to at least two types of antibiotics. Most commonly, the isolates were resistant to 8 different antibiotics(n = 30), followed by those resistant to 9 antibiotics(n = 19) and 5 antibiotics(n = 18). All 24 coagulase-negative strains of S. aureus were multidrug-resistant bacteria. In total, 12.5%(3/24) were resistant to four drug types and 33.33%(8/24) were resistant to 5 drug types. Only 4.17%(1/24) were resistant to 6 drug types, 4.17%(1/24) of strains were resistant to 7 drug types, and 45.83%(11/24) were resistant to 8 drug types.The S. aureus isolates were tested for 95 drug-resistant genes. Among these genes, 55(57.89%, 55/95) were detected, and 40(42.11%, 40/95) were not. Thirteen of the drug-resistant genes were detected in 100% of the samples(fem B, fem X, tet K, Inu A/A’, Inu A, grl A, grl B, ant4’-Ia, class 1, nor A, nor B, nor C, and mep A). Furthermore, 11 drug-resistant genes were carried by ≥ 90% of the samples(pbp4, erm B, erm C, erm T, tet38, gyr A, gyr B, cat:pc221, aph A3, dfr A(S1), and dfr G). The main types of antibiotics and resistance mechanisms of these drug-resistant genes were a-lactam penicillin, tetracyclines, lincosamides, fluoroquinolones, aminoglycosides, TMP, efflux pumps, and integrons.The S. aureus bacteria were tested for 66 virulence factors. Of these, 45(68.18%, 45/66) were detected and 21(31.82%, 21/66) were not. The following 12 virulence factors were carried in 100% of the isolated bacterial strains: hla, hlgv, hld, luk ED, lbp, bbp, sak, efb, fnb A, clf A, clf B, and ebp. The tst virulence factor associated with TSST-1(toxic shock syndrome toxin-1) was not detected.Based on an MLST analysis of the 85 S. aureus isolates, 6 STs were detected, ST 5(n = 30, 35.29%), ST 7(n = 24, 28.23%), ST 9(n = 16, 18.82%), ST 398(n = 7, 8.24%), ST 464(n = 1, 1.18%), and ST 2750(n = 1, 1.18%). A specific ST type was not determined for 6(7.06%) of the isolates. ST 5, ST 7, and ST 9 were the most prevalent types. Only ST 5 was present in isolates from all three animal sources. In coagulase-negative S. aureus bacteria, ST 5(54.17%) and ST 7(33.33%) were most prevalent.Four strains carried mec A and accordingly were classified as methicillin-resistant S. aureus(MRSA). The sources of these strains were as follows: 1 strain from chickens in Henan Province(this strain was also coagulase-negative), 1 strain from swine in Hubei, and 2 strains from swine in Hunan. Three MRSA strains were cap 5 and the remaining strain was non-cap 5 and non-cap 8. Two MRSA strains were ST 7 and 2 were ST 9. All four were multidrug-resistant. The Hunan chicken-derived MRSA demonstrated resistance to 8 drugs. The Hubei swine-derived MRSA was resistant to 5 drugs. The Hunan swine-derived MRSAs were both resistant to 9 drugs. This MRSA also carried 46(83.64%, 46/55) drug-resistant genes.Accounting for the serotype, ST type, and multidrug resistance of the 85 S. aureus strains, those with the cap 5 serotype exhibited 5 STs, most frequently ST 5 and ST 9. Strains with cap 8 serotypes were represented by 3 STs, especially ST 7. ST 5 strains mainly showed resistance to 4 or 5 drugs. ST 7 strains mainly showed resistance to 8. ST 9 and ST 398 strains mainly showed resistance to 9 drugs. 4. Prevalence and Molecule Character of Resistance and Virulence of Campylobacter jejuni isolates from livestock in Central ChinaIn the 2,185 samples collected from healthy animals, 18 strains of Campylobacter jejuni were identified. They came from chicken(n = 6) and swine(n = 12) sources. The agar dilution method was used to test the sensitivity of the C. jejuni bacteria to 27 antibiotics. The resistance rates to doxycycline and tetracycline were 100%. The resistance rates to ciprofloxacin and erythromycin were 66.67% and 88.87%, respectively. Relatively high resistance rates were observed for other antibiotics as well.In summary, Salmonella spp., Staphylococcus aureus, and C. jejuni can all be isolated from healthy chickens, swine, and dairy cows on farms in Henan, Hubei, and Hunan Provinces. They exhibit serotype diversity and have various sensitivities to different antibiotics currently used in veterinary medicine. Drug-resistant(and multidrug-resistant) bacteria were detected. Isolated strains carried a variety of drug-resistant genes and mobile genetic elements. They all had virulence genes with different functions. A variety of genotypes were observed. Therefore, long-term monitoring of antibiotics used in veterinary medicine is necessary. This study provides basic data related to the control and reasonable use of antibiotics, and has applications to animal health and source-based food safety. |
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