19 research outputs found
家畜由来大腸菌における第3世代セファロスポリンに対する薬剤感受性調査および分子遺伝学的解析に関する研究
第3世代セファロスポリン(TGC)はヒト用のみならず牛および豚用の動物用医薬品としても承認され、使用されている。家畜由来細菌の薬剤耐性モニタリング(JVARM, Japanese Veterinary Antimicrobial Resistance Monitoring System)では、1999年から家畜におけるTGCに対する耐性状況のモニタリングを開始しており、1999年から2002年に分離された家畜(牛、豚、採卵鶏および肉用鶏)由来の大腸菌のうち、TGCが承認されていない肉用鶏から分離された大腸菌において、TGCに対する耐性株が認められたことを報告した。そこで本研究は、(1)2004年から2009年に収集された家畜由来大腸菌におけるTGC耐性大腸菌の推移を調べるとともに、耐性菌の分子遺伝学的解析を行い、(2)TGCが承認されていない肉用鶏にTGC耐性大腸菌が出現した要因を、農場における抗菌性物質使用歴およびTGC耐性大腸菌に認められる特徴から調べ、(3)農場以外の要因が農場の肉用鶏由来大腸菌のTGC耐性に与えた影響を調べた。第1章 2004年から2009年に農場の家畜から分離された大腸菌のTGCに対する薬剤感受性調査JVARMで分離された大腸菌についてTGCに対する薬剤感受性調査を行い、TGC耐性大腸菌については分子遺伝学的解析を行った。計3,274株の大腸菌(牛由来1,095株、豚由来802株、採卵鶏由来699株および肉用鶏由来678株)に対し薬剤感受性試験を実施したところ、TGC耐性が108株(3.3 %)で認められた。畜種別では、牛由来株の2株(1.9 %)、豚由来株の2株(1.9 %)、採卵鶏由来株の15株(13.9 %)および肉用鶏由来大腸菌の89株(82.4 %)においてTGC耐性株が認められた。同一検体から分離された同一薬剤耐性遺伝子型および薬剤耐性型の2株は同一株として取扱い、最終的には69株(牛由来1株、豚由来1株、採卵鶏由来10株および肉用鶏由来57株)のTGC耐性株についてパルスフィールドゲル電気泳動を行った結果、69株のうち54株はそれぞれ80 %以下の低い相同性を示した。また、β-ラクタマーゼ遺伝子型別において、69株のうち45株(65.2 %、肉用鶏由来38株、採卵鶏由来6株および豚由来1株)はセファロスポリナーゼのblaCMY-2を保有していた。以上より、我が国の家畜には特に肉用鶏にTGC耐性大腸菌が多く認められ、TGC耐性株間では遺伝学的多様性が認められる一方で、TGCに対する耐性遺伝子は特定の型が分布していたことが示唆された。第2章 農場の肉用鶏由来大腸菌におけるTGC耐性出現要因の検討薬剤耐性菌は薬剤使用の選択圧によって選択されることから、TGC耐性大腸菌の出現要因として、①肉用鶏農場におけるTGCの適応外使用の可能性、②農場よりも上流の孵卵場におけるTGCの適応外使用の可能性、③TGC以外の薬剤の使用に起因する交差耐性または共耐性により、肉用鶏におけるTGC耐性大腸菌が選択された可能性が考えられた。このうち、②孵卵場における使用状況調査は、JVARMのモニタリング対象外であったことから、本研究では、農場の抗菌性物質使用歴調査を行うとともに、肉用鶏から分離された大腸菌に対し、TGC以外の薬剤についても薬剤感受性試験および遺伝学的性状解析を行った。2009年に肉用鶏から分離された大腸菌96株のうち、同一検体から分離された同一薬剤耐性型の2株は1株として取り扱い、最終的には78株についてさらなる解析を行った。78株中12株でTGCであるセフチオフル(CTF)に対する耐性が認められたが、セフェム系抗菌性物質はTGC耐性株が分離された鶏のみならず、いずれの鶏に対しても一切使用されていなかった。薬剤感受性ではTGC耐性株のアンピシリン(AMP)、セファゾリン(CFZ)、ジヒドロストレプトマイシン(DSM)およびクロラムフェニコール(CHL)の耐性率がTGC感受性株よりも有意に高く、また他の薬剤についても有意差は認められないものの感受性株と比較して高い傾向が認められた。このうち、AMPおよびCFZは、CTFと同系統の薬剤かつ大腸菌に対する抗菌力がCTFよりも弱いため、TGC耐性株で高くなったと考えられた。一方、DSMおよびCHLについてはCTFと系統が異なる薬剤であるため、CTFと系統が異なる薬剤の使用によってTGC耐性が選択される可能性が示唆されたが、DSM耐性またはCHL耐性を保有するTGC耐性株が分離された肉用鶏に対してDSMまたはCHLは投与されていなかった。以上より、農場の肉用鶏由来大腸菌におけるTGC耐性は農場におけるセフェム系抗菌性物質の適応外使用およびTGC以外の薬剤の使用が要因である可能性は低いと考えられた。第3章 孵卵場におけるセフチオフルの自主的使用禁止が農場の肉用鶏由来大腸菌のTGC耐性に与える影響2012年3月に生産者の協会から生産者に対して孵卵場におけるCTFの接種を中止するよう自主的な注意喚起がなされた。このため、注意喚起の前後における農場の肉用鶏由来大腸菌のTGC耐性の変動を調べることで、孵卵場におけるCTFの適応外使用と農場における肉用鶏由来大腸菌のTGC耐性との関係を検討できるものと考えられた。そこで孵卵場における注意喚起の前後2年における肉用鶏由来大腸菌のTGCに対する薬剤感受性調査を行い、孵卵場でのCTF接種中止が農場における肉用鶏由来大腸菌のTGC耐性に与える影響を調べた。2010年から2013年に農場の肉用鶏から分離された計693株(2010年195株、2011年161株、2012年206株および2013年131株)の大腸菌に対し薬剤感受性試験を実施したところ、TGCに対する耐性が693株中84株(12.1 %、2010年32株、2011年27株、2012年19株および2013年6株)で認められた。農場の肉用鶏由来大腸菌のCTFの接種中止前後のTGCの耐性率の変動は、2010年: 16.4 %(32/195)および2011年: 16.8 %(27/161)に対し、2012年: 9.2 %(19/206)および2013年: 4.6 %(6/131))であり、CTFの接種中止の前後で耐性率が有意に減少した。主要なβ-ラクタマーゼ遺伝子としては、CTF接種中止の前後のいずれの年もblaCMY-2が優勢であり(2010年: 62.5 %(20/32)、2011年: 81.5% (22/27)、2012年: 57.9 %(11/19)および2013年: 83.3 %(5/6))、84株のTGC耐性株のうち58株がblaCMY-2を保有していた。blaCMY-2以外にもblaCTX-M等の耐性遺伝子が認められたが、CTFの接種中止前後で耐性遺伝子保有割合の有意な変動は認められなかった。以上より、注意喚起後1年という短期間で肉用鶏由来大腸菌におけるTGC耐性率が主要なβ-ラクタマーゼを維持したまま有意に減少したことは、農場の肉用鶏におけるβ-ラクタマーゼ遺伝子の起源が孵卵場であることを示唆すると考えられた。以上、本研究は我が国の家畜分野におけるTGC耐性大腸菌の分布状況を明らかにするのみならず、農場の肉用鶏由来大腸菌におけるTGC耐性出現の要因についても明らかにし、また同時に農場よりも生産段階が上流の孵卵場における薬剤耐性に対する取り組みが、農場における薬剤耐性を効果的に減弱させた1例についても示した研究である。Third-generation cephalosporins (TGC) are approved for use in not only humans, but also cattle and swine. The Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM), which was established in 1999, monitors TGC resistance among bacterial isolates from livestock and between 1999 and 2002 detected TGC-resistant Escherichia coli from broiler chickens, an animal for which TGC was not approved.In this study, potential factors affecting the emergence of resistance to TGC among E. coli isolates from livestock in Japan were investigated. Firstly, TGC resistance among E. coli isolated from farm livestock (cattle, pigs, layer chickens and broiler chickens) between 2004 and 2009 was investigated and molecular genetic analysis of the TGC-resistant isolates was conducted. Secondly, to determine how TGC resistance had arisen among E. coli isolates from broiler chickens in which TGC was not approved for use, an investigation into antimicrobial use on the farms and the antimicrobial profiles of isolates was undertaken. Thirdly, other factors that may have affected the emergence of TGC resistance among E. coli isolates from boiler chickens were investigated. I. Investigation into the susceptibility to TGC of E. coli isolates from farm livestock between 2004 and 2009In total, 3274 E. coli isolates were obtained (cattle: 1095 strains; pigs: 802 strains; layer chickens: 699 strains; broiler chickens: 678 strains) from farm livestock between 2004 and 2009, and of these, 108 isolates were resistant to TGC (cattle: 2 strains; pigs: 2 strains; layer chickens: 15 strains; broiler chickens: 89 strains). When two isolates from a fecal sample exhibited the same β-lactamase gene and antimicrobial resistance type, they were considered to be a single isolate. Accordingly, 69 E. coli isolates (cattle: 1 strain; pigs: 1 strain; layer chickens: 10 strains; broiler chickens: 57 strains) were used for further analyses. Pulsed-field gel electrophoresis (PFGE) analyses with XbaI digestion of TGC-resistant E. coli showed high diversity among the PFGE profiles. Using the unweighted pair group method and the arithmetic mean, a low level of similarity (< 80%) was observed among all of the isolates with the exception of six broiler chicken isolates. Also, of 69 TGC-resistant isolates, 45 isolates (pigs: 1 strain; layer chickens: 6 strains; broiler chickens: 38 strains) harbored the blaCMY-2. II. Investigation of the factors that contribute to the appearance of TGC-resistant E. coli among broiler chickensIt is well established that the routine use of antimicrobial agents can lead to the emergence of antimicrobial-resistant bacteria. Therefore, the appearance of TGC-resistant E. coli strains at broiler chicken farms was investigated by considering whether the extra-label use of TGC had occurred on the farms or at the hatcheries, and whether other antimicrobial agents had been administered. It is worth noting that investigations by JVARM had not included the monitoring of hatcheries. In this study, the antimicrobial resistance profiles of 78 E. coli isolates obtained from broiler chickens at farms in 2009 were analyzed, and the use of antimicrobial agents at these farms was also investigated. Of these 78 strains, 12 exhibited resistance to TGC but interestingly, cephem antibiotic had never been used at these farms. When comparing the antimicrobial resistance profiles between TGC-resistant and TGC-susceptible strains, the resistance rates to dihydrostreptomycin and chloramphenicol were significantly higher for TGC-resistant strains than for TGC-susceptible strains (p<0.05); however, this was not the case for β-lactam antibiotics. However, dihydrostreptomycin and chloramphenicol had not been administered to the broiler chickens from which the dihydrostreptomycin- and chloramphenicol-resistant strains had been isolated. III. Voluntary withdrawal of ceftiofur (CTF) at hatcheries influenced TGC resistance among E. coli isolated from broiler chickens at farmsThe off-label use of CTF was voluntarily withdrawn by the farmer’s association around March 2012. To assess the effect of this, the percentage of TGC-resistant E. coli isolates from healthy broilers before and after the voluntary withdrawal of CTF was investigated. A total of 693 E. coli isolates (2010: 195 strains; 2011: 161 strains; 2012: 206 strains; 2013: 131 strains) were obtained from 362 fecal samples from broiler chickens from July to September between 2010 and 2013. TGC resistance was detected in 84 of these 693 isolates (12.1%). The percentage of TGC-resistant E. coli isolates significantly decreased after voluntary withdrawal from the off-label use of CTF from 16.4% (32/195) in 2010 and 16.8% (27/161) in 2011 to 9.2% (19/206) in 2012 and 4.6% (6/131) in 2013 (p<0.05). Regarding the detection of β-lactamase genes, 58 of the 84 TGC-resistant isolates harbored the blaCMY-2 (69.0%), and the blaCMY-2 was the predominant β-lactamase gene among TGC-resistant strains for all of the years examined (57.9–83.3%). Of the other β-lactamase genes detected, such as blaCTX-M, no significant changes in the rates of occurrence were detected among TGC-resistant E. coli before and after the withdrawal of CTF use. In conclusion, this study revealed the widespread distribution of TGC-resistant E. coli among livestock, including broiler chickens, at farms in Japan. Furthermore, the percentage of TGC-resistant E. coli isolated from broiler chickens at farms was found to be significantly decreased after voluntary withdrawal from the off-label use of CTF at hatcheries. These findings indicated that TGC resistance among E. coli isolates from broiler chickens could be controlled by restricting the use of CTF at the hatchery level.博士(獣医学)麻布大
Phylogenetic groups and cephalosporin resistance genes of Escherichia coli from diseased food-producing animals in Japan
A total of 318 Escherichia coli isolates obtained from different food-producing animals affected with colibacillosis between 2001 and 2006 were subjected to phylogenetic analysis: 72 bovine isolates, 89 poultry isolates and 157 porcine isolates. Overall, the phylogenetic group A was predominant in isolates from cattle (36/72, 50%) and pigs (101/157, 64.3%) whereas groups A (44/89, 49.4%) and D (40/89, 44.9%) were predominant in isolates from poultry. In addition, group B2 was not found among diseased food-producing animals except for a poultry isolate. Thus, the phylogenetic group distribution of E. coli from diseased animals was different by animal species. Among the 318 isolates, cefazolin resistance (minimum inhibitory concentrations: ≥32 μg/ml) was found in six bovine isolates, 29 poultry isolates and three porcine isolates. Of them, 11 isolates (nine from poultry and two from cattle) produced extended spectrum β-lactamase (ESBL). The two bovine isolates produced blaCTX-M-2, while the nine poultry isolates produced blaCTX-M-25 (4), blaSHV-2 (3), blaCTX-M-15 (1) and blaCTX-M-2 (1). Thus, our results showed that several types of ESBL were identified and three types of β-lactamase (SHV-2, CTX-M-25 and CTX-M-15) were observed for the first time in E. coli from diseased animals in Japan
Detection of aac(6')-Ib-cr in Avian Pathogenic Escherichia coli Isolates in Japan
We investigated the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in avian pathogenic Escherichia coli (APEC) strains in Japan. A total of 117 APEC strains collected between 2004 and 2007 were examined for PMQR genes (qnrA, qnrB, qnrC, qnrD, qnrS, aac(6’)-Ib-cr, qepA and oqxAB) by polymerase chain reaction. None of the APEC strains carried qnrA, qnrB, qnrC, qnrD, qnrS, qepA or oqxAB, but one of the isolates was identified as an AAC (6’)-Ib-cr producer. Phylogenetic grouping, multi-locus sequence typing and serotyping showed that this isolate belonged to phylogenetic group A, sequence type 167 and untypable serogroup. To our knowledge, this is the first report of the aac (6’)-Ib-cr gene in bacteria from food-producing animals in Japan
Effect of Antimicrobial Exposure on AcrAB Expression in Salmonella enterica Subspecies enterica Serovar Choleraesuis
Understanding the impact of antimicrobial use on the emergence of resistant bacteria is imperative to prevent its emergence. For instance, activation of the AcrAB efflux pumps is responsible for the emergence of antimicrobial-resistant Salmonella strains. Here, we examined the expression levels of acrB and its multiple regulator genes (RamA, SoxS, MarA, and Rob) in 17 field isolates of S. Choleraesuis by using quantitative PCR methods. The expression of acrB increased in eight of the field isolates (P < 0.05). The expression of acrB was associated with that of ramA in one isolate, soxS in one isolate, and both these genes in six isolates. Thereafter, to examine the effect of selected antimicrobials (enrofloxacin, ampicillin, oxytetracycline, kanamycin, and spectinomycin) on the expression of acrB and its regulator genes, mutants derived from five isolates of S. Choleraesuis were selected by culture on antimicrobial-containing plates. The expression of acrB and ramA was higher in the mutants selected using enrofloxacin (3.3-6.3- and 24.5-37.7-fold, respectively), ampicillin (1.8-7.7- and 16.1-55.9-fold, respectively), oxytetracycline (1.7-3.3- and 3.2-31.1-fold, respectively), and kanamycin (1.6-2.2- and 5.6-26.4-fold, respectively), which are AcrAB substrates, than in each of the parental strains (P < 0.05). In contrast, in AcrAB substrate-selected mutants, the expression of soxS, marA, and rob remained similar to that in parental strains. Of the four antimicrobials, the level of ramA expression was significantly higher in the enrofloxacin- and ampicillin-selected mutants than in the oxytetracycline- and kanamycin-selected mutants (P < 0.05), whereas the expression levels of acrB and multiple regulator genes in spectinomycin-selected mutants were similar to those in each parental strain. These data suggest that exposure to antimicrobials that are AcrAB substrates enhance the activation of the AcrAB efflux pump via RamA, but not via SoxS, MarA, or Rob in S. Choleraesuis
Effect of Antimicrobial Exposure on AcrAB Expression in Salmonella enterica Subspecies enterica Serovar Choleraesuis
Evaluation of Transferability of R-Plasmid in Bacteriocin-Producing Donors to Bacteriocin-Resistant Recipients
Presence of Staphylococcus aureus ST398 and ST9 in Swine in Japan
Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is mainly associated with swine and is capable of causing zoonotic infections. The methicillin-resistant S. aureus (MRSA) multilocus sequence type (ST) 398 of swine origin is predominant in Europe and North America, whereas ST9 is predominant in Asia. To evaluate the possible emergence of MRSA in swine, we examined the ST and spa type of 15 methicillin-susceptible S. aureus (MSSA) isolates obtained from swine in 8 different prefectures from north to south Japan between 2003 and 2009. Sequence analyses revealed that 6 porcine MSSA isolates belonged to ST398; 6 to ST9; and 1 each to ST5, ST97, and ST705. Of the 6 MSSA ST398 strains, 4 were classified as spa type t034. This study illustrated that there is a reservoir in Japanese swine of livestock-associated MSSA types
Detection of aac(6')-Ib-cr in Avian Pathogenic Escherichia coli Isolates in Japan
Este artigo pretende explorar uma política pública - a política científica e tecnológica (PCT) - debatendo algumas de suas principais características constitutivas (que a diferenciam significativamente das demais) e procurando evidenciar um de seus aspectos que raramente é explicitado: como política pública elaborada no âmbito do Estado capitalista, a PCT é uma política que favorece às classes dominantes. O artigo procura entender o porquê desse aspecto nem sempre ser reconhecido. E discorre também sobre algumas das principais causas e implicações disso. Para tanto, são abordados conceitos e definições gerais sobre as políticas públicas. Em seguida, são discutidas as principais características e processos associados à política científica e tecnológica. Também são abordadas as particularidades da PCT brasileira, que denunciam, em grande medida, seu caráter de classe. Por fim, são apresentados argumentos que se propõem a explicar o porquê desse caráter ser raramente reconhecido