Role of integrons in the proliferation of multiple drug resistance in selected bacteria occurring in poultry production

ABSTRACT

• 1. The increase in microbial resistance, and in particular multiple drug resistance (MDR), is an increasing threat to public health. The uncontrolled use of antibiotics and antibacterial chemotherapeutics in the poultry industry, especially in concentrations too low to cause inhibition, and the occurrence of residues in feed and in the environment play a significant role in the development of resistance among zoonotic food-borne microorganisms.

• 2. Determining the presence and transmission methods of resistance in bacteria is crucial for tracking and preventing antibiotic resistance. Horizontal transfer of genetic elements responsible for drug resistance is considered to be the main mechanism for the spread of antibiotic resistance.

• 3. Of the many well-known genetic elements responsible for horizontal gene transfer, integrons are among the most important factors contributing to multiple drug resistance. The mechanism of bacterial drug resistance acquisition through integrons is one of the essential elements of MDR prevention in animal production.

     

整合子-基因盒系统与细菌耐药性

整合子 基因盒系统在细菌中能捕获外来耐药基因,是细菌耐药性传播的机制之一。整合子携带着重组的基因盒插入到转座子或接合质粒中,在不同的细菌间运动而传播耐药性;同时一个整合子可以捕获多个基因盒,使细菌产生多重耐药性,细菌产生多重耐药性的能力取决于它们捕获新的抗生素耐药基因的能力。整合子是一种遗传因素,编码一个位点特异重组酶(IntI)负责基因盒在 attI位点的插入,同时整合子也提供一个启动子(Pant)负责基因盒耐药基因的表达。文章对整合子 基因盒的结构、种类、耐药基因盒的表达及耐药基因的获得和传播进行综述。     

环境中细菌抗药性的研究进展

抗生素抗性基因已经成为公认的环境污染物,威胁着生态安全和人类健康。抗生素在临床和养殖业中的大量应用,使生态环境中各微生物处于残留抗生素及抗药性遗传元件的影响中,导致抗生素抗性菌获得了竞争优势,破坏了微生态系统的稳定性。作者从宏观环境角度阐述了抗药性基因传播的微分子概念,通过从抗药性发展机制、抗药性引起的环境污染、抗药性对环境微生物影响3个方面进行分析,阐明环境在细菌抗药性的发展进程中起到的关键作用,同时分析环境抗药性,包括菌群生态多样性、抗药性细菌种类及传播、抗生素残留及抗药性基因传递富集的危害等。     

Research Progress on Bacterial Resistance in Environment

Antibiotic resistance genes have become a recognized environmental pollutant, threatening the ecological safety and human health. The application of antibiotics in the clinical and animals breeding, making the environmental microorganisms living with the impact of the residue of antibiotics and elements of resistance genetic and leading to antibiotic resistant bacteria to gain a competitive advantage and destroyed the stability of ecosystem. In this paper, we expounded the concept of resistance gene transmission by the view of macro environment. Through analyzing the mechanism of resistance development,environmental pollution caused by drug resistance and the impact of environmental microorganism for drug resistance, we clarified the key role of the environment in the development of the characteristics of bacterial resistance and analysis environmental resistance.Such as the ecological diversity of flora, the types and the spread of resistant bacteria, the residues of antibiotics and the transmission of the resistance genes.     

beta-Lactam resistance and beta-lactamases in bacteria of animal origin

beta-Lactams are among the most clinically important antimicrobials in both human and veterinary medicine. Bacterial resistance to beta-lactams has been increasingly observed in bacteria, including those of animal origin. The mechanisms of beta-lactam resistance include inaccessibility of the drugs to their target, target alterations and/or inactivation of the drugs by beta-lactamases. The latter contributes predominantly to beta-lactam resistance in Gram-negative bacteria. A variety of beta-lactamases have been identified in bacteria derived from food-producing and companion animals and may further serve as a reservoir for beta-lactamase-producing bacteria in humans. While this review mainly describes beta-lactamases from animal-derived Escherichia coli and Salmonella spp., beta-lactamases from animal-derived Campylobacter spp., Enterococcus spp., Staphylococcus spp. and other pathogens are also discussed. Of particular concern are the increasingly-isolated plasmid-encoded AmpC-type CMY and extended-spectrum CTX-M beta-lactamases, which mediate acquired resistance to extended-spectrum beta-lactams. The genes encoding these enzymes often coexist with other antimicrobial resistance determinants and can also be associated with transposons/integrons, increasing the potential enrichment of multidrug resistant bacteria by multiple antimicrobial agents as well as dissemination of the resistance determinants among bacterial species. Characterization of beta-lactam-resistant animal-derived bacteria warrants further investigation of the type and distribution of beta-lactamases in bacteria of animal origin and their potential impact on human medicine.     

Use of antimicrobials in veterinary medicine and mechanisms of resistance.

This review deals with the application of antimicrobial agents in veterinary medicine and food animal production and the possible consequences arising from the widespread and multipurpose use of antimicrobials. The various mechanisms that bacteria have developed to escape the inhibitory effects of the antimicrobials most frequently used in the veterinary field are reported in detail. Resistance of bacteria to tetracyclines, macrolide-lincosamide-streptogramin antibiotics, beta-lactam antibiotics, aminoglycosides, sulfonamides, trimethoprim, fluoroquinolones and chloramphenicol/florfenicol is described with regard to enzymatic inactivation, decreased intracellular drug accumulation and modification/protection/replacement of the target sites. In addition, basic information is given about mobile genetic elements which carry the respective resistance genes, such as plasmids, transposons, and gene cassettes/integrons, and their ways of spreading via conjugation, mobilisation, transduction, and transformation.     

Characterization of integron mediated antimicrobial resistance in Salmonella isolated from diseased swine

Forty-two Salmonella isolates obtained from diseased swine were genetically characterized for the presence of specific antimicrobial resistance mechanisms. Twenty of these isolates were characterized as S. Typhimurium DT104 strains. Pulsed-field gel electrophoresis was used to determine genetic relatedness and revealed 20 distinct genetic patterns among the 42 isolates. However, all DT104 isolates fell within 2 closely related genetic clusters. Other Salmonella isolates were genetically grouped together according to serotype. All DT104 isolates displayed the penta-resistance phenotype to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. Resistance to sulfamethoxazole, tetracycline, streptomycin, kanamycin, and ampicillin was most common among the non-DT104 Salmonella isolates. All DT104 strains contained 2 chromosomal integrons of 1000 and 1200 base pairs. The DNA sequencing revealed that the 2 integrons contained genes encoding a resistance to streptomycin and ampicillin, respectively. None of the non-DT104 strains showed the same pattern, although several strains possessed integrons of 1000 base pairs or larger. However, the majority of non-DT104 Salmonella strains did not possess any integrons. Two Salmonella isolates displayed tolerance to the organic solvent cyclohexane, indicating the possibility that they are overexpressing chromosomal regulatory genes marA or soxS or the associated multidrug efflux pump, acrAB. This research suggests that integrons contribute to antimicrobial resistance among specific swine Salmonella serotypes; however, they are not as widely disseminated among non-Typhimurium swine Salmonella serotypes as previously thought.     

Aspects of bacterial resistance to antimicrobials used in veterinary dermatological practice

Aspects of bacterial resistance to the major classes of antimicrobials used in veterinary dermatology are presented in this review. Resistance of gram-positive and gram-negative bacteria to tetracyclines, macrolide-lincosamide-streptogramin antibiotics, chloramphenicol, mupirocin, sulphonamides, trimethoprim, aminoglycosides, fluoroquinolones and β-lactam antibiotics are depicted with respect to the different mechanisms of acquired and intrinsic resistance. Examples are given for the three major resistance mechanisms, enzymatic inactivation, decreased intracellular drug accumulation and target modification. In addition, basic information about mobile genetic elements which carry resistance genes, such as plasmids, transposons and gene cassettes, and their modes of spreading via transduction, conjugation, mobilization and transformation is provided.     

Characteristics of extended-spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates from horses

The aim of the present study was to contribute to the knowledge on extended-spectrum beta-lactamases (ESBL's), AmpC beta-lactamases and integrons in Enterobacteriaceae isolated from horses, which is still limited. The susceptibility of 1581 clinical isolates from animals to ceftiofur was tested. Most of these isolates (n=1347) originated from horses. Seven ceftiofur-resistant equine isolates (four Escherichia coli and three Klebsiella pneumoniae) were identified and all seven were multidrug-resistant. These isolates were further studied for the presence of ESBL's, AmpC beta-lactamases and class 1 integrons. The potential for the horizontal transfer of resistance genes among these clinical isolates was also studied. ESBL-type resistance genes were found in five isolates, AmpC-type genes in one isolates and integrons in six isolates. Nucleotide sequence analysis revealed that the isolates carried the bla(CTX-M-1), bla(CMY-2), bla(TEM-1) and/or bla(SHV-1) genes. This is the first report describing the in vitro conjugal transfer of the bla(CTX-M-1) genes from a clinical E. coli isolate to Salmonella isolates. Gene cassettes encoding resistance to aminoglycosides (aadA1, aadA2 and aadA5), and trimethoprim (dfrA1, drfA12 and dfrA17) were found on the integrons present in the isolates. The cassette arrays of the dfrA17-aadA5 and dfrA1-aadA1 genes in the two integrons of a single E. coli isolate have not yet been described before. To our knowledge this is the first report on ESBL's and AmpC beta-lactamases in equine E. coli and Klebsiella isolates.     

Isolation,Identification and Drug Sensitivity Analysis of Shigella from Minks

In order to provide therapeutical guidance for drug admistration, the bacteria of three sick minks suffering from typical diarrhea symptoms provided by mink farms in Jilin province were isolated and identified, and the drug sensitivity was tested. The bacteria were isolated with TSA plates, and identified using biochemical methods and PCR assay. The virulence of the isolates was determined by infecting BALB/c mice. The antimicrobial susceptibility of the isolates to antimicrobial agents was investigated using the K-B method. PCR was used to detect the resistance genes and Ⅰ integrons. A total of 3 Shigella isolates were obtained from sick minks. The virulent determination showed that all isolates could cause mice diarrhea. The drug sensitivity results showed that 3 strains were sensitive to fluoroquinolone, cephalosporin, florfenicol and polymyxin, but they were resistant to aminoglycoside, tetracycline, chloramphenicol, penicillin and ampicillin. There were seven resistance genes were detected,bla_TEM-1,aadA1, aac(3')-Ⅱc, aac(6')-Ⅰb, aph(3')-Ⅶ, tet(M), cat2 and three class Ⅰ integrons carrying aadA 1 gene cassette. All of the isolates were virulent and caused the mice diarrhea. The resistance of the 3 strains were very serious and mainly for multiple drug resistance phenomenon. The resistance genes detected in the mink were various, and could bring enormous implications for clinical treatment.     

水貂志贺氏菌分离鉴定及药物敏感性分析

试验旨在对吉林省某水貂养殖场送检的3只具有典型腹泻症状的病死水貂的小肠和肠内容物样品进行细菌分离鉴定及耐药情况分析,为临床治疗提供参考。通过细菌分离纯化和PCR方法对分离菌株进行鉴定,对BALB/c小鼠进行菌液注射来检测菌株的致病性。采用K-B药敏法检测菌株对常用药物的敏感性,并通过PCR方法检测其耐药基因和Ⅰ类整合子的携带情况。结果显示,分离得到3株志贺氏菌,致病性检测试验显示分离菌可引起小鼠腹泻。药物敏感性试验结果显示,3株志贺氏菌对氟喹诺酮类药物、头孢类药物、氟苯尼考和多黏菌素较敏感,对氨基糖苷类、四环素、氯霉素、青霉素、氨苄西林耐药。耐药基因检测结果显示,3株志贺氏菌共检测出7种耐药基因bla_TEM-1、aadA1、aac（3'）-Ⅱc、aac（6'）-Ⅰb、aph（3'）-Ⅶ、tet（M）、cat2及携带aadA1基因盒的Ⅰ类整合子。结果表明,分离的3株志贺氏菌均具有致病性,可引起小鼠腹泻,主要表现为多重耐药现象,携带的耐药基因呈多样性,为临床治疗带来巨大影响。     

Isolation and molecular characterization of multidrug-resistant Gram-negative bacteria from imported flamingos in Japan

Imported animals, especially those from developing countries, may constitute a potential hazard to native animals and to public health. In this study, a new flock of lesser flamingos imported from Tanzania to Hiroshima Zoological Park were screened for multidrug-resistant Gram-negative bacteria, integrons and antimicrobial resistance genes. Thirty-seven Gram-negative bacterial isolates were obtained from the flamingos. Seven isolates (18.9%) showed multidrug resistance phenotypes, the most common being against: ampicillin, streptomycin, tetracycline, trimethoprim/sulfamethoxazole and nalidixic acid. Molecular analyses identified class 1 and class 2 integrons, β-lactamase-encoding genes, bla_TEM-1 and bla_CTX-M-2 and the plasmid-mediated quinolone resistance genes, qnrS and qnrB. This study highlights the role of animal importation in the dissemination of multidrug-resistant bacteria, integrons and antimicrobial resistance genes from one country to another.     

犬脓皮病耐甲氧西林伪中间型葡萄球菌抗菌药耐药性及消毒剂抗性研究进展

犬脓皮病是一类主要由耐甲氧西林伪中间型葡萄球菌（MRSP）感染而引起的化脓性皮肤病。葡萄球菌是一种人与动物均易感的细菌，常引起各种化脓性疾病，其中，MRSP作为一种动物源葡萄球菌还会成为耐药基因贮存库，可将耐药基因通过环境或食物链传给人类。近年来MRSP造成的皮肤疾病病例大幅上升，给感染的控制带来挑战。笔者综合了犬脓皮病致病菌的抗菌药耐药性及其消毒剂抗性的相关研究，从MRSP的致病机制出发，总结了MRSP通过破坏细胞免疫系统的功能导致感染发生的相关机制，简述了多个国家MRSP对抗菌药的显著耐药性与相关耐药基因，如mecA和cat基因等，介绍了MRSP对胍类消毒剂与季铵盐类消毒剂的抗性及抗性机制，对外排泵、基因调控与抗性基因的可转移性等多种机制进行了论述，同时为避免MRSP对抗菌药与消毒剂的共同耐药性对犬脓皮病的治疗造成干扰，笔者从移动遗传元件介导的获得性抗性与依赖于细菌细胞结构的固有抗性等方面系统地分析了MRSP对消毒剂抗性和抗菌药耐药性之间的争议与联系，以期寻找一种科学合理的治疗方案，为犬脓皮病的临床用药提供参考。     

临床16S rRNA甲基化酶的研究新进展

16SrRNA甲基化酶是近年来出现于临床的一种新耐药决定因子,介导细菌对多种氨基糖苷类高水平耐药,最初发现于肠杆菌科中,目前在革兰阴性菌中已发现至少由12种等位基因编码的10种16SrRNA甲基化酶。由于大多编码16SrRNA甲基化酶的基因常位于可动遗传因子如接合型质粒、整合子、转座子、插入序列共同区上,易引起耐药性和耐药基因的传播,导致临床抗感染治疗的失败。本文综述了临床16SrRNA甲基化酶的新发现,其介导的耐药性、作用机制、临床流行特点、传播特点及分子遗传背景、来源及进化,为临床合理应用氨基糖苷类抗生素、开发16SrRNA甲基化酶抑制剂奠定基础。     

中草药对细菌耐药质粒的消除作用研究

动物养殖中常见病原菌对抗菌药物的耐药性呈逐年上升的趋势,引起了广大科研工作者的极大关注。病原菌耐药性的产生主要与质粒有关,质粒能够将耐药基因传递给环境中的其他细菌,进而引起多重耐药菌株的出现。本文就细菌耐药现状、细菌耐药性与质粒关系、中草药对细菌耐药质粒的消除效果、中草药消除耐药质粒的机理、存在的问题、中草药对耐药质粒消除的展望这些方面进行简单综述。     

Role of coresistance in the development of resistance to chloramphenicol in Escherichia coli isolated from sick cattle and pigs

OBJECTIVE: To determine the cause of persistent resistance to chloramphenicol (CP) after the ban on its use in food-producing animals in several countries. SAMPLE POPULATION: 71 CP-resistant and 104 CP-susceptible Escherichia coli strains isolated from sick cattle and pigs in Japan. PROCEDURE: Susceptibility of all bacterial strains to thiamphenicol (TP) and florfenicol (FFC) was tested by use of an agar dilution method. The CP-resistance genes and variable region within class 1 integrons in CP-resistant strains were identified by use of a PCR assay. RESULTS: The CP acetyltransferase gene (ie, cat1) was identified as the predominant CP-resistance gene in strains isolated from cattle, and the cat1and nonenzymatic CP-resistance gene (ie, cmlA) were the predominant CP-resistance genes in strains isolated from pigs. Additionally, strains with cat1 isolated from cattle often were resistant to ampicillin, dihydrostreptomycin (DSM), oxytetracycline, and trimethoprim (TMP), whereas strains with cat1 or cmlA isolated from pigs often were resistant to DSM and TMP. Class 1 integrons were significantly more prevalent in strains with CP-resistance genes, compared with prevalence in strains without CP-resistance genes. All gene cassettes within the integrons were involved in resistance to DSM, TMP, or both. CONCLUSIONS AND CLINICAL RELEVANCE: Coresistance that develops because of the use of DSM and TMP in cattle and pigs apparently contributes to the selection of CP-resistant strains of E coli. Thus, it is possible that bacterial resistance to CP in animals would persist despite a ban on the use of CP in cattle and pigs.     

Spread of resistant bacteria and resistance genes from animals to humans--the public health consequences

The paper reviews the lines of evidence which link the use of antimicrobial drugs for food animals with the emergence of antimicrobial drug resistance in bacteria pathogenic to humans, with a particular focus on the public health aspects. Deductions from the epidemiology of food-borne infections, ecological studies, outbreak investigations, typing studies and direct epidemiological observations show that resistant bacteria are transferred from food animals to man. In addition to transfer in the food chain, exchange of mobile genetic elements among commensal and pathogenic bacteria contributes to the emergence of drug resistance. There is growing evidence that this has measurable consequences for human public health. One consequence is increased transmission supported by unrelated use of anti-microbials in humans. Other consequences are related to reduced efficacy of early empirical treatment, limitations in the choices for treatment after confirmed microbiological diagnosis, and finally a possible coselection of virulence traits. Recent epidemiological studies have measured these consequences in terms of excess mortality associated with resistance, increased duration of illness, and increased risk of invasive illness or hospitalization following infections with resistant Salmonella.     

Characterization of antibiotic-resistant bacteria in rendered animal products.

Antibiotics are used in food animal production to treat diseases and also to improve performance. Antibiotics are not used on all farms, and antibiotic resistance is occasionally found on farms that do not use antibiotics. Rendered animal protein products are often included in poultry feeds and could potentially serve as a source of antibiotic-resistant bacteria. One hundred sixty-five rendered animal protein products from cattle, poultry, and fish were aseptically collected from poultry feed mills. Fifty-five percent of the poultry meal samples had detectable levels of gram-negative bacteria ranging from 40 to 10,440 colony-forming units/g of sample. Poultry meal and meat and bone meal had the greatest number of samples with bacteria resistant to five or more antibiotics. A high percentage of feed samples (85%) contained bacteria resistant to amoxicillin, ampicillin, clavulanic acid, or cephalothin, whereas few samples contained bacteria resistant to ciprofloxacin, kanamycin, or trimethoprim/sulfamethoxazole. Acinetobacter calcoaceticus, Citrobacter freundii, and Enterobacter cloacae were the most commonly isolated antibiotic-resistant bacteria. Isolation for Salmonella was also performed, with 14% of the meat and bone meal samples containing Salmonella sp. Only one of the meat and bone meal isolates, Salmonella livingstone, was resistant to five or more antibiotics. Many of the antibiotic-resistant bacteria contained integrons, genetic elements that mediate multiple drug resistance.     

细菌耐药性产生的分子机制及对细菌毒力的影响研究进展

抗菌药物选择压力导致细菌耐药性日趋严重,一些细菌菌株的分子耐药机制会引起细菌致病性的改变,说明两者之间存在一定的相关性。论文从细菌胞壁、胞膜、胞质和染色体4个部分概述了其相关主要大分子物质参与细菌耐药的机制,介绍了与耐药性密切相关的生物大分子参与细菌致病的过程,分析细菌耐药性的产生对细菌毒力变化的影响,以期为解决现今较严重的细菌耐药性及细菌性疾病的防治难题找到新方法和突破点。     

盐源县养鸭场主要病原菌分离鉴定及其耐药性分析

为掌握养鸭场病原菌种类及其耐药情况,本实验采用传统细菌学方法进行病原菌分离鉴定和药敏试验,并采用PCR方法进行耐药基因检测,结果显示：养鸭场病原菌主要是大肠埃希氏菌、金黄色葡萄球菌和鸭里默氏杆菌;主要病原菌对20种抗生素呈现多重耐药现象,在大肠埃希氏菌分离株中检出TEM和CTX-M耐药基因,在金黄色葡萄球菌分离株中检出mecA和blaZ耐药基因。这些结果为盐源县养鸭场细菌性疾病的预防与控制提供了科学依据。