Prevalence and characteristics of quinolone resistance in Escherichia coli in veal calves

Quinolone resistance is studied and reported increasingly in isolates from humans, food-producing animals and companion animals. Resistance can be caused by chromosomal mutations in topoisomerase genes, plasmid-mediated resistance genes, and active transport through efflux pumps. Cross sectional data on quinolone resistance mechanisms in non-pathogenic bacteria from healthy veal calves is limited. The purpose of this study was to determine the prevalence and characteristics of quinolone resistance mechanisms in Escherichia coli isolates from veal calves, after more than 20 years of quinolone usage in veal calves. MIC values were determined for all isolates collected as part of a national surveillance program on antimicrobial resistance in commensal bacteria in food-producing animals in The Netherlands. From the strains collected from veal calves in 2007 (n=175) all isolates with ciprofloxacin MIC ≥ 0.125 mg/L (n=25) were selected for this study, and screened for the presence of known quinolone resistance determinants. In this selection only chromosomal mutations in the topoisomerase type II and IV genes were detected. The number of mutations found per isolate correlated with an increasing ciprofloxacin MIC. No plasmid-mediated quinolone resistance genes were found. The contribution of efflux pumps varied from no contribution to a 16-fold increase in susceptibility. No correlation was found with the presence of resistance genes of other antimicrobial classes, even though all quinolone non-wild type isolates were resistant to 3 or more classes of antibiotics other than quinolones. Over twenty years of quinolone usage in veal calves in The Netherlands did not result in a widespread occurrence of plasmid-mediated quinolone resistance, limiting the transmission of quinolone resistance to clonal distribution.     

耐氟喹诺酮类药物禽源大肠埃希菌gyrA基因的检测分析

探讨不同禽源大肠埃希菌中喹诺酮类药物的耐药情况及耐药基因gyrA的分布和突变特征。采用K-B药敏纸片法、gyrA基因的PCR扩增,对9株大肠埃希菌进行喹诺酮类药物试验,并将gyrA基因的PCR产物测序,对测序结果采用DNA MAN、DNA Star、MEGA6等软件分析。药敏试验结果表明,C1、C2、C3菌株对左氧沙星、氧氟沙星、环丙沙星、诺氟沙星敏感,D1、D2、D3、B1、B2和B3菌株对左氧沙星、氧氟沙星、环丙沙星、诺氟沙星均表现为耐药和中介;gyrA基因的测序结果表明,除B1菌株有1处核苷酸突变位点和B2菌株有14处核苷酸突变位点;B2菌株gyrA基因的氨基酸突变发生在87位Ile→Val替代、101位Leu→Met替代、102位Ala→Ser替代、129位Lys→Gln替代。9株禽源大肠埃希菌的同源性和进化树分析表明,不同禽源耐氟喹诺酮类药物的大肠埃希菌菌株中B2菌株gyrA基因与其他9株菌株相比,同源性在90%左右,进化树不在一个分支上,研究中的B2菌株将为大肠埃希菌的氟喹诺酮类耐药机制的研究提供候选菌株。     

喹诺酮类药物耐药机制研究进展

喹诺酮类属于合成的广谱抗菌药,用于治疗与肠杆菌科相关的各种感染性疾病.近几十年来,喹诺酮类药物的广泛使用和过度使用导致了喹诺酮耐药菌株的出现.喹诺酮类药物耐药的产生是一个复杂的多因素过程,主要的耐药机制包括染色体介导的一个或多个靶点基因突变改变靶点酶的药物结合力;Ac-rAB-tolC多耐药外排泵的过表达和孔蛋白的改变...     

质粒介导喹诺酮类耐药机制研究进展

质粒介导喹诺酮类耐药(PMQR)基因的出现,迅速提高了细菌对喹诺酮类药物的耐药性,给临床细菌性疾病的治疗带来了严重的威胁。目前虽然认为喹诺酮类耐药基因(qnr)只引起低水平耐药,但低水平耐药性可使细菌数量达到出现突变所需的浓度,从而出现高水平耐药。因此,对质粒介导该类药物耐药机制的研究,及耐药基因的分子传播机制的研究不仅能指导临床合理用药,而且有助于控制耐药菌株的产生和传播。     

Molecular basis of quinolone resistance in Escherichia coli from wild birds

Nine quinolone resistant (minimal inhibitory concentration [MIC] was > 32 microg/mL for nalidixic acid, > 1 microg/mL for ciprofloxacin) isolates of Escherichia coli have been found in wild birds with septicemia. All of the isolates were aerobactin positive. The mechanisms of resistance were characterised by sequencing the quinolone resistance-determining region (QRDR) of the gyrA, gyrB, parC, and parE genes. Sequence analysis of the gyrA gene in all isolates identified only 1 nucleotide substitution at codon Serine-83 for Leucine-83. Sequence analysis of the gyrB, parC, and parE QRDR genes revealed no mutations in any of the isolates. This study was conducted to determine the importance of these genes in the susceptibility of E. coli strains isolated from wild birds to quinolones.     

豫北地区猪源大肠杆菌耐药性及基因多态性分析

为了解豫北地区规模化养殖场猪源大肠杆菌的耐药性及基因多态性,对分离的21株猪源大肠杆菌进行药物敏感实验,采用PCR技术对其耐药基因进行检测,RAPD技术进行基因多态性分析。结果表明:豫北地区规模化养殖场分离的猪源性大肠杆菌菌株对常见16种抗生素存在不同情况的耐药性。其中对四环素、红霉素、麦迪霉素、阿莫西林、磺胺异恶唑、环丙沙星、新生霉素、复方新诺明、恩诺沙星、甲氧苄啶的耐药率均为100%,对庆大霉素、洛美沙星的耐药率为85%以上,对其他药物的耐药率相对较低;PCR检测得到8种耐药基因的条带;对菌株进行RAPD分析得到7种不同的基因型。大肠杆菌极易产生耐药性,耐药基因广泛存在于耐药菌株中,但耐药表型与耐药基因之间无绝对相关性。豫北地区猪源大肠杆菌感染多重耐药十分严重,严重影响了该地区猪源大肠杆菌病的诊治和预防。     

广西地区禽致病性大肠杆菌的耐药性分析

禽致病性大肠杆菌(APEC)是一种能引起鸡、火鸡和其他鸟类肠外感染的致病性大肠杆菌,可以导致肉鸡气囊炎、败血型全身感染、蜂窝织炎和蛋鸡输卵管炎、腹膜炎。为了了解广西地区禽致病性大肠杆菌的耐药表型以及耐药基因的携带情况,本实验室对2019年从广西分离到的69株APEC采取K-B药敏纸片法进行药敏试验,药敏结果显示,69株APEC对氧氟沙星(56.5%)、恩诺沙星(69.6%)、氟苯尼考(79.7%)、氨苄西林(91.3%)、四环素(98.6%)耐药率较高,而对美罗培南、丁胺卡那霉素、呋喃妥因均不耐药;其中,多重耐药现象严重,对10种抗菌药物以耐4种、5种、6种的情况居多。同时用PCR扩增的方法对其耐药基因,包括碳青霉稀类、β-内酰胺类、氨基糖苷类、黏菌素类、喹诺酮类、四环素类在内的6大类共计17种耐药基因进行了检测。特别值得关注的是,发现了7株携带mcr-1基因的多黏菌素耐药APEC。药敏纸片法检测菌株的耐药表型和耐药基因存在一定关联度。本研究可为养禽场临床用药提供参考,同时为减缓耐药菌传播、降低对人类健康和公共卫生安全威胁提供依据。     

动物源性大肠杆菌耐药性分析

目的:对吉林省长春市伊通地区分离出的113株动物源性大肠杆菌耐药性进行分析,了解猪源和鸡源大肠杆菌耐药程度及其耐药谱。方法:采用K-B纸片扩散法对大肠杆菌耐药性初步定性分析,并从中选出十株耐药谱不同的大肠杆菌测定其抗生素的最小抑菌浓度(MIC)。结果:113株均表现出不同程度的耐药,耐药谱广,且多表现为多重耐药。结论:动物源性大肠杆菌对抗生素的耐药性以多重耐药为主。113株对喹诺酮类、磺胺类、利福平耐药率达到90%以上,其中鸡源、猪源大肠杆菌耐药情况各不相同,二者均对丁胺卡那霉素、先锋必/舒巴坦较敏感,耐药率低于15%,可知丁胺卡那霉素、先锋必/舒巴坦可作为这些地区防治致病性大肠杆菌的首选药物。本实验结果对临床科学合理用药有重要指导意义。     

长治鸡群大肠杆菌耐药状况的研究

研究长治鸡群大肠杆菌耐药状况、交叉耐药状况、多重耐药状况及是否出现超级大肠杆菌,同时为临床用药提供参考,减少用药成本,控制大肠杆菌的危害,增加养殖户的收入.剖解病死鸡,采集可疑病料进行细菌分离培养鉴定,通过药敏试验确定其耐药情况.试验结果:长治鸡群大肠杆菌对氨苄青霉素耐药程度83%;对红霉素耐药程度67%;对复方新诺明耐药程度50%;先锋V耐药程度50%;对诺氟沙星耐药程度50%;对万古霉素耐药程度50%;对氯霉素耐药程度33%;对庆大霉素耐药程度33%;对阿米卡星耐药程度17%;对环丙沙星耐药程度17%;对利福平耐药程度17%;对复达欣耐药程度17%;对新生霉素耐药程度为0.结论:长治鸡群分离出的6株大肠杆菌中的5株为多重耐药性大肠杆菌,目前未出现超级大肠杆菌.对常用抗菌药物中的青霉素类、大环内酯类、头孢类、喹诺酮类耐药程度达到50%以上,分别为83%、67%、50%、50%;对新药万古霉素的耐药程度也达到了50%.     

主要动物性食品生产链大肠杆菌的耐药性分析

为了解主要动物性食品生产链大肠杆菌(Escherichia coli)的耐药情况,采用美国临床和实验室标准协会（CLSI）推荐的微量肉汤稀释法,对源自猪肉、鸡蛋、鸡肉生产链的350株大肠杆菌进行常用10种抗菌药(组合)的敏感性测定,参考CLSI标准（2010）判定药敏结果。结果显示,350株大肠杆菌对四环素（TET）耐药率（82.0%）最高,其次是甲氧苄啶/磺胺甲噁唑（SXT）（75.4%）,对庆大霉素（GEN）耐药率（26.9%）最低。尽管不同来源大肠杆菌对10种抗菌药中大部分药物耐药率较一致,但不同生产链环节的菌株耐药率却存在差异。76.2%（267株）受试菌株多重耐药,其中以8重耐药菌株（17.7%）最多。菌株共产生108种耐药谱,猪肉、鸡肉和鸡蛋生产链中大肠杆菌的耐药谱分别为40、46和55种,但优势耐药谱不明显。菌株数较多的耐药谱为：NAL-CIP-AMP-AMC-CEF-SPT-GEN-SXT-TET（21/350）、TET（20/350）、NAL-CIP-AMP-AMC-CEF-SPT-SXT-TET（16/350）、NAL-CIP-AMP-CEF-FLO-SPT-SXT-TET（16/350）及SPT-SXT-TET（15/350）。提示,主要动物性食品生产链大肠杆菌耐药情况较严重,应加强其耐药性连续监测与控制。     

Prevalence and Genetic Relatedness of Antimicrobial‐Resistant Escherichia coli Isolated From Animals,Foods and Humans in Iceland

The prevalence of resistant bacteria in food products in Iceland is unknown, and little is known of the prevalence in production animals. The aim of this study was to investigate the prevalence and genetic relatedness of antimicrobial‐resistant Escherichia coli from healthy pigs and broiler chicken, pork, broiler meat, slaughterhouse personnel and outpatients in Iceland. A total of 419 E. coli isolates were tested for antimicrobial susceptibility using a microbroth dilution method (VetMIC), and resistant strains were compared using pulsed‐field gel electrophoresis (PFGE). All samples were screened for enrofloxacin‐resistant strains with selective agar plates. The resistance rates among E. coli isolates were moderate to high from caecal and meat samples of pigs (54.1% and 28%), broilers (33.6% and 52%) and slaughterhouse personnel (39.1%), whereas isolates from outpatients showed moderate resistance rates (23.1%). Of notice was resistance to quinolones (minimum inhibitory concentrations: nalidixic acid ≥ 32, ciprofloxacin ≥ 0.12 and enrofloxacin ≥ 0.5), particularly among broiler and broiler meat isolates (18.2% and 36%), as there is no known antimicrobial selection pressure in the broiler production in Iceland. The majority (78.6%) of the resistant E. coli isolates was genotypically different, based on PFGE fingerprint analyses and clustering was limited. However, the same resistance pattern and pulsotype were found among isolates from broiler meat and a slaughterhouse worker, indicating spread of antimicrobial‐resistant E. coli from animals to humans. Diverse resistance patterns and pulsotypes suggest the presence of a large population of resistant E. coli in production animals in Iceland. This study gives baseline information on the prevalence of antimicrobial‐resistant E. coli from production animals, and their food products in Iceland and the moderate to high resistance rates emphasize the need for continuing surveillance. Further studies on the origin of the resistant strains and the genetic relatedness of strains of different origin are needed.     

Microarray based comparative genotyping of gentamicin resistant Escherichia coli strains from food animals and humans

Recent data from the European and Hungarian Antimicrobial Resistance Monitoring Systems have indicated that the routine use of gentamicin in human and veterinary medicine frequently leads to the selection of gentamicin resistance in Escherichia coli. The aim of this study was to provide molecular characterization of gentamicin resistance in clinical and commensal E. coli strains representing humans and food producing animals by genotyping for antimicrobial resistance and virulence using a miniaturized microarray. All 50 strains tested proved to be multidrug resistant defined as resistance to three or more antimicrobial classes. Antimicrobial resistances genes such as aadA1-like, strB, bla(TEM), sul1 and tet(A) or tet(B), and corresponding phenotypes (streptomycin-, ampicillin-, sulfamethoxazole- and tetracycline resistance) were detected in >50% of isolates regardless of the host or clinical background. However, certain genes encoding gentamicin resistance such as aac(6')-Ib and ant(2″)-Ia as well as catB3-like genes for phenicol resistance were only detected in human isolates. Among virulence genes, the increased serum survival gene iss was predominant in all host groups. Although the majority of gentamicin resistant E. coli strains were characterized by diverse antimicrobial resistance, and virulence gene patterns, accentuated links between catB3-like, aac(6')-Ib, bla(CTX-M-1) and sat genes could be detected in human strains. Further resistance/virulence gene associations (tet(A) with iroN and iss) were detected in poultry strains. In conclusion, the simultaneous characterization of antimicrobial resistance and virulence genotypes of representative clinical and commensal strains of E. coli should be useful for the identification of emerging genotypes with human and or animal health implications.     

THE ISOLATION OF ANTIBIOTIC RESISTANT COLIFORMS FROM MEAT AND SEWAGE

Beef and pig carcases, meat products, frozen chickens, and sewage were examined in 3 separate surveys for antibiotic resistant coliforms. Escherichia coli was isolated from 18 of 50 beef carcases; the numbers were low and resistance was found only to tetracycline. E. coli was isolated from 45 of 50 pig carcases; the numbers were high and showed a range of patterns of multiple antibiotic resistance. In meat products, the proportion of E. coli in contaminating organisms was low, and most resistance found was to tetracycline and streptomycin. E. coli was isolated from 66 of 75 chickens and these gave 23 patterns of antibiotic resistance, often multiple. Sewage from hospital or domestic origin and abattoir effluent yielded approximately 10⁶ coliforms/ml, most of which were resistant to one or more antibiotics; few of those from hospital or domestic origin however, were classified as E. coli of faecal origin. Twenty-four patterns of resistance were found in coliforms from domestic sewage, 19 from hospital sewage and 11 from abattoir effluent. Transfer of resistance, often multiple, was achieved from 55% of 447 resistant strains to an E. coli K₁₂ recipient. Much more information is required on the prevalence of R-factors in bacteria associated with food producing animals and their products.     

宁夏部分地区腹泻羔羊源大肠埃希菌的分离鉴定与药敏试验

为掌握宁夏地区羔羊腹泻源大肠埃希菌的耐药性,采集腹泻羔羊肛拭子90份,开展大肠埃希菌的分离鉴定及药敏试验。结果分离到78株大肠埃希菌,对头孢类、氨基糖苷类、喹诺酮类、氟苯尼考、青霉素类、四环素等抗菌药物有较高的敏感性,对万古霉素、复方新诺明、克林霉素耐药(耐药率78.21%~94.87%)。结果表明,宁夏地区羔羊腹泻源大肠埃希菌对抗菌药物保持较高的敏感率,耐药现象并不严重。     

不同动物源大肠杆菌的耐药性调查

为了解畜禽源大肠杆菌的耐药状况,2011—2012年从患病和健康食品动物中分离鉴定大肠杆菌935株,包括健康猪源606株、患病猪源114株、患病鸡源51株、患病水禽源(鸭和鹅)164株。试验采用琼脂稀释法测定了不同动物来源的大肠杆菌对15种抗菌药物的敏感性。结果发现,大肠杆菌耐药严重,对大部分药物的耐药率超过70.0%,其中氨苄西林、复方新诺明和四环素的耐药率均达到了80.0%以上;仅对头孢西丁、黏菌素和阿米卡星较敏感,但患病水禽源大肠杆菌对阿米卡星的耐药率达到了52.4%,明显高于其他动物。健康畜禽源大肠杆菌对多数抗菌药的耐药率低于患病畜禽源大肠杆菌,表明抗菌药物的使用增加了病原菌对第3代头孢类药物和氟喹诺酮类药物的耐药率,势必增加临床治疗难度。     

PREVALENCE AND CHARACTERIZATION OF QUINOLONE RESISTANCE GENES IN PROTEUS SPECIES ISOLATED FROM PET TURTLES

Proteus spp. are widely recognized as opportunistic pathogens causing urinary tract and septic infections in humans and animals. The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance genes and mutations in the quinolone resistance determining region in association with the detection of quinolone susceptibility of 24 strains of pet turtle-borne Proteus spp. Susceptibility of 4 antimicrobials including nalidixic acid, ciprofloxacin, ofloxacin, and levofloxacin was examined by disk diffusion and minimum inhibitory concentration test. Six isolates were resistant to nalidixic acid showing either intermediate resistance or resistance to other quinolones. All nalidixic acid, resistant isolates harbored mutations in gyrB (N440T/A401G/Q411S). Two of the isolates had both gyrA (S83I) and parC (S80I) mutations. Twenty-one isolates were positive for the presence of plasmid-mediated quinolone resistance genes; the qnrD gene had the highest prevalence with 19 (79.2%), while qnrS, qnrA, qnrB, and aac(6′), Ib-cr genes were present in 9 (37.5%), 2 (8.3%), 1 (4.2%), and 11 (45.8%) isolates, respectively. These results suggest that pet turtle-associated Proteus spp. should be considered a potential source of antimicrobial resistance determinants.     

Increase of bacterial resistance in human medicine by resistance genes of bacteria from meat supplying animals

Two different groups of bacteria carrying genes encoding for resistance to antibiotics may be transmitted from animals to humans via food products: a.) obligate infectious agents (enteric pathogens, e.g. Salmonella enterica spp., Campylobacter spp., EHEC) and b) facultative pathogenic species (e.g. E. coli, enterococci). Thus far, it is unknown whether genes encoding for resistance to antibiotics from these bacteria may be transferred to bacteria in normal flora of the host. The transfers of genes encoding for resistance to vancomycin from animal sources to the mucosa of humans has been suggested. Thus, there is a threat that these plasmid-encoded resistance genes may also be transferred to other gram-positive organisms present in the human flora. Vancomycin is the antibiotic in reserve for treatment of infections caused by oxacillin (methicillin) resistant strains of S. aureus and by strains of pneumococcus resistant to penicillin.     

Characterization of fluoroquinolone resistance in Escherichia coli strains from ruminants.

Thirty-seven fluoroquinolone-resistant Escherichia coli strains from ruminants (according to Clinical and Laboratory Standards Institute guidelines) were screened by molecular methods for mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes and for the presence of the qnrA gene. One of the strains studied was an enterohemorrhagic E. coli (EHEC) strain potentially pathogenic for humans. Three E. coli strains resistant to enrofloxacin (minimal inhibitory concentration [MIC] = 2 microg/ml) but not to ciprofloxacin (MIC = 1 microg/ml) presented single mutations in the gyrA and parC genes, while 34 strains resistant to both fluoroquinolones presented double and single mutations in gyrA and parC, respectively (31 strains), or double mutations in gyrA and parC (3 strains). The EHEC strain presented a double amino acid substitution in the GyrA protein (Ser-83-->Leu and Asp-87-->Gly) and a double amino acid substitution in the ParC protein (Gly-78-->Cys and Ser-80-->Arg), one of which has not been previously described. The present study shows that most of the mutations in the QRDR of the gyrA and parC genes of fluoroquinolone-resistant E. coli strains from ruminants are the same as those seen in E. coli strains from other animal species and humans and that there are no differences in mutation patterns in the QRDR of E. coli strains from healthy ruminants and those with diarrhea. No strains carried qnrA, which indicates that this gene does not play an important role in the selection of fluoroquinolone-resistant E. coli strains from ruminants.     

宠物犬源大肠杆菌氟苯尼考耐药基因floR的检测及药物敏感性分析

floR是氟苯尼考特异性耐药基因之一,当前宠物犬源大肠杆菌氟苯尼考耐药基因floR的研究报道很少,故本试验利用PCR方法对宠物犬源大肠杆菌氟苯尼考耐药基因floR进行检测,并采用微量肉汤稀释法测定宠物犬源大肠杆菌对10种抗菌药物的最小抑菌浓度(MIC)。结果显示,20株宠物犬源大肠杆菌中floR耐药基因的阳性菌株数为9株,阳性检出率为45%;对氟苯尼考的耐药率为65%,并呈现3~7重的多重耐药性。结果表明,随着氟苯尼考在兽医临床的广泛应用,其耐药率越来越高,需不断加强对氟苯尼考等抗菌药物的耐药性监控。