In vitro comparison of bovine mastitis and fecal Escherichia coli isolates

In vitro methods were used to test the hypothesis that Escherichia coli from bovine mastitis are essentially no different from isolates from bovine feces. Fifty E. coli isolates from bovine mastitic milk, 50 from feces of mastitic cows and 50 from feces of healthy cows were compared with respect to biochemical properties and certain potential virulence factors. There were no significant differences among the groups in tests for biotype; production of colicins, colicin V, or Vero cell cytotocity; and growth in 90% gnotobiotic calf serum or 90% normal milk whey. Resistance to killing in 90% gnotobiotic calf serum varied from 66 to 84%. Most isolates grew in normal whey: the percentage in a group varied from 86 to 96. Mastitic milk isolates were significantly different from the fecal isolates in adonitol fermentation (P0.006), production of aerobactin (P0.026), and ability to grow in 90% mastitic whey (P0.00004). However, only 40% of mastitis E. coli fermented adonitol and only 20% produced aerobactin. Ninety-six percent of mastitic milk E. coli grew in mastitic whey, whereas 64% and 60%, respectively, of mastitic fecal and normal fecal isolates grew in this medium. It is concluded that none of the properties that were investigated constitute potential virulence factors or markers for ability to induce mastitis; the data are consistent with the hypothesis that mastitic E. coli are simply opportunistic pathogens.     

Enterotoxigenic Escherichia coli (ETEC) and Klebsiella pneumoniae isolated from dogs with diarrhoea

Faecal samples from 148 dogs with diarrhoea and from 15 healthy dogs were cultured for bacterial pathogens with enterotoxigenic properties. The aim of the study was to define the toxin profile (production of heat-labile [LT] and heat-stable [ST] toxins) and possible surface fimbrial antigens. Enterotoxigenic bacteria were isolated from 6 (4.1%) dogs with diarrhoea, four of these were Escherichia coli and two were Klebsiella pneumoniae. The E. coli strains and K. pneumoniae strains were producing both LT and ST toxins. The LT toxin from these strains was not neutralized by human anti-LT serum or anti-choleragen and did not cause coagglutination with Staphylococcus aureus coated with anti-human-LT. This suggests that the LT toxin produced by these canine isolates is non-identical to LT toxin from human strains.

Three of the ETEC strains were haemagglutinating and showed surface hydrophobic properties. Electron microscopy showed that canine ETEC isolates possessed fimbriae of two different types: thick (5–5.5 nm) and thin (2–3 nm).     

Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli isolates in faecal samples of broilers

Seventy-six faecal samples were obtained from broilers at slaughterhouse level in Portugal. Samples were inoculated on cefotaxime-supplemented Levine agar plates. Cefotaxime-resistant Escherichia coli isolates were recovered from 32 samples (42.1%), obtaining a total of 34 E. coli isolates (one or two isolates per sample). Susceptibility to 16 antibiotics was studied by disk diffusion method, and 85% of the isolates presented a phenotype of multi-resistance that included antimicrobial agents of at least four different families. Extended-spectrum-beta-lactamases (ESBL) of the TEM and CTX-M groups were detected in 31 ESBL-positive E. coli isolates. Twenty-six isolates harboured the bla_TEM-52 gene and two of them also harboured bla_TEM-1b. The bla_CTX-M-14 gene was identified in three isolates (in association with bla_TEM-1b in one of them), and bla_CTX-M-32 was demonstrated in two additional isolates. Three of the 34 cefotaxime-resistant isolates (9%) did not produce ESBLs, and two of them presented mutations at positions −42 (C → T), −18 (G → A), −1 (C → T), and +58(C → T) of the promoter/attenuator region of ampC gene. tet(A) and/or tet(B) genes were detected in all 34 tetracycline-resistant isolates, aadA in all 26 streptomycin-resistant isolates; cmlA in 3 of 6 chloramphenicol-resistant isolates, and aac(3)-II or aac(3)-I + aac(3)-IV genes in all 4 gentamicin-resistant isolates. Different combinations of sul1, sul2 and sul3 genes were demonstrated among the 22 trimethoprim–sulfamethoxazole-resistant isolates. Amino acid changes in GyrA and ParC proteins were identified in all 18 ciprofloxacin-resistant isolates. The results of this study indicate that the intestinal tract of healthy poultry is a reservoir of ESBL-positive E. coli isolates.     

In vitro antimicrobial activity of marbofloxacin and enrofloxacin against bacterial strains isolated from companion animals

Fluoroquinolones were originally developed for the Gram-negative aerobic spectrum, but the newer generation agents are also highly effective against some Gram-positive pathogens and cause few adverse effects. Owing to these characteristics, fluoroquinolones are often used in first line therapy in small animal practice. However, their widespread use has raised concern over emerging bacterial resistance. In this study we evaluated the in vitro efficacy of two fluoroquinolones, marbofloxacin and enrofloxacin, on field strains isolated from clinical infections between 2002 and 2005. Our data show that most of the isolates are still sensitive to both antimicrobials and marbofloxacin was more effective than enrofloxacin, especially against P. aeruginosa and beta-Streptococci (P < 0.01). beta-Streptococci demonstrated the greatest resistance to the two study drugs.     

Susceptibility of Pseudomonas isolates from the ears and skin of dogs to enrofloxacin, marbofloxacin, and ciprofloxacin

The purpose of this study was to compare susceptibilities of ear and skin Pseudomonas spp. isolates to enrofloxacin, marbofloxacin, and ciprofloxacin. Specimens were obtained from dogs examined in a veterinary dermatology referral hospital. Susceptibilities of ear isolates to enrofloxacin, marbofloxacin, and ciprofloxacin were 46.9%, 66.7%, and 75.0%, respectively. Susceptibilities of skin isolates to the same drugs were 76.2%, 81.0%, and 80.0%, respectively. Ear isolates were significantly less susceptible to enrofloxacin than to ciprofloxacin (P=0.021), and ear isolates were significantly less susceptible to enrofloxacin than were skin isolates (P=0.034). When fluoroquinolone resistance was present, ear isolates were significantly less susceptible to enrofloxacin than to ciprofloxacin (P<0.001) and marbofloxacin (P=0.014).     

Effect of bacitracin on tetracycline resistance in Escherichia coli and Salmonella

Tetracyclines and bacitracin are used extensively as a growth promotant and a therapeutic, respectively in livestock. In this study, we test whether there is an interaction between bacitracin and tetracycline that can select for an increase in microbial tetracycline sensitivity, using Escherichia coli and Salmonella as a model. We found via in vitro studies that bacitracin at sublethal concentrations preferentially selects for outgrowth of tetracycline-sensitive bacteria from among a population of tetracycline-resistant and tetracycline-sensitive E. coli and Salmonella strains. Most of the bacterial strains employed in our study were shown by PCR to possess the tetracycline resistance gene tet(A) or tet(C), either of whose activity is associated with tetracycline efflux. Bacitracin could be substituted for the lipophylic chelator, fusaric acid, used as a positive selective agent for tetracycline sensitivity. Together, these observations suggest that bacitracin-mediated selection for tetracycline sensitivity alters the function of tetracycline efflux proteins. Using bacitracin and tetracycline simultaneously may improve the effectiveness of the antibiotics, while decreasing the risk of selecting for a population of tetracycline-resistant microorganisms.     

Characterization of shiga toxin-producing Escherichia coli strains isolated from calves in Poland

Fecal samples from 67 3–5-months-old calves with diarrhea were screened for the presence of shiga toxin-producing Escherichia coli (STEC). Several accessory virulence factors genes were also tested. Among 192 E.coli isolates tested, 15 (7.6%) were found to harbour the shiga toxin 1 or 2 (stx1 or stx2) genes. The stx2-carrying samples were further subtyped by PCR for the stx2c, stx2d, and stx2e toxin variants. It was shown that stx2-positive bacteria mainly possessed the stx2c shiga toxin type gene. The enterohemolysin (hlyA) and intimin (eae) genes were found in seven (46.7%) STEC strains whereas the cytotoxic necrotizin factor 1 and 2 or the P fimbrial genes were detected in two isolates only. This study confirmed that calves are a reservoir of STEC strains (with all pathogenicity genes) that may be virulent for humans.     

Relationship between level of antibiotic use and resistance among Escherichia coli isolates from integrated multi-site cohorts of humans and swine

The objective of this longitudinal ecological study was to examine the relationship between the prevalence of antibiotic-resistant (AR) commensal Escherichia coli isolates from both monthly human wastewater and composite swine fecal samples and the concurrent aggregated monthly antibiotic use recorded within each host species in multi-site vertically integrated swine and human populations. In addition, human vocation (swine worker versus non-swine worker), swine production group, and season were examined as potential confounding variables. Human and swine E. coli isolates (n = 2469 human and 2310 swine, respectively) were tested for antimicrobial susceptibility using a commercial broth microdilution system. In the human population, among swine workers the relative odds of tetracycline resistance were increased significantly for tetracycline (class) drug use at the third quartile and above of mean monthly dosage (MMD) (OR = 1.8) as compared to the referent category (non-use). The relative odds of ciprofloxacin resistance were significantly increased for ciprofloxacin use in non-swine workers (OR = 5.5) as compared to the referent (non-use). The relative odds of tetracycline resistance were increased significantly for chlortetracycline use in medicated feed for the upper tertile of MMD category (OR = 2.9) as compared to the referent category (no use) across all swine production groups. While high variability among seasonal samples over the 3-year period was observed, no common seasonal trends relating to antibiotic use and prevalence of resistance over the 3-year period were apparent. The overall effects of concurrent human and swine antibiotic use on AR E. coli levels were inconsistent and modest in this study.     

In vitro activity of difloxacin against canine bacterial isolates.

The in vitro activity of difloxacin against canine bacterial isolates from clinical cases was studied in the United States and The Netherlands. Minimal inhibitory concentrations (MIC), the postantibiotic effect, the effect of pH on antimicrobial activity, and the bacterial killing rate tests were determined according to standard techniques. The MICs of American and Dutch isolates agreed in general. The MICs of the American gram-negative isolates ranged from 0.06 to 2.0 microg/ml, and the MICs of the Dutch gram-negative isolates ranged from 0.016 to 8.0 microg/ml. A few European strains of Proteus mirabilis and Klebsiella pneumoniae had relatively high MICs. Bordetella bronchiseptica also was less susceptible to difloxacin. The MICs of the American gram-positive cocci ranged from 0.125 to 4.0 microg/ml, and the MICs of Dutch isolates ranged from 0.125 to 2.0 microg/ ml. Difloxacin induced a concentration-dependent postantibiotic effect that lasted 0.2-3 hours in cultures with Escherichia coli, Staphylococcus intermedius, Streptococcus canis, Proteus spp., and Klebsiella pneumoniae. There was no postantibiotic effect observed against canine Pseudomonas aeruginosa. Decreasing the pH of the medium increased the MIC of Proteus mirabilis for difloxacin. The MICs of Escherichia coli and Klebsiella pneumoniae were lowest at neutral pH and were slightly increased in acid or alkaline media. At a neutral pH, most tested bacterial species were killed at a difloxacin concentration of 4 times the MIC. Similar results were obtained when these same bacteria were tested against enrofloxacin. A Klebsiella pneumoniae strain in an acidic environment was readily killed at difloxacin or enrofloxacin MIC, but at neutral pH the drug concentration had to be raised to 4 times the MIC for a bactericidal effect. After 24 hours of incubation at pH 7.1, difloxacin and enrofloxacin had similar bactericidal activity for all bacteria tested except Staphylococcus intermedius. Against S. intermedius, difloxacin was more bactericidal than enrofloxacin.     

Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin after intravenous and oral administration of enrofloxacin in dogs

Rung, K., Riond, J.-L. & Wanner, M. Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin after intravenous and oral administration of enrofloxacin in dogs. J. vet
Four dogs were given 5 mg/kg body weight enrofloxacin intravenously (i.v.) and orally (p.o.) in a cross-over study. Plasma concentrations of the active ingredient enrofloxacin and its main metabolite ciprofloxacin were determined by a reversed phase liquid chromatographic method. Pharmacokinetic parameters of both substances were calculated by use of statistical moments and were compared to those of enrofloxacin described in the veterinary literature. Mean enrofloxacin t _½λZ was 2.4 h, mean Cl_s was 27.1 ml/min-kg, and mean V_ss was 7.0 1/kg. After i.v. and p.o. administration, concentrations of ciprofloxacin exceeding minimal inhibitory concentrations of several microorganisms were reached (C_max= 0.2 ng/ml, max = 2.2 h after intravenous administration; C_max= 0.2 (ig/ml, t _max= 3.6 h after oral administration). A considerable part of the antimicrobial activity is due to ciprofloxacin, the main metabolite of enrofloxacin.     

Heme-iron and ecology of Escherichia coli within the porcine gut

Growth of Escherichia coli was followed in sow whey by a turbidometric technique. Heme-iron clearly promoted bacterial growth, therefore free heme would be dangerous in small intestine by enhancing bacterial growth: duodenal brush border membrane (BBM) was seen to bind heme-iron, thus abolishing the growth promoting effect of this form of iron. Scatchard analysis of heme-binding onto BBM showed that heme became bound by a specific mechanism (K_D ≈ 10^-7 M) as well as non-specifically. The absorption system for iron within the enterocytes should be considered an important antibacterial system within the gut.     

Field trial evaluating changes in prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers medicated with enrofloxacin, apramycin and amoxicillin

The present study investigates, under field conditions, the influence of antimicrobial administration on prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers. For this purpose, a group of 16,000 commercial broiler chickens was treated with enrofloxacin from day 1 to day 3, gentamicin from day 19 to day 21, and ampicillin from day 26 to day 28. A control group of 16,000 broilers was placed in the same controlled environment poultry house. Fecal (from both groups) and feed samples were collected at regular intervals. Few E. coli isolates were obtained from either farm environment or poultry feed samples, while enterococci were found to be ubiquitous among these samples. The frequency of resistance against most antimicrobials tested was significantly higher (P < 0.05) in E. coli isolated from broilers receiving intermittent antimicrobial pressure than that from non-medicated broilers, whereas in enterococci these differences were only observed among structurally related antimicrobial drugs and over a short period of time. By the time the broilers reached market age (33 days), several multi-resistant E. coli and enterococci were detected in the feces of the medicated group. Results suggest that antimicrobial resistance in E. coli was mainly medication-dependent, whereas among enterococci, changes observed over time were apparently influenced by factors apart from antimicrobial exposure, namely the resistance organisms previously present in farm environment and those present in feedstuffs.     

恩诺沙星与其他抗菌药对鸡毒支原体的体外联合抑菌试验

采用2倍稀释法测定了恩诺沙星及其他8种抗菌药对鸡毒支原体BG44T株的最小抑菌浓度（MIC）,再以棋盘法测定恩诺沙星分别与其他8种抗菌药不同联合对鸡毒支原体BG44T株的敏感性。结果显示：恩诺沙星、替米考星、泰乐菌素、吉他霉素、林可霉素、沃尼妙林、泰妙菌素、氯霉素及氟苯尼考对鸡毒支原体BG44T株的MIC分别为0．063、0．004、0．016、0．063、16、〈0．004、0．008、8、8μg／mL。在8种不同联合用药对鸡毒支原体BG44T株的药敏试验中,恩诺沙星＋替米考星、恩诺沙星＋泰乐菌素、恩诺沙星＋吉他霉素、恩诺沙星＋林可霉素、恩诺沙星＋沃尼妙林、恩诺沙星＋泰妙菌素联合表现出相加作用,恩诺沙星＋氯霉素、恩诺沙星＋氟苯尼考表现出拮抗作用。     

阿莫西林和硫酸粘菌素对猪鸡大肠杆菌和沙门氏菌的体外联合抗菌作用

探讨了阿莫西林与硫酸粘菌素对猪鸡的大肠杆菌和沙门氏菌的联合抗菌效果。采用微量稀释法分别测定阿莫西林与硫酸粘菌素单药对大肠杆菌和沙门氏菌的最低抑菌浓度（MIC）,采用棋盘稀释法测定阿莫西林与硫酸粘菌素联用对大肠杆菌和沙门氏菌的MIC并计算联合指数（FIC）。试验结果显示,对14株试验菌株体外联合抗菌呈协同与相加作用的占78.6%,呈无关作用的占21.4%,无拮抗作用,表明阿莫西林和硫酸粘菌素可联合应用于治疗猪鸡大肠杆菌和沙门氏菌感染。     

In vitro susceptibility of selected veterinary bacterial pathogens to ciprofloxacin, enrofloxacin and norfloxacin.

The minimum inhibitory concentrations (MIC) of ciprofloxacin, enrofloxacin, and norfloxacin were tested for approximately ten clinical isolates of each of Actinobacillus pleuropneumoniae, Actinobacillus suis, Actinomyces pyogenes, Corynebacterium pseudotuberculosis, Erysipelothrix rhusiopathiae, Haemophilus parasuis, Haemophilus somnus, Pasteurella haemolytica, Pasteurella multocida, Rhodococcus equi, Streptococcus equi, Streptococcus suis and Streptococcus zooepidemicus. Ciprofloxacin and enrofloxacin had similar activity and were more active than norfloxacin. All isolates had an MIC of 1.0 microgram/mL or less for ciprofloxacin and enrofloxacin, and these drugs had particularly marked activity against the gram-negative bacteria tested.     

牛肉源大肠杆菌的耐药性检测及相关耐药基因分布

为了解牛肉源大肠杆菌的耐药性并检测其相关耐药基因分布,本研究选取了117株牛肉源大肠杆菌,经药敏纸片法对11种抗菌药物进行了药敏检测,并根据耐药表型利用普通和(或)多重PCR技术对耐四环素菌中tet(A)、tet(B)和tet(C)基因,耐氨苄西林菌中blaTEM1、blaPSE1和blaOXA1基因,耐链霉素菌中strA-strB、aadA1基因,耐磺胺类药菌中sul1、sul2和sul3基因进行了调查分析。结果显示,117株大肠杆菌对四环素、氨苄西林、链霉素和磺胺异恶唑的耐药率较高,分别为89%、42%、38%和22%。tet(A)基因是所有四环素耐药基因中最为流行的一种基因(55%);在检测的3个β-内酰胺类药物耐药基因中,最流行的为blaTEM1基因(73%);链霉素的耐药性主要由strA-strB基因(38%)编码;sul2基因在耐磺胺异恶唑菌中的检出率最高(77%)。结果表明本次分离的牛肉源大肠杆菌耐药非常严重,进一步肯定了肉牛业生产中抗菌药的使用对大肠杆菌耐药性的产生和发展所发挥的重要作用,提示食品动物养殖应严格控制饲料中抗菌药的滥用。