In vitro activity of chloropolysporin-C, a new glycopeptide-group antibiotic, on Clostridium perfringens isolates and its in vivo activity against C perfringens and other intestinal microflora of the caeca of broiler chickens

The influence of chloropolysporin-C, a new glycopeptide antibiotic, on in vitro activity of Clostridium perfringens isolates and its effects on the intestinal microflora of broiler chickens was examined. The in vitro sensitivity of 88 isolates of C perfringens to four antimicrobial agents, including chloropolysporin-C, was tested by an agar dilution method. The antibiotics used all had minimum inhibitory concentration levels of 6.25 micrograms ml-1 or less against this organism. Changes were examined in the intestinal microflora of broiler chickens fed a diet containing chloropolysporin-C to obtain basic data on the mechanisms by which the antibiotic aided livestock production. No clinical findings were recognised in chickens tested during the period of antibiotic administration. A decrease in viable cells of the clostridia was the principal response recognised during the period of drug administration in feed. Among the other microflora, chloropolysporin-C led to a significant response among Gram-positive bacteria, but no changes in the total bacterial count.     

Characterisation and antimicrobial sensitivity of haemophili isolated from pigs

A total of 70 haemophili from Australia pigs was compared with a range of reference strains of porcine haemophili. Forty-eight of the isolates were identified as Actinobacillus pleuropneumoniae biovar 1 and the remaining 22 isolates as Haemophilus parasuis. Forty one of the A. pleuropneumoniae isolates were used in a study to determine to the minimal inhibitory concentration (MIC) of 12 antimicrobial agents, or combinations of agents. Penicillin, neomycin, trimethoprim, trimethoprim-sulphamethoxazole and tetracycline all showed low MIC values, indicating their potential for the treatment of porcine pleuropneumonia, although 2 isolates showed resistance to tetracycline. A wide range of MIC values was encountered with the sulphonamides.     

山东省部分地区禽源致病性大肠杆菌的血清流行病学调查与耐药性检测

为调查山东省禽源致病性大肠杆菌流行的血清型及耐药性,从山东部分地区的45家养禽场分离到致病性大肠杆菌96株,应用微量平板凝集试验进行了血清型鉴定,共鉴定出18种血清型,其中优势血清型6种,分别为O78、O2、O15、O18、O143、O88,占定型致病性菌株的64％。抗菌药物敏感性试验发现,96株大肠杆菌对20种药物有不同程度的耐药性。75％以上的菌株对氨苄青霉素、阿莫西林、土霉素等5种抗菌药耐药,50％以上的菌株对卡那霉素、多西环素、环丙沙星等7种抗菌药表现为耐药;所有分离株存在多重耐药现象,75％的受检菌对9种或9种以上的被测药物耐药。结果表明,O78、O2、O15、O18、O143、O886种血清型是山东省部分地区近年来禽源致病性大肠杆菌的优势血清型,且禽源致病性大肠杆菌的耐药现象严重,有必要加强耐药性检测,以指导兽医临床合理使用抗菌药物。     

Antimicrobial susceptibility of Escherichia coli isolates from wild mice in a forest of a natural park in Hokkaido, Japan

To reveal the antimicrobial susceptibilities of Escherichia coli isolates from wild mice, 81 E. coli isolates were obtained from 109 voles (Clethrionomys spp.), 52 large Japanese field mice (Apodemus speciosus) and 19 small Japanese field mice (A. argenteus) captured in a forest of a natural park in Hokkaido, Japan. Seventy-eight of the 81 E. coli isolates were susceptible to all 10 antimicrobial agents tested. One E. coli isolate was resistant to ampicillin, dihydrostreptomycin, kanamycin, chloramphenicol and oxytetracycline. Two isolates were resistant to oxytetracycline. A low prevalence of antimicrobial resistance was maintained among wild mice that inhabited the forest.     

Inhibitory effect of different enterocins against fecal bacterial isolates

Anti-microbial activity of five different enterocins produced by ruminal and environmental enterococci against fecal bacterial isolates was tested. The majority of all 61 strains (80.4%) were inhibited by all enterocins used. The remaining strains were inhibited at least by one enterocin. The highest activity, 51,200 arbitrary units per ml-1 (AU ml-1) was measured when crude extracts of enterocin V24 (CE V24) were used against enterococci. CE AL41 and EK13 showed an activity of 25,600 AU ml-1. The lowest activity was obtained with Enterocin CE EC24 (100-400 AU ml-1). Crude extract of enterocin CCM4231 inhibited the indicator strains with an activity ranging from 100 up to 6400 AU ml-1, which is in accordance with our previous results. It further indicates the probable use of enterocins or its producers in environmental biotechnology with the aim to increase the effectiveness of sanitation of animal excrements after standard treatment.     

The isolation, propagation and characterization of tissue-cultured equine rotaviruses

From 105 field cases of diarrhea in neonatal or young foals, rotavirus was detected by electron microscopy (EM) and/or by enzyme-linked immunosorbent assay (ELISA) in the feces of 65 foals on 16 different premises. ELISA was performed with Rotazyme test kits developed by Abbot and Company for the detection of rotaviruses. Twenty-four field isolates from the feces of diarrheic foals with equine rotavirus infection as ascertained by EM were placed in MA-104 cell cultures after pretreatment of the viral suspension with 10 micrograms ml-1 of trypsin and incorporation of 0.5 micrograms ml-1 or 1 microgram ml-1 of trypsin in Earle's minimal essential medium (MEM), 2% lactalbumen hydrolysate, and antibiotics. The isolates that replicated in cell culture produced varying degrees of cytopathic effect. After the 24 isolates had been transferred 5 or 7 times in cell culture, viral particles were observed in 17 by EM, and 22 had positive ELISA tests as determined by visual color chart and spectrophotometric readings. Concentrated tissue-cultured viral antigen of 9 isolates fixed complement using Nebraska calf diarrhea rotavirus calf antiserum while four isolates gave negative results. The same 13 tissue-cultured viral suspensions failed to fix complement using reovirus antiserum. The 9th passages of two isolates (EID1 and EID2) yielded titers of 10(4.45) ml-1 TCID50 and of 10(4.95) ml-1 TCID50, respectively, as measured by cytopathic effect. After 13 tissue-cultured passages, 2 other isolates, EID3 and EID4, each had titers of 10(6.2) ml-1 TCID50 and of 10(5.95) ml-1 TCID, respectively. Cytoplasmic or intranuclear inclusions were not seen in any cells of the MA-104 infected cell cultures. Small, but distinct, plaques in MA-104 cell cultures were produced by the EID1 isolate. Polyacrylamide gel electrophoresis tests of EID1 and EID2 isolates at the 9th cell passage and EID3 and EID4 isolates at the 13th cell passage each showed that the RNA genome had 11 segments with a migrating pattern that was identical for each isolate and characteristic of rotaviruses. These 4 equine tissue-cultured isolates when tested by ELISA, utilizing a monoclonal antibody serum pool that cross-reacted with many rotavirus isolates, each gave positive values comparable to rotavirus antigen controls.     

Prevalence of antimicrobial resistance and resistance genes in faecal Escherichia coli isolates recovered from healthy pets

Faecal samples of healthy dogs (n=39) and cats (n=36) obtained in Northern Portugal were seeded on Levine agar plates, and two Escherichia coli isolates per sample were recovered (78 of dogs and 66 of cats). The susceptibility to 16 antimicrobial agents was tested in this series of 144 E. coli isolates. Almost 20% of them showed tetracycline resistance and 12 and 15% presented ampicillin or streptomycin resistance, respectively. The percentage of resistance to the other antimicrobial agents was in all cases below 4%, and no resistant isolates were detected for ceftazidime, imipenem, cefoxitin or amikacin. Two isolates (from one dog) showed cefotaxime-resistance and harboured both the CTX-M-1 and OXA-30 beta-lactamases. A bla(TEM) gene was detected in 12 of 17 ampicillin-resistant isolates, the aac(3)-II gene in the three gentamicin-resistant isolates, aadA in 7 of 22 streptomycin-resistant isolates, and tet(A) and/or tet(B) gene in all 28 tetracycline-resistant isolates. The gene encoding class 1 integrase was detected in six E. coli isolates, including the four trimethoprim-sulfamethoxazole-resistant isolates and those two harbouring CTX-M-1 and OXA-30 beta-lactamases; different gene cassette arrangements were identified: dfrA1+aadA1 (two isolates), dfrA12+orfF+aadA2 (two isolates) and bla(OXA30)+aadA1 (two isolates). One amino acid change in GyrA protein (Ser83Leu or Asp87Tyr) was detected in four nalidixic acid-resistant and ciprofloxacin-susceptible isolates and two amino acid changes in GyrA (Ser83Leu+Asp87Asn) and one in ParC (Ser80Ile) were identified in one nalidixic acid- and ciprofloxacin-resistant isolate. Faecal E. coli isolates of healthy pets could be a reservoir of antimicrobial resistance genes.     

In vitro susceptibility and a new point mutation associated with tylosin-resistance in Japanese canine intestinal spirochetes

The in vitro susceptibilities of six commonly used antimicrobial agents against 29 isolates of intestinal spirochetes isolated from dogs in Japan were examined by the agar dilution technique. In addition, the genetic basis of tylosin resistance in in vitro selected resistant mutants of two reference strains and three tylosin-susceptible field isolates obtained by three successive subcultures on blood agar containing 1 microg/ml of tylosin was investigated. Carbadox was the most active (MIC: < 0.00625) of all the antimicrobial agents. Although all the isolates were susceptible to tylosin, some were resistant to erythromycin. Tiamulin, lincomycin and dimetridazole were also very active against the isolates. All the resistant isolates did not harbor any plasmids. In vitro selected tylosin-resistant mutants of previously tylosin-susceptible isolates showed a new mutation in which their adenine at the base position equivalent to 2062 of 23S rDNA of Escherichia coli has been replaced by cytosine. These findings may both provide guidance towards the proper choice of antimicrobial agents for the treatment of canine intestinal spirochetosis, and add to the understanding of the genetic basis of tylosin resistance.     

内蒙古地区羊源大肠杆菌耐药性研究

为确定内蒙古地区羊源大肠杆菌的耐药表型及其耐药基因的流行情况,本研究采用微量稀释法测定了内蒙古地区108株羊源大肠杆菌对13种临床常用抗菌药物的最小抑菌浓度。结果显示,分离菌株对阿莫西林、头孢噻吩、磺胺甲唑、黏菌素的耐药率最高,均达100.0%,对阿莫西林/克拉维酸、四环素、环丙沙星的耐药率在50%~80%之间,对头孢噻肟、美洛培南、新霉素的耐药率均低于10%,较为敏感。108株羊源大肠杆菌中耐7种以上药物的菌株占94.4%,其中15.6%菌株对13种抗菌药物耐药,只有1株菌对所有抗菌药物敏感。采用PCR方法对羊源大肠杆菌分离株所携带的6种相关耐药基因进行检测,结果显示,6种耐药基因中的4种耐药基因bla_TEM、proP-2、sul-Ⅰ、ampG检出率超过50%,耐药基因aph（3'）-Ⅰ携带率达40%,只有耐药基因aac（3）-Ⅱ检出率仅为5.5%。由此可见,内蒙古地区羊源大肠杆菌对13种抗菌药物产生了不同程度的耐药性,且存在严重的多重耐药情况,羊源大肠杆菌分离株携带aph（3'）-Ⅰ、sul-Ⅰ、ampG、bla_TEM、proP-2、aac（3）-Ⅱ耐药基因。     

Study on the Drug-resistance of Escherichia coli Isolated from Sheep in Inner Mongolia

In order to determine the drug resistance phenotype and prevalence of drug resistance genes in Escherichia coli (E.coli) isolated from sheep in Inner Mongolia, the minimum inhibitory concentrations (MICs) of the isolates to antibiotics commonly used in veterinary clinical were detected by micro-dilution method in vitro. The results showed that the highest resistance rates of the isolates to amoxicillin,cephalothin,sulfamethoxazole and polymyxin were up to 100.0%,respectively.To amoxicillin-clavulanic, tetracycline and ciprofloxacin were 50% to 80%. These isolates were sensitive to cefotaxime, meropenem trihydrate, neomycin, and their resistance rates were all less than 10%. Among the 108 strains of E.coli from sheep, 94.4% of them were resistant to more than 7 antimicrobial agents,15.6% of them were resistant to 13 antimicrobial agents, only one strain was sensitive to all antimicrobial agents. Six kinds of resistance genes among the 108 E.coli isolates were detected by PCR method.The results showed that detection rates of 4 kinds of drug resistance genes including bla_TEM, proP-2, sul-Ⅰ and ampG were all over 50%,the detection rate of resistance gene aph (3')-Ⅰ was up to 40%, only resistance gene aac(3)-Ⅱ detection rate was 5.5%. Thus, the sheep E.coli isolates in Inner Mongolia produced various degrees of resistance to 13 kinds of antibiotics, and their multi-drug resistances were very serious. They carried aph(3')-Ⅰ, sul-Ⅰ, ampG, bla_TEM, proP-2 and aac(3)-Ⅱ resistance genes.     

Antimicrobial susceptibility pattern of Helicobacter suis strains

Helicobacter suis is a very fastidious porcine gastric pathogen, which is also considered to be of zoonotic importance. In vitro antimicrobial susceptibility cannot be determined using standard assays, as this agent only grows in a biphasic medium with an acidic pH. Therefore, a combined agar and broth dilution method was used to analyse the activity of nine antimicrobial agents against nine H. suis isolates. After 48 h microaerobic incubation, minimal inhibitory concentrations (MICs) were determined by software-assisted calculation of bacterial growth. Only for enrofloxacin a bimodal distribution of MICs was demonstrated, indicating acquired resistance in one strain, which showed an AGT→AGG (Ser→Arg) substitution at codon 99 of gyrA. In conclusion, the assay developed here is suitable for determination of the antimicrobial susceptibility of H. suis isolates, although activity of acid sensitive antimicrobial agents may be higher than predicted from MIC endpoints.     

Antimicrobial susceptibility patterns of thermotolerant Campylobacter strains isolated from food animals in Ethiopia

Thermotolerant Campylobacter spp. are frequent causes of diarrhoea in humans worldwide mostly originating from poultry. It has been suggested that extensive veterinary use of antibiotics is largely responsible for resistance in human isolates. During a 4-month period from January to April 2004, 192 Campylobacter spp. were isolated from fecal samples of 485 healthy food animals. The in vitro susceptibility to 12 antibiotics was determined by the agar disk diffusion method. Among the 192 Campylobacter spp. isolated, 135 (70.3%) were identified to be C. jejuni, 51 (26.6%) were C. coli and 6 (3.1%) were C. lari. C. jejuni was the most prevalent species in chickens (80.8%) versus 16.2% C. coli and 3.0% C. lari. All isolates found in pigs were C. coli. All strains were sensitive to chloramphenicol and ciprofloxacin and all were resistant to cephalothin. More than 90% of the strains were sensitive to clindamycin, erythromycin, gentamicin, nalidixic acid, norfloxacin, streptomycin and tetracycline. Resistance was found against ampicillin in 20% and trimethoprim-sulphamethoxazole in 37.5%. Resistance was not statistically different among C. jejuni, C. coli and C. lari (p>0.05). Multidrug resistance to two or more drugs was detected in 14.5% of strains. In conclusion, the study showed that antimicrobial resistance is found only at relatively low frequencies for most antimicrobial agents tested except for ampicillin and trimethoprim-sulphamethoxazole. The low percentages of resistance to most antimicrobial agents tested in this study may be the result of low/no usage of these agents as a growth promoters or treatment in the Ethiopian animal farm setting. The detection of multidrug resistant isolates may pose a threat to humans and further limits therapeutic options.     

鸡源印第安纳沙门菌多重耐药性分析

采集山东不同地区鸡源沙门菌,根据Kauffmann-White方法测定分离株血清型,采用肉汤微量稀释法测试分离株对16种抗菌药物的敏感性,PCR方法检测10种耐药基因,分析耐药表型和耐药基因之间的关系。结果显示：共鉴定出沙门菌80株,其中印第安纳沙门菌60株。药敏试验证实：60株印第安纳沙门菌对阿莫西林-克拉维酸、头孢唑啉、多黏菌素、氨苄西林、多西环素和甲氧苄啶等16种抗菌药物普遍耐药。88.33%菌株多重耐药分布在12~15耐,未发现3耐以下的菌株。PCR扩增出int1,blaTEM,aac（6’）-Ib-cr,floR,catA1,tetA,strA,cmlA 8种耐药基因。超过90%的菌株携带int1,blaTEM,floR和aac（6’）-Ib-cr耐药基因。上述结果表明,耐药表型及耐药基因的符合呈现相关性。     

Antimicrobial susceptibility, plasmid profiles and haemocin activities of Avibacterium paragallinarum strains

In this study, 18 Avibacterium paragallinarum isolates collected in Taiwan from 1990 to 2003 were serotyped and tested for resistance to antimicrobial agents. Serotyping revealed that 13 isolates were Page serovar A and 5 isolates were Page serovar C. More than 75% of the isolates were resistant to neomycin, streptomycin and erythromycin. The most common resistance pattern (15 isolates, 83.3%) was neomycin-streptomycin. Furthermore, 88.9% of the isolates were resistant to two or more antibiotics. About 72% of isolates contained plasmids (pYMH5 and/or pA14). Plasmid pYMH5 encoded functional streptomycin, sulfonamide, kanamycin and neomycin resistance genes and revealed significant homology to a broad host-range plasmid, pLS88. Plasmid pA14 encoded a putative MglA protein and RNase II, both of which might be associated with virulence. Furthermore, seven isolates showed haemocin activity. Plasmid pYMH5 is the first multidrug-resistance plasmid reported in A. paragallinarum and it may facilitate the spread of antibiotic-resistance genes between bacteria. The putative virulence plasmid pA14 and haemocin-like activity in A. paragallinarum indicate two possible mechanisms which might be responsible for the pathogenesis.     

Antimicrobial susceptibility of Bordetella bronchiseptica isolates from cats and a comparison of the agar dilution and E-test methods

One hundred and fifty-two predominantly feline isolates of Bordetella bronchiseptica were tested for their susceptibility to seven antimicrobial agents using an agar dilution method. The majority of isolates tested by the agar dilution method were resistant to trimethoprim (MIC₉₀ 500 μg/ml) and ampicillin (MIC₉₀ > 32 μg/ml) but sensitive to tetracycline, doxycycline and enrofloxacin (MIC₉₀ 2 μg/ml for all three agents). The isolates showed a spectrum of susceptibility to sulphadiazine and clavulanate potentiated amoxycillin. The MIC's of twenty-nine of the 152 isolates were then compared for five of the antimicrobial agents using the E-test (AB Biodisk, Sweden), a recently introduced method for measuring the MIC's of antimicrobial agents based on the diffusion of a pre-defined antibiotic gradient from a plastic strip. Comparisons with the E-test demonstrated an overall agreement (±1 log₂ dilution) with the agar dilution method of 79.4% and an agreement within ±2 log₂ dilutions of 96.2%.     

Antibacterial activity of thionin Thi2.1 from Arabidopsis thaliana expressed by bovine endothelial cells against Staphylococcus aureus isolates from bovine mastitis

Bovine mastitis is mainly caused by Staphylococcus aureus and antimicrobial therapy, commonly used for its control, has resulted in an increase in the frequency of resistant staphylococci in recent years. Thus, alternative therapies are desirable and the antimicrobial peptides represent attractive control agents. In this work, we expressed the antimicrobial peptide thionin Thi2.1 cDNA from Arabidopsis thaliana in the bovine endothelial cell line BVE-E6E7 and evaluated its activity against bovine mastitis S. aureus isolates. A polyclonal population from BVE-E6E7 cells transfected with the pThi2.1 construct was obtained and thionin Thi2.1 expression was confirmed by RT-PCR. From this population, eight stably transfected cell clones were obtained and their conditioned media (CM) were evaluated against the S. aureus ATCC 27543 strain. Clones showed high antibacterial activity (>95%) relative to the activity of the polyclonal population. The C8 clone showed the highest antibacterial activity (>99%) and its CM was evaluated against eleven bovine mastitis S. aureus isolates. A 2.5microg aliquot of total protein from the C8 clone's CM inhibited the growth of S. aureus isolates (>40%) relative to the CM from BVE-E6E7 cells used as control. Growth inhibition of S. aureus isolates was dose-dependent, showing a total inhibition at concentrations higher than 3.12microg/ml. These results suggest that thionin Thi2.1 antimicrobial peptide could be use in the treatment of bovine mastitis.     

Antimicrobial susceptibility of Mycoplasma hyorhinis

A broth microdilution technique was used to determine the antimicrobial susceptibility of 15 field isolates of Mycoplasma hyorhinis to 10 antimicrobial agents, representative of different classes, and contrasting newer agents to existing ones. For the macrolides, the MIC(90) for tylosin and tilmicosin was 1 and 4 microg/ml, respectively, but was > or = 16 microg/ml for erythromycin. Tetracycline, lincomycin and enrofloxacin each had an MIC(90) of 2 microg/ml. The mycoplasma had similar levels of susceptibility to the aminoglycoside and aminocyclictol classes exhibiting an MIC(90) of 4 microg/ml for gentamicin and 2 microg/ml for spectinomycin. The isolates exhibited high MICs to trimethoprim/sulfamethoxazole with an MIC(90) > or = 16/304 microg/ml. In summary, M. hyorhinis isolates from the US had low MICs against a variety of antimicrobials tested, with the exception of erythromycin and trimethoprim/sulfamethoxazole.     

Influence of intensive and extensive breeding on lactic acid bacteria isolated from Gallus gallus domesticus ceca

In the present study, lactic acid bacteria (LAB) from the cecum of chickens bred either under intensive (commercial broilers) or extensive (free-range) conditions were isolated, identified and some of their probiotic characteristics determined. The LAB identified by 16S-23S rRNA PCR-ARDRA were mainly of Lactobacillus species and to a lesser extent of Enterococcus spp. for all animals. Free-range chickens showed a higher presence of Lactobacillus acidophilus while Lactobacillus reuteri and Lactobacillus johnsonii were more frequently recovered from commercial broilers. Lactobacillus crispatus was found only in commercial broilers, Lactobacillus vaginalis and Lactobacillus agilis only in free-range chickens and Lactobacillus salivarius in both types. Enterococcus isolates from ceca of commercial broilers showed a higher resistance to antimicrobial drugs. Lactobacillus isolates from free-range chickens presented a higher frequency of in vitro antagonistic activity against selected pathogens than from commercial broilers. All LAB isolates had predominantly non-hydrophobic surfaces, but with variations depending on age of the chickens and breeding conditions. Animal breeding caused variation on composition, antimicrobial susceptibility, antagonistic activity and surface hydrophobicity of LAB from chicken cecum. LAB isolates from ceca of free-range chickens have potential as probiotic agents, which may be used in the future as replacing the use of antimicrobials as growth promoters.     

Antimicrobial susceptibility of Pasteurella multocida isolated from cattle and pigs

Minimum inhibitory concentrations (MICs) of 10 antimicrobial agents were determined for Pasteurella multocida from cattle and pigs (72 and 68 isolates, respectively). Higher MICs were observed with oxytetracycline, doxycycline, tilmicosin and thiamphenicol for porcine isolates than for bovine isolates. Enrofloxacin was the most active, with an MIC for 90% of the isolates (MIC90) of 0.05 microg/ml for both bovine and porcine isolates. Aspoxicillin exhibited the same excellent activity against penicillin-susceptible isolates as ceftiofur, with MICs ranging from < or = 0.025 to 0.1 microg/ml. Aminoglycosides were less active, with an MIC90 of > 100 microg/ml for both bovine and porcine isolates.