Effects of bovine lactoferrin hydrolysate on the in vitro antimicrobial susceptibility of Escherichia coli strains isolated from baby pigs

OBJECTIVE: To determine the antibacterial activity of bovine lactoferrin hydrolysate (bLf-lysate) alone or in combination with other antimicrobials against antimicrobial-resistant Escherichia coli strains isolated from baby pigs. SAMPLE POPULATION: 3 clinical strains of E coli were isolated from baby pigs with severe diarrhea and designated as strains 9061, 9062, and 9065. PROCEDURE: The broth microdilution checkerboard and fractional inhibitory (or bactericidal) concentration index were used to evaluate the antibacterial effect elicited by bLf-lysate in combination with kanamycin, gentamicin, cephalothin, cefamandole, penicillin G, ampicillin, tetracycline, erythromycin, or rifampicin against the 3 strains of E coli. RESULTS: The 3 strains of E coli were susceptible to gentamicin and rifampicin but highly resistant to most of the other antimicrobials tested, except for strain 9061 that was also susceptible to cephalothin but intermediately inhibited by kanamycin and cefamandole. Synergistic growth-inhibitory activity was observed between bLf-lysate and gentamicin against 1 strain of E coli (strain 9062); synergistic bactericidal activity was found between bLf-lysate and rifampicin against all 3 strains of E coli. Moreover, partial synergy was observed between bLf-lysate and kanamycin, gentamicin, cephalothin, or cefamandole against the strains of E coli, but this partial synergistic activity was mostly seen against only 1 of the strains. Little interaction between bLf-lysate and tetracycline, ampicillin, penicillin G, or erythromycin was observed against the clinical strains of E coli. CONCLUSIONS AND CLINICAL RELEVANCE: A combination of bLf-lysate and certain antimicrobials may prove clinically effective against antimicrobial-resistant strains of E coli.     

In vitro growth of mastitis-inducing Escherichia coli in milk and milk fractions of dairy cows

The outcome of E. coli mastitis in cows ranges from mild to severe in individual animals. This study explored the hypothesis that milk from individual cows differs in its growth medium properties for E. coli, and whether possible variation could be related to specific milk constituents. To mimic the early phase of intramammary E. coli infection, a low inoculum size and a short incubation period were used. Cell-reduced, cell- and fat-free (skim) and cell- and fat-free and protein-reduced (whey) fractions were prepared from whole milk samples (n=18). Ten ml of whole milk, milk fractions and brain heart infusion broth (BHI) were inoculated with approximately 100cfu E. coli. After 6h of incubation, bacterial counts were assessed by dilution plating in triplicate. Bacterial counts in whole milk differed up to a 100-fold between cows, which was not associated with SCC. Bacterial counts were significantly higher in whey fractions than in whole milk, cell-reduced and skim fractions and variation in whey was smaller, indicating that the acid-precipitable protein fraction contains the milk constituents of major relevance for inhibition of and variation in bacterial growth. The presence of fat and cells added to bacterial growth inhibition to a lesser extent. In conclusion, in vitro growth of E. coli in milk differs substantially between individual cows within an incubation period comparable with the early phase of intramammary infection. This suggests that the growth medium properties of milk could be of importance in the pathogenesis of E. coli mastitis and subsequent outcome of disease.     

In vitro antimicrobial activity of nitrofurantoin against Escherichia coli and Staphylococcus pseudintermedius isolated from dogs and cats

Minimum inhibitory concentrations (MIC) of nitrofurantoin were determined by agar dilution in 269 canine and feline isolates of Escherichia coli and Staphylococcus pseudintermedius, two of the most common bacterial species associated with urinary tract infection (UTI) in small animals. The MIC90 for E. coli and S. pseudintermedius were 32 and 16 μg/ml, respectively. All isolates, including multidrug-resistant strains of known genetic background, displayed MICs below the drug concentrations reported in canine urine following oral administration of nitrofurantoin. Preliminary data on mutant prevention concentration (MPC) and many years of nitrofurantoin usage in human medicine suggest that emergence of resistant mutants during treatment is not a critical issue for this drug. The study provides species-specific data on nitrofurantoin MIC distribution that can be used for setting dog- and cat-specific breakpoints. Although nitrofurantoin is not an appropriate first-line agent for empirical treatment of canine UTI due to toxicity and poor pharmacokinetic properties, it may be indicated for treatment of UTI caused by multidrug-resistant bacteria, which are otherwise difficult to treat using conventional veterinary antimicrobial agents.     

In vitro susceptibility of Escherichia coli strains isolated from diarrhoeic dairy calves to 15 antimicrobial agents

The in vitro activities of 15 antimicrobial agents against 195 strains of Escherichia coli isolated from dairy calves affected by neonatal diarrhoea were determined. Of these strains 137 produced one or more potential virulence factors (F5, F41, F17, cytotoxic necrotizing factor, verotoxin and the eae gene), but the remaining 58 strains did not produce any of these factors. The overall percentage of resistant strains to streptomycin, tylosin and tetracycline was very high (above 65%). A high level of resistance (from 23 to 50%) to ampicillin, neomycin, kanamycin, spectinomycin, chloramphenicol, sulphadimethoxine and trimethoprim was also detected. The E. coli strains were very susceptible (89-95%) to apramycin and gentamicin and highly susceptible (99-100%) to polymyxin B, florfenicol and nitrofurazone. Some significant differences (P < 0.05) in the frequencies of resistance to some of the antimicrobials tested and in the rates of multi-drug resistance among the strains producing potential virulence factors and non-fimbriated, non-toxigenic, eae-negative strains were found. Most of the strains showed multi-resistance: 76.9% of the isolates were resistant to at least two antibiotics, 67.7% were resistant to at least four antibiotics and 50.3% were resistant to at least six antibiotics.     

Characteristics of verotoxigenic Escherichia coli from pigs.

Porcine verotoxigenic Escherichia coli were characterized with respect to frequency of occurrence, serogroup, and association with disease, weaning, and selected properties of the bacterium. Of 668 strains of E. coli from southern Ontario pigs with enteric disease, 32 (4.8%) produced verotoxin at 10(3)-10(7) cytotoxic doses per mL of culture supernatant. Of 22 isolates which belonged to O serogroups 138, 139 and 141, 15 produced verotoxin. Among other enterotoxigenic types of E. coli, two of 57 isolates of O157:K"V17" and two of 96 isolates of O149:K91 were verotoxigenic. The remaining 13 verotoxigenic E. coli belonged to O groups 2, 107, 120, 121 and 130. An additional 21 verotoxigenic E. coli belonging to O groups 138, 139 and 141 and three to O157:K"V17" were identified in a collection of 47 E. coli recovered from weaned pigs with enteric disease. Verotoxigenic E. coli were associated with postweaning diarrhea, bloody stools, sudden death and edema disease. They were isolated at similar frequencies (14%) from healthy weaned pigs, and from weaned pigs with enteric disease. Isolation rates from neonates were low and significantly different from rates in weaned pigs. Neutralizing antibody to verotoxin was not detected in the sera of 45 pigs, which included pigs from herds with a history of edema disease. Verotoxin was not associated with production of colicin, hemolysin, or enterotoxins or with any of 23 biochemical properties of the organisms. The serological data indicate that porcine verotoxigenic E. coli are not a common source of verotoxigenic E. coli for humans. Porcine verotoxin may play a role in postweaning diarrhea and absence of detectable neutralizing antibody in serum may be an important aspect of pathogenesis.     

Antibody titres to lamb rotavirus in colostrum and milk of vaccinated ewes.

Ewes were vaccinated two to three weeks prior to mating with a formalin-treated preparation of lamb rotavirus. The colostrum and milk produced by vaccinated ewes after the subsequent pregnancy were shown to contain significantly higher titres of antibody to the virus than did mammary secretions from non-vaccinates. The virus neutralising antibody activity was associated with IgG in both colostrum and milk. However, IgG concentrations in the mammary secretions of vaccinates and non-vaccinates did not differ. It is suggested that vaccination of the dam may be of value in protecting the suckled neonatal lamb against rotavirus infection.     

Experimental intramammary inoculation with Mycoplasma bovis in vaccinated and unvaccinated cows: effect on milk production and milk quality.

The effect of vaccination on milk production was evaluated in vaccinated and control cows experimentally challenged in two of four quarters with live Mycoplasma bovis. During the first three weeks after experimental challenge, six of eight unchallenged quarters on vaccinated cows and seven of eight unchallenged quarters on control cows became infected. Most of these quarters secreted normal milk, with negative California Mastitis Test scores and maintained normal milk production throughout most of the study (although some quarters on control cows remained infected). All challenged quarters became infected, had strong California Mastitis Test reactions, and had a drastic (greater than 85%) loss in milk production. Thereafter, four of eight challenged quarters on control cows remained infected, had mostly positive California Mastitis Test scores, produced mostly normal-appearing milk, and recovered some productive capabilities. By the end of the study no M. bovis could be recovered from challenged quarters on vaccinated cows and the milk appeared mostly normal. The California Mastitis Test scores on these quarters, however, remained elevated and milk production remained very low.     

In vitro susceptibility of Escherichia coli strains isolated from diarrhoeic lambs and goat kids to 14 antimicrobial agents

The in vitro activities of 14 anti-microbial agents were determined against 92 strains of E. coli isolated from lambs (60 strains) and kids (32 strains) affected by neonatal diarrhoea. The overall percentage of resistant strains to streptomycin, sulphadimethoxine and tetracycline was very high (above 70%). A high level of resistance (from 30% to 50%) to ampicillin, kanamycin, neomycin and chloramphenicol was also detected. The E. coli strains were highly susceptible to cephalosporins, polymyxin and quinolones. Most of the strains showed multiresistance: 77.2% of isolates were resistant to at least two antibiotics, 55.4% were resistant to at least four antibiotics and 33.7% were resistant to at least six antibiotics. A total of 34 antibiotypes could be distinguished.     

Observations on production of hemolysin, heat-labile enterotoxin and antimicrobial drug resistance among enterotoxigenic Escherichia coli from pigs.

A total of 52 isolates of Escherichia coli belonging to enterotoxigenic serotypes from piglets with diarrhea were examined for hemolysis, production of cholera-like enterotoxin (LT) and susceptibility to 10 antimicrobial drugs. A strong association between production of LT and hemolysis was seen. Ninety percent of 29 hemolylic isolates were LT⁺ whereas 100% of 23 nonhemolytic isolates were LT⁻ in a commercial latex agglutination assay. Antimicrobial susceptibility tests employing disc diffusion showed that resistance to trimethoprim-sulfamethoxazole (TMS), neomycin and tetracycline was significantly less among LT⁺ isolates compared to LT⁻ ones. Enrofloxacin was the only antimicrobial drug to which all the 52 isolates were susceptible.     

猪致病性大肠杆菌耐药性与整合子-基因盒的相关性

为了解猪致病性大肠杆菌耐药性与整合子-基因盒的相关性,本研究采用腹腔注射法测定52株大肠杆菌对昆明小鼠的致病性;采用K-B法测定致病性大肠杆菌对15种抗菌药物的敏感性;对致病性大肠杆菌Ⅰ、Ⅱ和Ⅲ型整合子及其基因盒进行PCR检测及测序分析,并分析其整合子-基因盒与耐药性的相关性。结果显示,52株大肠杆菌对小鼠均具有较强致病性,96.15%分离株对15种抗菌药物表现为多重耐药性,61.54%分离株耐药性均在八重耐药以上。Ⅰ、Ⅱ和Ⅲ型整合子阳性率分别为84.62%,15.38%和3.85%。Ⅰ型整合子检测到dfrA17-aadA5(18.18%)、dfrA12-orfF-aadA2(4.55%)和dfrA1-catB3-aacA4(4.55%)基因盒,Ⅱ型整合子检测到dfrA1-sat2-aadA1(50.00%)基因盒,Ⅲ型整合子未检测到基因盒,基因盒阳性率与细菌耐药性不存在相关性。     

In vitro effects of a mixture of Escherichia coli heat-stable enterotoxins on chloride flux in everted jejunal sacs of male pigs

In vitro effects of a mixture of Escherichia coli heat-stable enterotoxins (STa and STb) on isolated jejunum of 3-week-old male pigs were studied, using everted intestinal sac techniques. Heat-stable enterotoxins increased chloride secretion and chloride absorption in everted intestinal sacs. The increase of secretory flux was greater than that for absorptive flux. Vasoactive intestinal peptide (6 x 10(-9) M) increased chloride secretion, but had no effect on chloride absorption. Neither vasoactive intestinal peptide nor pilocarpine (10(-5) M) had additive effect to ST. Secretory effects of ST were not blocked by atropine (2 x 10(-5) M), clonidine (10(-6) M), or morphine (4.2 x 10(-6) M).     

Chicken egg antibodies for prophylaxis and therapy of infectious intestinal diseases. V. In vivo studies on protective effects against Escherichia coli diarrhea in pigs

A field study and a controlled infection trial showed the protective effect of egg yolk lyophilisate and whole egg lyophilisate against enterotoxic E. coli germs. The lyophilisates were gained from eggs of hens immunized against pilus antigen of porcine-enterotoxic E. coli. In a first field study using egg yolk antibodies, 92% of 299 diarrhea affected piglets were cured. In a further field study diarrhea affected piglets were cured after 3 days by application of egg yolk lyophilisate from immunized hens. Piglets treated only with egg yolk of not immunized hens showed no signs of recovery. The infection trial showed, that whole egg lyophilisate of immunized hens was as successful as a common antibiotic therapy in curing piglets, orally infected with 5 x 10(10) E. coli/feeding and animal. The present data show that chicken egg antibodies can be used for treatment of infectious diarrheal diseases in young animals. So far they represent a good alternative to the common used antibiotic therapy.     

Effect of antimicrobial agents on the production and release of shiga toxin by enterotoxaemic Escherichia coli isolates from pigs

Edema disease (ED) of pigs is an enterotoxaemic disease caused by enterotoxaemic Escherichia coli (ETEEC) infection. Antimicrobial therapy for pigs with ED is controversial because it may induce death of sickish piglets. In this study, we investigated the effects in vitro of 7 antimicrobial agents, ampicillin, gentamicin, colistin, bicozamycin, fosfomycin, sulfamethoxazole-trimethoprim and enrofloxacin, on the release and production of shiga toxin (Stx) 2e by ETEEC strains. We found that more Stx 2e accumulated in the bacterial cells than was released into supernatant. Associated with inhibition of cell wall synthesis, the exposure to ampicillin or fosfomycin increased the release of Stx 2e. The production levels of Stx 2e in all antimicrobial-treated cultures were equal to the level in the control or less than in the control. These results suggest that cell wall synthesis inhibitors, such as ampicillin and fosfomycin, may change for the worse in the signs in ETEEC infectious pigs. On the other hand, gentamicin, colistin, bicozamycin and enrofloxacin may be useful for the treatment of pigs with ED.     

Associations of antimicrobial uses with antimicrobial resistance of fecal Escherichia coli from pigs on 47 farrow-to-finish farms in Ontario and British Columbia

This study assessed the associations between antimicrobial use and other management practices in pigs and antimicrobial resistance in generic Escherichia coli recovered from feces of weaner and finisher pigs on 39 purposefully selected farrow-to-finish farms in Ontario and 8 in British Columbia. Antimicrobials (n = 13), most frequently penicillins and tetracycline, wer dministered to different age groups of pigs on study farms through various routes of administration. Logistic regression was used to model risk factors to antimicrobial resistance in fecal E. coli of pigs for the following antimicrobials: ampicillin, apramycin, carbadox, cephalothin, chloramphenicol, kanamycin, neomycin, nitrofurantoin, spectinomycin, streptomycin, sulfamethoxazole tetracycline, and cotrimoxazole (trimethoprim and sulfamethoxazole). Use of antimicrobials in weaner pigs compared with use in finisher pigs was associated with resistance in most models. There was phenotypic evidence of different mechanisms of resistance selection, including direct selection [use of carbadox was associated with carbadox resistance (OR = 6.48)]; cross-resistance [use of spectinomycin was associated with streptomycin resistance (OR = 2.29)]; and possible co-selection [ceftiofu use was associated with tetracycline resistance (OR = 6.12)]. These results provide further evidence that use of antimicrobials in pigs selects for resistance among fecal E. coli within and between classes of antimicrobials.