Effect of bambermycins, in vitro, on plasmid-mediated antimicrobial resistance

The potential of bambermycins (a growth-promoting antimicrobial approved for turkeys, broilers, and swine) to overcome or control plasmid-mediated antimicrobial resistance was determined in a series of in vitro experiments. Four possible modes of action of bambermycins were studied: synergistic effect with 12 other antimicrobials, elimination of resistance (R) plasmids from Escherichia coli, selective killing or inhibition of E coli carrying R plasmids, and inhibition of R plasmid transfer. Bambermycins had no synergistic activity with the other drugs tested and had little effect on eliminating plasmids from host bacteria. Dependent on plasmid type, bambermycins decreased or increased transfer frequency of R plasmids. Bambermycins also selectively inhibited growth of bacteria harboring certain R plasmids.     

Bacterial growth during the early phase of infection determines the severity of experimental Escherichia coli mastitis in dairy cows

The aim of this study was to investigate the importance of bacterial growth for the severity of experimental Escherichia coli mastitis, indirectly expressed as the area under the curve of bacterial counts in milk over time. The association of pre-infusion somatic cell count and post-infusion influx of inflammatory cells in milk with severity of infection was also examined. Bacterial growth was studied through culture in milk samples (in vitro) and through monitoring of bacterial counts in milk during the early phase of infection (in vivo) in 36 cows. Individual variation in bacterial counts was more than 2 x 10(2)-fold after 6 h of in vitro incubation, and more than 8 x 10(2)-fold 6 h after intramammary infusion. In vitro growth in milk was not associated with in vivo growth during the early phase of infection, nor with severity of E. coli mastitis. Somatic cell count before experimental E. coli mastitis was negatively associated with in vivo bacterial growth during the early phase of infection (R2 = 0.28), but was not associated with severity of E. coli mastitis (R2 = 0.06). In vivo bacterial growth during the early phase of infection (positive association; R2 = 0.41), together with influx of inflammatory cells in milk, expressed as mean hourly increase of somatic cell count between 6 and 12 h post-infusion (negative association; R2 = 0.11), are major determinants for the severity of experimental E. coli mastitis (R2 = 0.56).     

关中奶山羊乳房炎灭活菌苗的制备及其免疫效果测定

对自制乳房炎疫苗免疫后的抗体效价进行评估.用甲醛37℃过夜灭活大肠埃希菌、葡萄球菌,分别制备蜂胶灭活疫苗、转移因子灭活苗及无佐剂疫苗,接种泌乳期奶山羊后,分别在不同时间采集免疫羊的血清和乳汁,ELISA测定血清大肠埃希菌和葡萄球菌的抗体效价.研究结果显示,大肠埃希菌和葡萄球菌经甲醛灭活彻底;与免疫前相比,血清和乳汁中的...     

In vivo transfer of an Escherichia coli enterotoxin plasmid possessing genes for drug resistance.

Experiments were conducted to study transfer of an enterotoxin (Ent) plasmid from a porcine enteropathogenic Escherichia coli to an E coli K12 strain in the intestine of newly weaned pigs. The Ent plasmid carried genes for resistance to tetracycline, streptomycin, and sulfonamides, thereby permitting a selection for tetracycline-resistant exconjugants in the feces of the pigs. In vivo transfer of the Ent plasmid was demonstrated to occur when the pigs were given large oral inocula of donor and recipient cultures, 1 hour apart. Differences in extent of transfer were not detected in pigs given antibiotic-free feed compared with littermates on feed containing oxytetracycline at 50 g/ton. In one experiment, tetracycline-resistant Ent- exconjugants were found which appeared to have received an R plasmid from an enteropathogenic type of E coli resident in the intestine.     

Hydrogen Sulphide Producing Variants of Escherichia Coli: Widespread Occurrence in Animals and Humans within a Confined Environment

Of 178 strains of E. coli isolated on non-selective medium from faeces of pigs and humans living in the same farm 14 produced hydrogen sulphide. The H₂S producing variants all belonged to different clones suggesting that the H₂S character was plasmid-mediated. Attempts to transfer the H₂S character were not successful nor were attempts to motilize the gene by introduction of a transmissible R factor.     

The incidence of antibiotic resistance among coliform bacteria isolated from food.

Three-hundred-and-seventy-eight strains of coliform bacteria were isolated from specimens of commonly sold milk and food products. Klebsiella and Enterobacter spp. were predominating. Resistance to sulphonamides, streptomycin, and chloramphenicol occurred in only 5, 1, and 2 strains, respectively. No tetracycline-resistant strains were found. Two-hundred-and-two strains (54 %) were resistant to ampicillin. In genetic crosses with a sensitive strain of E. coli Κ 12 W 3132 transmissible R factors could not be demonstrated in any of the resistant coliform strains.It is concluded that food is not a significant source of antibiotic resistant enteric bacteria. It may, however, be suggested that food is a source of potentially pathogenic gram-negative bacteria which points out the importance of strict hygienic surveillance of food production.     

Plasmid transfer and plasmid-mediated genetic exchange in Brucella abortus.

Naturally-occurring plasmids and gene transfer mechanisms have not yet been reported in brucellae. Here we show that Brucella abortus is capable of maintaining and transferring the broad-host-range plasmids pTH10 (IncP), pSa (IncW) and R751 (IncP), and describe pTH10-mediated transfer of B. abortus chromosomal genes to Escherichia coli. All three plasmids transferred by conjugation from E. coli to B. abortus S19, and from B. abortus S19 to B. abortus 292 (biovar 4). They were stably maintained with no effect on biotyping characteristics. Plasmid pTH10 is a Tn1-containing derivative of RP4. It confers temperature-sensitive resistance to kanamycin, tetracycline and ampicillin to E. coli, but its tetracycline resistance and temperature sensitivity were poorly expressed in B. abortus. Plasmids pTH10 and pSa both transferred from B. abortus to E. coli DP50, a strain that is auxotrophic for diaminopimelic acid (DAP) Plasmid pTH10 (but not pSa) mobilized Brucella chromosomal gene(s) for DAP synthesis to DP50, yielding non-DAP-requiring (NDR) transconjugants. Neither plasmid transferred the NDR marker from their original E. coli host strains, nor did pTH10 transfer it from NDR transconjugants. Escherichia coli NDR transconjugant EP8.11 was cured of pTH10 by passage at the nonpermissive temperature, but retained the NDR marker and the Tn1-encoded resistance to ampicillin, indicating Tn1-mediated integration of Brucella chromosomal DNA into the E. coli chromosome.     

Analysis of the AIDA-I gene sequence and prevalence in Escherichia coli isolates from pigs with post-weaning diarrhoea and oedema disease

Three hundred and twenty-four strains of Escherichia coli isolated from weaned pigs with diarrhoea or oedema disease in Eastern China were screened by multiplex PCR for the presence of the gene encoding adhesin involved in diffuse adhesion I (AIDA-I). Two AIDA-I positive strains were subjected to analysis of the nucleotide sequence of the complete orfA and orfB of the AIDA gene. The AIDA-I positive E. coli isolates were also assessed for five fimbriae (F4, F5, F6, F18 and F41) by monoclonal antibodies and for toxin genes (STa, STb, LT, EAST1, Stx2e) by PCR. Twenty-one (6.5%) of the isolates possessed AIDA-I genes. Of these isolates, two carried AIDA-I genes as the only demonstrated virulence factors, and the remaining isolates carried other virulence factor genes. Comparing the AIDA-I sequence from porcine and human sources, a high homology of orfA both in porcine E. coli and human E. coli was observed. However, each orfB of the two porcine E. coli isolates was 3864 nucleotides long compared with 3861 for the E. coli 2787 orfB, and showed 96.5% homology to E. coli 2787. The data indicated (1) that AIDA-I may be an occasional virulence factor in post-weaning diarrhoea and oedema disease in pigs, (2) that it has the potential to transfer between porcine and human E. coli, and (3) that there is a genetic diversity in orfB between human and porcine E. coli.     

Analysis of Transfer Modes of 16S rRNA Methylase in Chicken Intestinal E.coli

To study the transfer mode of rmtB and armA in chicken intestinal E.coli,plasmid conjugation experiment was carried out to study the horizontal transfer mode of rmtB and armA genes, and conjugative transfer frequencies were calculated. Broth microdilution method was applied to test susceptibility of 16S rRNA emthylase-positive isolates and their transconjugants to antimicrobial agents. As a result, all the rmtB genes located in five rmtB-positive isolates were easily transferred to recipients of E.coli C600. Conjugative transfer frequencies varied from 4.30×10^-10 to 5.5×10^-6 per recipient cell, and the armA gene located in one armA-positive isolate was not easily transferred to the recipient of E.coli C600. The results suggested that horizontal transfer was responsible for the dissemination of rmtB gene in chicken intestinal E.coli, but not for armA.     

Distribution of lipopolysaccharide core types among avian pathogenic Escherichia coli in relation to the major phylogenetic groups

Five distinct lipopolysaccharide (LPS) core types, namely R1-R4 and K12 have been identified in Escherichia coli. The aims of this study were to determine, primarily by means of PCR, the distribution of those oligosaccharide core types among avian pathogenic E. coli and their relationship to phylogenetic groups. To identify putative avian pathogenic E. coli, serum resistance and the presence of three virulence genes encoding temperature sensitive haemagglutinin (tsh), increased serum survival (iss) and colicin V (cvaC) were determined. Of the 143 clinical isolates examined 62% possessed the R1 core, 22% were R3, 13% were R4 and 3% were R2. Fifty commensal isolates consisted of 58% with R1 core, 38% with R3 core, 4% with R4 core, and none with R2. None of the isolates were of K12 core type. The distribution of core oligosaccharide types in clinical and commensal isolates were not statistically significant (P=0.51). Three genes, tsh, iss and cvaC were found in E. coli of all four core types. The genes tsh (P<0.001) and iss (P=0.03412) were significantly associated with the R4 core oligosaccharide type. The isolates containing R4 core type LPS were mainly confined to phylogenetic group D. The widespread R1 core type showed less ability to possess virulence genes and 83% were in the phylogenetic group A. Results of this study indicated that E. coli with R1, R2, R3 and R4 were important in causing infections in chickens and further, the E. coli with R4 core type were less common among commensals, possessed more virulence genes and were related to phylogenetic groups pathogenic for poultry.     

Susceptibility of sows to experimentally induced Escherichia coli mastitis

A study was conducted to compare susceptibility of sows from 2 herds to experimentally induced Escherichia coli mastitis. Four sows each from herds R and S were inoculated intramammarily at postpartum hour 8 with a strain of E coli shown previously to be capable of producing mastitis. After inoculation with E coli, sows from herd S had higher temperatures, lower WBC counts, and lower plasma protein:fibrinogen ratios than did sows from herd R. Inoculated sows from herd S lost 83% of newborn pigs due to starvation by 14 days after inoculation, whereas sows from herd R lost none. Control, noninoculated sows from both herds had normal temperatures, normal hematologic values, and minimal mortality of piglets. Levels of antibodies (complement fixing, enzyme-linked immunosorbent assay, and agglutinating) to E coli in preinoculation sera from the 2 populations of sows did not differ. Assay of lactoferrin by radial immunoassay revealed comparable concentrations in milk of sows from both herds during the first 24 hours after sows had delivered, but significantly higher values were detected in milk from sows of herd S at postpartum days 2 and 3. The basis for the marked difference in susceptibility to E coli-induced mastitis was not determined except that "susceptible" sows (herd S) were from a conventional herd and "resistant" sows (herd R) were from a specific-pathogen-free herd.     

Effects of Tryptophan Analogs, Mitomycin c and Acridine Orange on the Production of Filtrable Hemolysin by Escherichia coli

Bacteriostatic doses of 5-methyltryptophan and of 7-azatryptophan exert a complete inhibition on the in vitro production of filtrable hemolysin by Escherichia coli. This inhibition is readily overcome by L-tryptophan, and does not seem to be specific but secondary to an interference with de novo protein synthesis and cell multiplication as is observed with chloramphenicol in sensitive strains. Although the influence of the two tryptophan analogs on hemolysin production and cell multiplication is similar, their mechanism of action at the molecular level appears to be different.

The addition of 50 µg/ml 7-azatryptophan which causes an unbalanced growth characterized by an arrest of the cellular division and an increase of cell size, blocks the production of an active hemolysin. Exposure to 4 µg/ml 5-methyltryptophan also prevents cell multiplication and hemolysin production but no sign of unbalanced growth is evident. Mitomycin C in concentrations sufficient to prevent increase in the number of viable units provokes an extreme elongation of E. coli cells and, apparently, does not stop the synthesis of hemolysin. In blocking the production of hemolysin the three inhibitors of protein synthesis used in this study were more effective than mitomycin C an agent known to affect deoxyribonucleic acid synthesis and to induce extrachromosomic genetic factors. Results of conjugation experiments also described here support the finding of other workers that the genetic factor that controls the production of the filtrable hemolysin in E. coli can be transmitted by conjugation. Acridine orange eliminated the hemolytic property from a large proportion of the population of a hemolytic strain which did not carry the R factor, but was little effective in the strains which had received both the R factor and the hemolytic character by conjugation.

     

Effect of endocytic and metabolic inhibitors on the internalization and intracellular growth of Brucella abortus in Vero cells.

Uptake, transfer to rough endoplasmic reticulum, and intracellular growth of Brucella abortus were studied in Vero cells treated with endocytic and metabolic inhibitors. Infection of Vero cells was suppressed when inhibitors of energy metabolism (iodoacetate, dinitrophenol), receptor-mediated endocytosis (monodansylcadaverine, amantadine, methylamine), or endosomal acidification (chloroquine, ammonium chloride, monensin) were added to the inoculum. Inhibition was not observed when these drugs were added after the inoculation period. Infection of Vero cells by B abortus was inhibited by dibutyryl-cyclic adenosine monophosphate and Vibrio cholerae enterotoxin, but was stimulated by dibutyryl-cyclic guanosine monophosphate and escherichia coli heat-stable enterotoxin a. Uptake of B abortus by Vero cells was not prevented by colchicine, but was abolished by cytochalasin B. Uptake of heat-killed B abortus and noninvasive E coli was similar to that of viable brucellae. Intracellular growth of B abortus was not affected by cycloheximide. Results indicate that: B abortus may be internalized by a receptor-mediated phagocytic process; transfer of B abortus from phagosomes to rough endoplasmic reticulum may require endosomal acidification; and replication of B abortus within the rough endoplasmic reticulum may not depend on protein synthesis by the host cell.     

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.     

Location of increased serum survival gene and selected virulence traits on a conjugative R plasmid in an avian Escherichia coli isolate

Avian colibacillosis is a costly disease for the poultry industry. The mechanisms of virulence employed by the etiologic agent of this disease remain ill defined. However, accumulated evidence suggests that complement resistance and the presence of the increased serum survival gene (iss) in an avian Escherichia coli isolate may be indicative of its ability to cause disease. This association of iss with the E. coli implicated in avian disease may mean that iss and/or, perhaps, the genes associated with it are important contributors to avian E. coli virulence. For this reason, we have begun a search for iss's location in the bacterial genome. Thus far, iss in an avian E coli isolate has been localized to a conjugative R plasmid and estimated to be about 100 kilobase (kb) in size, encoding resistance to tetracycline and ampicillin. Hybridization studies have revealed that this plasmid contains sequences with homology to tsh, a gene associated with virulence of avian E coli; intI 1, a gene encoding the integrase of Class 1 integrons; and certain genes of the aerobactin- and CoIV-encoding operons. Sequences homologous to merA, a gene of the mercury resistance operon, were not identified on this R plasmid. This plasmid, when transferred into an avirulent, recipient strain by conjugation, enhanced the transconjugant's resistance to complement but not its virulence, in spite of the plasmid's possession of several putative virulence genes and traits. Such results may reflect the multifactorial nature of virulence, the degree of the recipient's impairment for virulence, or an inability of the embryo assay used here to detect this plasmid's contribution to virulence. Additionally, this plasmid contains genes encoding antimicrobial resistances, which may provide a selective advantage to virulent E. coli in the production environment. Further study will be needed to determine whether this plasmid is widespread among virulent E. coli and to ascertain the implications that this link between virulence and antimicrobial resistance genes may have for poultry management.     

Antimicrobial resistance in commensal faecal Escherichia coli of hospitalised horses

The objective of this study was to examine the impact of hospitalisation and antimicrobial drug administration on the prevalence of resistance in commensal faecal E. coli of horses. Faecal samples were collected from ten hospitalised horses treated with antimicrobials, ten hospitalised horses not treated with antimicrobials and nine non-hospitalised horses over a consecutive five day period and susceptibility testing was performed on isolated E. coli. Results revealed that hospitalisation alone was associated with increased prevalence of antimicrobial resistance and multidrug resistance in commensal E. coli of horses. Due to the risk of transfer of resistance between commensal and pathogenic bacteria, veterinarians need to be aware of possible resistance in commensal bacteria when treating hospitalised horses.     

Effect of chlortetracycline on the spread of R-100 plasmid-containing Escherichia coli BEL15R from experimentally infected pigs to uninfected pigs and chicks

Swine from two herds with different histories of antibiotic exposure were fed diets containing 0 or 55 mg of chlortetracycline (CTC)/kg. One of five pigs in each herd-diet treatment group was infected orally with Escherichia coli strain BEL15R that was resistant to nalidixic acid (NA), chloramphenicol (C), streptomycin (S), sulfamethizole (TH) and tetracycline (TE). Effects of CTC on the quantity and duration of fecal shedding of E. coli BEL15R and on the transmission of strain BEL15R and its R-100 plasmid from infected pigs to uninfected pigs and chicks were determined. Quantity and duration of shedding were greater in infected antibiotic-herd pigs than in infected nonantibiotic-herd pigs. Feeding on CTC increased the duration of shedding in infected pigs from both herds. Strain BEL15R colonized and was shed in one uninfected antibiotic pig in each treatment group, but it did not colonize in any of the uninfected nonantibiotic-herd pigs or in the uninfected chicks. In vivo transfer of resistance to C, S, TH and TE occurred in the infected antibiotic-herd pigs but not in the infected nonantibiotic-herd pigs. Transfer of the R-100 plasmid occurred from the infected to the uninfected antibiotic-herd pigs and to the uninfected chicks housed near the antibiotic-herd pigs fed CTC, but not to the chicks housed with the antibiotic-herd pigs fed the control diet. No transfer of resistance occurred from the infected nonantibiotic-herd pigs fed either CTC or control diet.     

The age-dependent expression of the F18+ E. coli receptor on porcine gut epithelial cells is positively correlated with the presence of histo-blood group antigens

F18(+)Escherichia coli have the ability to colonize the gut and cause oedema disease or post-weaning diarrhoea by adhering to specific F18 receptors (F18R) on the porcine epithelium. Although it is well established that a DNA polymorphism on base pair 307 of the FUT1 gene, encoding an alpha(1,2)fucosyltransferase, accounts for the F18R phenotype, the F18R nature is not elucidated yet. The aim of the present study was to investigate the correlation between the presence of H-2 histo-blood group antigens (HBGAs) or its derivative A-2 HBGAs on the porcine gut epithelium and F18(+)E. coli adherence. A significant positive correlation was found between expression of both the H-2 (r=0.586, P<0.01) and A-2 (r=0.775, P<0.01) HBGAs and F18(+)E. coli adherence after examination of 74 pigs aged from 0 to 23 weeks. The majority of the genetically resistant pigs (FUT1M307(A/A)) showed no HBGA expression (91.7%) and no F18(+)E. coli adherence (83.3%). In addition, it was found that F18R expression levels rise with increasing age during the first 3 weeks after birth and that F18R expression is maintained in older pigs (3-23 weeks old). Taken together, these data suggest that, apart from H-2 HBGAs, A-2 HBGAs might be involved in F18(+)E. coli adherence.     

Influence of oral antibiotics on resistance and enterotoxigenicity of Escherichia coli.

Three groups of five piglets were formed and 1390 Escherichia coli isolates were obtained during the 45-day period of observation. One of the groups received feed without antibiotic whereas the second received feed containing 100 ppm neomycin and the third feed with 100 ppm neomycin plus 100 ppm tetracycline. Rectal swabbings for bacterial isolation were repeated ten times, twice during an adaptation period and eight times during the treatment period. Resistance among the isolates to tetracycline, streptomycin and triple sulfas remained high throughout this experiment whereas resistance to neomycin, chloramphenicol and ampicillin were found to increase significantly under the influence of antibiotic supplemented feed. This increase of antibiotic resistance was associated with an increase of the percentage of isolates harboring an R. factor. When comparing the ability of strains harboring an R factor to receive the plasmid Ent from the E. coli K12 (P155) with isolates not harboring such a plasmid, no significant difference was observed in their ability to receive the Ent plasmid.     

Comparison of antimicrobial resistant Escherichia coli in wild and captive Japanese serows.

The fecal Escherichia coli isolated from wild Japanese serows living in mountainous areas away from humans and those from captive serows kept in human areas were examined for antimicrobial resistance and the possession of transferable R plasmids. Of 874 E. coli strains isolated from 283 wild serows in 1980-1981, only 11 (1.3%) were resistant to at least one of 6 antimicrobial drugs; ampicillin, streptomycin, tetracycline, chloramphenicol, kanamycin and sulfadimethoxin. Seven (2.5%) individuals were found to carry resistant E. coli. To heighten the isolation frequency of drug-resistant strains, fecal samples of 244 wild serows in 1983-1984 were cultured directly onto drug-supplemented media. Only 12 (4.9%) serows were shown to have drug-resistant E. coli. No transferable R plasmid was detected among a total of 87 resistant strains from wild serows. In contrast, all 33 captive serows except one which was kept only one day after capture, showed resistant E. coli and 20 (60.6%) serows were excreting R plasmid-carrying E. coli. Of 161 drug-resistant strains from captive serows, 50 (31.1%) were found to carry R plasmids. Wild serows seemed to readily change to harbor resistant E. coli almost as soon they were reared in human areas without direct exposure to drugs. These results lead to the conclusion that drug-resistant E. coli can probably be used as microbial indicator for natural environmental pollution.