The in vitro efficacy of a quaternary ammonia disinfectant and/or ethylenediaminetetraacetic acid-tris against commercial broiler hatchery isolates of Pseudomonas aeruginosa.

Studies have indicated variations in the degree of efficacy of certain commercial disinfectants used in poultry production facilities. We used an adequate method of in vitro testing to compare the efficacy of disinfectants while testing them in conditions similar to those of the poultry facilities. BioSentry 904, ethylenediaminetetracetic acid (EDTA)-Tris, and a combination of the two were tested by this method against five field isolates of Pseudomonas aeruginosa at 10(3), 10(6), and 10(9) colony-forming units (CFU)/ml. At the 10(9) CFU/ml concentration, most compounds failed to achieve a total kill with a contact time of 15 min. When tested at bacterial concentrations of 10(3) CFU/ml, the combination of EDTA-Tris mixed at a 1:1 ratio with BioSentry 904 killed the bacteria upon initial contact (< or = 0.05 min). This disinfectant mixture exhibited antagonistic, indifferent, or synergetic effects when exposed to different bacterial isolates at a concentration of 10(6) CFU/ml.

In vitro inhibition of Salmonella organisms isolated from reptiles by an inactivated culture of microcin-producing Escherichia coli.

OBJECTIVE: To determine whether an inactivated culture of a microcin-producing avian Escherichia coli was capable of killing Salmonella isolates from reptiles in an in vitro test system. SAMPLE POPULATION: 57 Salmonella isolate from reptiles. PROCEDURE: A wild-type avian E. coli electrotransformed with a plasmid coding for the production of microcin 24 was tested in an in vitro microassay system for its ability to kill 57 Salmonella spp isolated from reptiles. The reptile population included snakes, iguana, frilled lizards, turtles, other lizards, and unspecified reptiles. RESULTS: 44 of the Salmonella isolates were inhibited strongly, compared with the in vitro assay controls; 12 had weak inhibition, and 1 was not inhibited by the microcin-producing E. coli. Thirteen of the 57 isolates had resistance to at least 1 antibiotic, primarily streptomycin. There were 9 O serogroups identified in the 57 isolates, with serogroup H being the most prevalent (18 to 57). CONCLUSION AND CLINICAL RELEVANCE: Antibiotics are not recommended to eliminate Salmonella organisms from reptiles because of the development of antibiotic resistance. Further studies are necessary to determine whether the use of microcin-producing bacteria will be effective in controlling Salmonella infections in companion reptiles.

Chicken embryo lethality assay for determining the virulence of avian Escherichia coli isolates.

Multiple isolates of Escherichia coli from clinical cases of colibacillosis and E. coli from the intestinal tracts of normal broilers at slaughter were assayed by the embryo lethality test to determine their virulence. The assay was repeated five times in order to establish reproducibility and determine the statistical parameters of the test. This study showed that the inoculation of approximately 100 colony-forming units in the allantoic cavity of 12-day-old embryos discriminated between virulent and avirulent E. coli isolates. Gross lesions included cranial and skin hemorrhages in addition to encephalomalacia in embryos inoculated with virulent isolates. Abnormalities were observed by microscopic examination of the heart, brain, and liver in embryos inoculated with virulent isolates. Analysis of data indicated that the length of the test should be 4 days. In the virulent group, day 2 postinoculation had the most significant death patterns. Sample size calculations indicated that 11 embryos are sufficient for the assay. On the basis of death rates, isolates considered to be avirulent had an embryo death rate of <10%, moderately or secondary pathogens had a 10%-29% death rate, and virulent isolates had a death rate of >29%. An important aspect of this assay is the accessibility of good-quality fertile embryonated eggs.

In vitro effect of ethylenediaminetetraacetic acid-Tris on the efficacy of hatchery disinfectants.

Solutions of ethylenediaminetetraacetic acid (EDTA)-Tris showed synergistic or additive effects on gram-negative bacteria when combined with hatchery disinfectants consisting of phenol and detergent (Magnaphen-100), quaternary ammonium compound (QAC) and glutaraldehyde (Synergize), QAC (BioSentry 904), and hydrogen peroxide. The gram-positive bacteria reacted less favorably, with reaction mixtures showing all three levels of potentiation (synergistie, additive, and antagonistic). Combinations of EDTA-Tris and a commercial glutaraldehyde solution (Glutracide), when mixed with the test organisms, showed mostly antagonistic effects. Solutions of EDTA-Tris decreased the concentration of hatchery disinfectants required for bacterial killing by 75% in those situations in which synergistic potentiation occurred. EDTA-Tris is nontoxic to 12-day-old embryos. Serial passage of the test organisms in solutions of EDTA-Tris did not result in the development of resistant forms.

Inhibition of Salmonella typhimurium in the chicken intestinal tract by a transformed avirulent avian Escherichia coli.

An avirulent, wild-type avian Escherichia coli (E. coli Av) was electrotransformed with a plasmid coding for the production of microcin 24 (pGOB18) and was designated E. coli AvGOB18. The transformant inhibited the growth of seven serotypes of Salmonella commonly associated with colonization and contamination of poultry products and seven strains of E. coli O157:H7 in the in vitro colicin/microcin assay. The transformant did not inhibit the replication of multiple isolates of Listeria monocytogenes or Campylobacter jejuni in similar assays. The transformant is nonconjugative, indicating that the plasmid would not be transmitted to other intestinal microflora in the environment. The transformant also survived in sterile tap and deionized water incubated at 25 C and 37 C in the laboratory for 30 days and was recovered from drinkers and birds in in vivo floor pen studies. In in vivo studies, E. coli AvGOB18 did not colonize the intestinal tract of broiler chicks when given as a single or multiple dose and did not reduce the Salmonella load in the broilers. But Salmonella typhimurium was reduced significantly in the intestinal tracts of broiler chickens when E. coli AvGOB18 was administered continually in the water supply.

Mutation rate of avian intestinal coliform bacteria when pressured with fluoroquinolones.

The purpose of this study was to determine the rate at which resistance developed in avian coliform bacteria when exposed to nalidixic acid, sarafloxacin, or enrofloxacin. In in vitro studies, the rates of mutation of avian isolates of Escherichia coli and Salmonella were determined following nalidixic acid, sarafloxacin, or enrofloxacin pressure. The rates of mutation were similar for nalidixic acid and sarafloxacin, whereas a lower rate of mutation was seen after enrofloxacin pressure. In in vivo studies, the quinolones were administered in the drinking water to broiler chickens at a concentration of 40 ppm for five consecutive days. Samples of feces were inoculated onto appropriate media and the frequency of resistance was determined. The frequency rates of resistance to nalidixic acid and sarafloxacin were similar. Enrofloxacin-medicated birds did not develop enrofloxacin-resistant coliform bacteria. The in vitro and in vivo data appear to correlate.

Colonization of the chicken trachea by an avirulent avian Escherichia coli transformed with plasmid pHK11.

Recombinant plasmid pHK11 was transformed into an avirulent, wild-type avian Escherichia coli (E. coli Av) in order to study the plasmid's effect on colonization of the chicken trachea. The transformant (E. coli Av + pHK11) produced colicin V (ColV), had type F1 fimbriae, and was motile. The E. coli Av recipient possessed type F1 fimbriae but was nonmotile; it did not produce ColV. Four-day-old chicks were inoculated in the trachea with 100 microliters of an overnight culture (approximately 10(8) colony-forming units) of E. coli Av, E. coli Av + pHK11, or sterile brain-heart infusion (BHI) broth. A group of uninoculated chicks was also included. Samples of the trachea were taken on days 4 and 10 postinoculation and compared histologically and bacteriologically. Birds inoculated with E. coli Av + pHK11 had enhanced tracheal colonization and showed increased histologic changes as compared with those inoculated with E. coli Av or BHI broth or uninoculated controls. These results indicate that production of ColV and motility enhance the colonization of the trachea and may be involved in the cause of pathologic lesions.

Analysis of plasmids cloned from a virulent avian Escherichia coli and transformed into Escherichia coli DH5 alpha.

Three of four plasmids from a virulent wild-type avian Escherichia coli were cloned or transformed into an avirulent laboratory recipient E. coli DH5 alpha and tested for the ability to confer a virulence phenotype. The three plasmids transformed into E. coli DH5 alpha were 5, 6, and 56 kb. A fourth plasmid of 64 kb was not successfully transformed. Parameters used to measure virulence included presence of type 1 pili and a smooth lipopolysaccharide (LPS) layer, motility, production of Colicin V, resistance to host complement, and embryo lethality. The 5-kb plasmid encoded for ampicillin resistance, whereas the 6-kb plasmid encoded for tetracycline resistance. The 56-kb plasmid encoded for streptomycin, sulfisoxazole, and tetracycline resistance. Twelve-day embryos inoculated with 467 colony-forming units of E. coli DH5 alpha containing the 56-kb plasmid had increased death rates (45%) in the embryo lethality assay and a decreased weight of surviving embryos with cranial hemorrhages as compared with embryos inoculated with similar amounts of E. coli DH5 alpha (0%) and phosphate-buffered saline (0%). Embryos inoculated with the wild-type virulent E. coli had 90% deaths. The 56-kb plasmid also had homology with a probe for Colicin V production (cvaC). No differences in LPS layer, complement resistance, motility, Colicin V activity, type 1 pili, cell-free supernatant proteins, or outer membrane proteins were observed in the transformants when compared with nontransformed E. coli DH5 alpha.

The effect of plasmid acquisition on potential virulence attributes of Pasteurella multocida.

Several plasmids that were isolated from complement-resistant Pasteurella multocida or Escherichia coli were evaluated for phenotypic markers. Plasmid p2267, isolated from a tetracycline-resistant, complement-resistant fowl cholera field isolate of P. multocida (PM2267), was used to transform a K-12 E. coli (C600); this resulted in increased complement resistance, which was eliminated by curing. Either of two plasmids (p1870 or p70-1, isolated from P. multocida and E. coli, respectively) conferred an increase in complement resistance and invasiveness to turkey epithelial cells when expressed in the Clemson University (CU) vaccine strain of P. multocida. Additionally, the CU strain containing p1870 was more virulent in turkey challenge, and the plasmid appeared amplified in vivo. No detectable differences in major outer-membrane proteins, capsule, or carbohydrate fermentation were found to be associated with the acquisition of these plasmids.

Antimicrobial effect of combinations of EDTA-Tris and amikacin or neomycin on the microorganisms associated with otitis externa in dogs.

Combinations of EDTA-Tris and two aminoglycoside antibiotics (amikacin and neomycin) were tested for synergistic activities against the microorganisms associated with otitis externa in dogs and for the solutions' stability over time. Synergistic activity was observed when EDTA-Tris plus amikacin and EDTA-Tris plus neomycin were tested against Staphylococcus intermedius, Proteus mirabilis, Pseudomonas aeruginosa, and Escherichia coli, but not against Candida albicans. Stability studies over a 3-month period indicated that the test solutions were stable at room temperature and that their antimicrobial activity was maintained.

Phenotypic expression of recombinant plasmids pKT107 and pHK11 in an avirulent avian Escherichia coli.

An avirulent wild-type avian Escherichia coli strain (Av) was electrotransformed with plasmids coding for complement resistance (pKT107) and Colicin V (ColV) production (pHK11) in order to study the effects of complement resistance and ColV production on virulence. Transformants were also compared with the wild type for embryo lethality, uptake by macrophages, motility, growth rate, plasmid content, and hemolysis. Growth rates and complement resistance patterns of strain Av and transformant Av+pHK11 were similar, but Av+pHK11 caused a significantly greater number of deaths in embryos and acquired motility. Transformant Av+pKT107 had a lower rate of phagocytosis, a slower growth rate, and a greater sensitivity to complement, and it changed from being non-hemolytic to expressing alpha-hemolytic action. The 35-kb plasmid present in the wild type was not present in the transformants. Although some of the results demonstrate the difficulties encountered in using wild-type organisms as recipients in virulence studies, the results with Av+pHK11 indicate that ColV production plus the acquisition of motility contributes to the virulence of avian E. coli.

Effect of normal intestinal flora of chickens on colonization by virulent colicin V-producing, avirulent, and mutant colicin V-producing avian Escherichia coli.

Colonization of the intestinal tracts of newly hatched chicks with Escherichia coli was attempted by swabbing test organisms onto the air-shell of 19-day-old embryos. Test organisms consisted of two virulent E. coli isolates, one avirulent isolate, and one laboratory-derived mutant of the avirulent isolate carrying a recombinant plasmid coding for Colicin V production. Chicks were cultured weekly for 3 weeks for total E. coli and for the test organisms using selective media. Control chicks were sampled on weeks 1 and 5, and the normal E. coli intestinal microflora were examined for the production of colicins. The two virulent E. coli isolates maintained colonization of the chicks for the 3-week test period, with titers decreasing from 10' to 10'- colony-forming units (CFU)/g of intestine. The avirulent isolate and laboratory mutant did not consistently colonize the intestinal tracts. The majority of intestinal samples taken from the control chicks at 1 and 5 weeks had colicin-producing E. coli that were inhibitory to the test organisms.

Non-association of macrophage phagocytosis and oxidant stimulation with complement resistance and colicin V production by avian Escherichia coli.

Avian isolates of Escherichia coli were classified as virulent based on their isolation from chickens with natural cases of colisepticemia, production of colicin V, and complement resistance. A second group of isolates was designated as avirulent based on their isolation from healthy chickens, their inability to produce colicin, and their classification as sensitive or intermediate to the action of complement. In vitro assays of phagocytosis and oxidant production were performed in an attempt to correlate these activities with the ability of each group of bacteria to escape the specific host defense mechanisms of phagocytosis and killing. Although oxidant production regressed with significant linearity on percent phagocytosis, neither group (virulent or avirulent) differed in ability to stimulate peritoneal macrophage phagocytic and oxidant activity when opsonized with normal chicken serum. These results differ from those in mammalian species.

Characterization of an avirulent mutant of a virulent avian Escherichia coli isolate.

A virulent, complement-resistant avian Escherichia coli isolate and its avirulent, complement-sensitive, transposon-insertion mutant were compared for the purpose of revealing structures associated with complement resistance. Both had a smooth lipopolysaccharide layer, contained traT, and lacked a capsule, but the mutant possessed a 16.2-kilodalton outer-membrane protein (OMP) not present in the wild-type. This protein may be the product of a coding region interrupted by transposon insertion. Such results suggest that an OMP greater than 16.2 kilodaltons in size may be responsible for the complement resistance and virulence of this wild-type E. coli.

Invasion of epithelial cell monolayers by turkey strains of Pasteurella multocida.

Two serotype 3,4:A strains of Pasteurella multocida that differ in virulence in turkeys were examined for their ability to invade epithelial cell monolayers grown in tissue culture. Both organisms were comparably adherent to cells of turkey kidney origin. However, the virulent strain (86-1913) penetrated primary turkey kidney epithelial cell monolayers at 10 times the level of the low-virulence vaccine strain. The virulent strain was also able to invade porcine epithelial cells (PK15) and feline epithelial cells (CRFK) in cell culture. Neither organism invaded rabbit epithelial cells (RK13). Invasion of turkey cells was prevented by inhibition of bacterial protein or RNA synthesis but not by pretreatment of the monolayers with periodate, trypsin, or neuraminidase. Invasion might be a mechanism of pathogenicity for this organism, contributing to colonization or virulence.

Association of K-1 capsule, smooth lipopolysaccharides, traT gene, and Colicin V production with complement resistance and virulence of avian Escherichia coli.

A group of complement-resistant, virulent avian Escherichia coli isolates were compared with a group of complement-sensitive, avirulent avian isolates for the presence of K-1 capsule, smooth lipopolysaccharides (LPS), the traT gene, and Colicin V (ColV) production. These parameters were selected because of their reported association with complement resistance and virulence in E. coli. Lethality in chicken embryos has also been shown to be correlated with virulence of avian E. coli for chickens. The complement-resistant, virulent E. coli isolates did not possess a K-1 capsule. Production of ColV and the presence of smooth LPS were significantly correlated with embryo lethality. There was no correlation between the presence of traT and embryo lethality. These results suggest that complement resistance and virulence in avian E. coli are associated with ColV production and smooth LPS but not with K-1 antigen or traT.

Failure of the Congo red dye uptake test to discriminate between virulent and avirulent avian Escherichia coli.

Twenty avian Escherichia coli isolates from normal and diseased chickens were compared by use of three virulence tests. These tests included the uptake of Congo red dye, an embryo lethality test, and a quantitative microtiter complement resistance test. A direct correlation was seen between the results of the complement resistance test and the embryo lethality test. The results of the Congo red test did not correlate with the two other tests.

Relationship of complement resistance and selected virulence factors in pathogenic avian Escherichia coli.

Complement resistance, antibiotic resistance profiles, and virulence profiles of 80 Escherichia coli isolates from the intestines of normal chickens (40 isolates) and chickens diagnosed as having colisepticemia (40 isolates) were compared. Differences were observed between the two groups for antibiotic resistance, siderophore production, presence of type 1 pili, complement resistance, motility, and size of plasmids. The systemic isolates were more likely to have siderophores and type 1 pili, and to be complement-resistant and motile than were the intestinal isolates. No differences between the two groups were observed for colicin production. Further comparison of the 10 most complement-resistant isolates from the systemic group and 10 most complement-sensitive isolates from the intestinal group revealed a correlation between an isolate's resistance to complement and its ability to kill embryos, express type 1 pili, and be motile. Virulence of avian E. coli strains appears to be correlated with complement resistance and the interaction of this resistance with the ability to produce type 1 pili and be motile.

Factors affecting endotoxin release from the cell surface of avian strains of Pasteurella multocida.

Two avian strains of Pasteurella multocida, a vaccine strain and a virulent field isolate, were investigated to determine their propensity to release endotoxin from the cell surface. Both organisms released comparable amounts of endotoxin when plasma complement proteins were present, however the virulent strain did so without the loss of viability that occurred in the vaccine strain. Blocking complement activity decreased the ability of plasma to elicit endotoxin release from the bacteria. When the cells were treated with divalent metal chelators such as trans-1, 2-diaminocyclohexane-N,N,N1,N1-tetraacetic acid (CDTA), more endotoxin was released from the vaccine strain than from the virulent isolate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of purified lipopolysaccharide (LPS) from both strains revealed virtually identical patterns. Both had patterns considered typical of rough LPS. Challenge studies in 8 weeks old turkeys showed that the field strain induced endotoxemia of longer duration than the vaccine strain and produced greater mortality.

Transposon mutagenesis used to study the role of complement resistance in the virulence of an avian Escherichia coli isolate.

The role of complement resistance in the virulence of an avian Escherichia coli isolate was examined with transposon mutagenesis. A suicide plasmid containing a kanamycin-encoding mini-transposon was used to transform a virulent complement-resistant avian E. coli isolate. A less resistant mutant was identified that contained a transposon insertion in a plasmid and in the chromosome. This loss of complement resistance was associated with a drop in virulence in an embryo assay. No other phenotypic changes were detected in the mutant. These results suggest that complement resistance is associated with the virulence of this organism.