Antimicrobial resistance of gram-negative enteric bacteria from pigs in a nonantimicrobial-exposed herd before and after transportation.

Loading pigs onto trucks and transporting them for 30 min resulted in a significant increase in proportion of antimicrobial resistance of gram-negative enteric bacteria in fecal material. Similarly, the mean number of antimicrobial agents in the resistance patterns of these bacteria increased during loading and transportation. However, the increases were of a transient nature, as resistance values were similar to those of a nontransported control group 1 day after the pigs had been transported.

Cytolethal distending toxin genes in Campylobacter jejuni and Campylobacter coli isolates: detection and analysis by PCR.

Campylobacter jejuni produces a toxin called cytolethal distending toxin (CDT). Knowledge of the prevalence and homogeneity of Campylobacter sp. cdt genes is incomplete. In this work, we identified four PCR primer pairs that collectively amplified cdt genes in all of the C. jejuni and Campylobacter coli strains tested. Restriction analyses of the cdt PCR products showed clear differences between the cdt genes of these two species, yet there were few heterogeneities noted between members of the same species. Consequently, it may be possible to speciate C. jejuni and C. coli isolates on the basis of restriction patterns within their cdt genes.

Detection of cytolethal distending toxin activity and cdt genes in Campylobacter spp. isolated from chicken carcasses.

This study was designed to determine whether isolates from chicken carcasses, the primary source of Campylobacter jejuni and Campylobacter coli in human infections, commonly carry the cdt genes and also whether active cytolethal distending toxin (CDT) is produced by these isolates. Campylobacter spp. were isolated from all 91 fresh chicken carcasses purchased from local supermarkets. Campylobacter spp. were identified on the basis of both biochemical and PCR tests. Of the 105 isolates, 70 (67%) were identified as C. jejuni, and 35 (33%) were identified as C. coli. PCR tests amplified portions of the cdt genes from all 105 isolates. Restriction analysis of PCR products indicated that there appeared to be species-specific differences between the C. jejuni and C. coli cdt genes, but that the restriction patterns of the cdt genes within strains of the same species were almost invariant. Quantitation of active CDT levels produced by the isolates indicated that all C. jejuni strains except four (94%) had mean CDT titers greater than 100. Only one C. jejuni strain appeared to produce no active CDT. C. coli isolates produced little or no toxin. These results confirm the high rate of Campylobacter sp. contamination of fresh chicken carcasses and indicate that cdt genes may be universally present in C. jejuni and C. coli isolates from chicken carcasses.

Microbial populations of Botrytis cinerea-inoculated strawberry fruit exposed to four volatile compounds.

Aerobic, microaerophilic, coliform, and mold populations of Botrytis cinerea-inoculated strawberry fruit not exposed (control) or exposed to low and high quantities of four volatile compounds during storage at 2 degrees C were determined after storage for 7 days and after removal of the volatile and transfer to 22 degrees C for 3 days. Fruit harvested at the ripe stage were inoculated with 10(6) conidia B. cinerea per ml and were placed in plastic containers containing no volatile compound (control) or two quantities of (E)-2-hexenal (10 or 100 microliters), (E)-2-hexenal diethyl acetal (30 or 300 microliters), benzaldehyde (30 or 300 microliters), or methyl benzoate (12 or 60 microliters). The fruit containers were overwrapped with a low-density polyethylene film, sealed, stored at 2 degrees C for 7 days, and then transferred to 22 degrees C for 3 days. Aerobic, microaerophilic, and coliform populations of fruit exposed to volatile compounds tended to be lower than the controls after storage at 2 degrees C for 7 days and, depending on the volatile compound, similar, lower, or higher than the controls after transfer and storage at 22 degrees C. However, due to variability in initial aerobic, microaerophilic, and coliform populations of the fruit used in the different trials (P < 0.05), none of the differences between control and treatment and between treatments within a sample time were significant (P > 0.05). Strawberry fruit exposed to 100 microliters of (E)-2-hexenal was the only treatment that did not show a significant increase in mold populations after transfer and storage at 22 degrees C for 3 days. Additional studies are needed to determine if (E)-2-hexenal can be used in combination with other postharvest storage conditions, such as low temperature and controlled/modified atmosphere, to delay mold spoilage and extend the shelf life of the strawberry.

Lecithinase reaction of Staphylococcus aureus strains of different origin on Baird-Parker medium.

Staphylococcus aureus produces one or more enzymes with lipolytic activity, but differences between strains have been reported (Owens and John 1975; O'Toole 1987; Rollof et al. 1987). The biological and biochemical properties of these enzymes have been investigated and results were recently reviewed (Kötting et al. 1984). Baird-Parker medium (Baird-Parker 1962) is a selective medium commonly used for the isolation of Staph. aureus. The presence of egg yolk in this medium permits the detection of two reactions due to lipolytic activity of staphylococci: (1) Lecithinase reaction, a zone of precipitate in the medium surrounding the colonies; and (2) Lipase reaction or 'pearly layer', an iridescent film in and immediately surrounding colonies, visible by reflected light (iridescent sheen or 'oil in water'). In this study, human and bovine strains, previously biotyped according to the scheme of Devriese et al. (1984), were compared for production of a zone of precipitation, lecithinase reaction, on Baird-Parker medium. Bovine and human strains of Staph. aureus were compared for production of the egg yolk reaction (lecithinase reaction) on Baird-Parker medium and the results were related to their biotypes and site of origin of the sample. Human strains and strains biotyped as human biotypes had higher percentage of positive results than bovine isolates and/or biotypes. However, all strains isolated from body sites of heifers produced a positive reaction regardless of the biotype.

Detection of tetracycline resistance determinants in pig isolates from three herds with different histories of antimicrobial agent exposure.

A total of 114 gram-negative fecal isolates from domestic pigs in herds with different histories of antimicrobial agent exposure were screened for the presence of plasmid DNA and specific tetracycline resistance determinants. More than 84% of the isolates harbored plasmid DNA, which ranged in size from 2.1 to 186 kb. A total of 78 isolates (68.4%) were resistant to tetracycline at concentrations greater than 4 micrograms/ml. Plasmid DNAs from about 56% of the tetracycline-resistant isolates hybridized with DNA probes for class A, B, C, and D tetracycline resistance determinants. The class B determinant was the most common determinant (35% of the isolates), followed by the class C determinant (12%) and the class A determinant (1%). About 9% of the isolates contained two determinants on plasmids. None of the plasmids from isolates hybridized with the class D determinant probe. The class C determinant was the most prevalent determinant on plasmids in isolates from pigs not exposed to antimicrobial agents for more than 146 months, while the class B determinant was more prevalent on plasmids in isolates from pigs exposed to either subtherapeutic or therapeutic levels of antimicrobial agents. Most tetracycline resistance determinants were localized on plasmids which were more than 30 kb long. A great number of wild-type tetracycline-resistant Escherichia coli strains were found with the class E determinant on their chromosomes. This study revealed a high prevalence of tetracycline resistance determinants in the fecal flora of pig herds whether or not they were fed with antibiotics.

Beta-glucuronidase activities of fecal isolates from healthy swine.

Research has shown that various percentages of fecal Escherichia coli isolates obtained from healthy subjects may be beta-glucuronidase negative. The ability to detect beta-glucuronidase activity among fecal E. coli isolates from healthy subjects may be affected by assay conditions. A study was conducted in which agar and broth media containing 4-methylumbelliferyl-beta-D-glucuronide (MUG) were used to examine beta-glucuronidase activities of fecal isolates from healthy swine. Rectal swabs were plated on MacConkey agar plus 100 mg of MUG per liter (MAC-MUG) and incubated at 35 degrees C for 24 h. Each of 986 isolates picked from MAC-MUG was inoculated into duplicate tubes of lauryl tryptose broth plus 50 mg of MUG per liter (LT-MUG). One set of tubes was incubated at 35 degrees C and the other set of tubes was incubated at 44.5 degrees C. Gas production and hydrolysis of MUG, indicated by fluorescence when observed with UV light with a wavelength of 360 nm, were determined after incubation for 24 and 48 h. A higher percentage (P less than 0.01) of isolates was MUG positive at 44.5 degrees C than at 35 degrees C after 24 h of incubation in LT-MUG. A higher percentage (P less than 0.01) of isolates was MUG positive after 48 h than after 24 h of incubation at both 35 and 44.5 degrees C. A lower percentage of isolates (P less than 0.05) was observed to be MUG positive on MAC-MUG agar compared with their MUG reactions in LT-MUG at 35 and 44.5 degrees C. Approximately 89% of the isolates identified were beta-glucuronidase-positive E. coli. The largest proportion of MUG-positive E. coli was detected with LT-MUG at 35 degrees C after 48 h of incubation.

Prevalence of Staphylococcus species during the periparturient period in primiparous and multiparous cows.

During a 14-mo period, 77 multiparous and 36 primiparous cows were sampled to determine the prevalence of staphylococci during the periparturient period. Distal streak canal swabs were taken at 14 d prepartum, and foremilk was sampled the first 5 consecutive wk of lactation. Staphylococcus aureus was isolated from 7.6% of quarters of primiparous cows but from only .6% of quarters of multiparous cows at parturition. Prevalence in primiparous cows declined to 3.5% by the wk-1 sampling. Quarter prevalence of coagulase-negative Staphylococcus species prepartum, at parturition, and wk 1 to 5 in primiparous cows was 38.9, 27.8, 15.3, 14.6, 13.2, 15.3, and 14.6%, respectively. In multiparous cows, prevalence at these times was 50.3, 12.3, 6.2, 8.1, 10.7, 7.1, and 8.1%. Staphylococcus chromogenes was the predominant species isolated, accounting for over 50% of the staphylococci isolated at each sampling time. Results suggest that high prevalence of staphylococci isolated prepartum is a reflection of natural skin flora and that a higher postpartum prevalence of these organisms was observed in primiparous cows than in multiparous cows. These data suggest also that the peripartum heifer could be a source of Staphylococcus aureus in the lactating herd.

Effect of naturally occurring coagulase-negative staphylococci infections on new infections by mastitis pathogens in the bovine.

Microbiological data from 1123 uninfected quarters and 216 quarters with preexisting coagulase-negative staphylococci infections were analyzed to determine the influence of infection status on subsequent new infection rate. Overall, prevalence of new infections in uninfected quarters was approximately two times that in quarters already harboring a coagulase-negative Staphylococcus infection. New infections by coagulase-negative staphylococci were greater in uninfected quarters than in quarters with preexisting coagulase-negative staphylococci infections. However, no differences were observed between uninfected and infected quarters in number of new infections by major pathogens (Staphylococcus aureus, streptococci, and coliforms). No differences were observed in uninfected or coagulase-negative Staphylococcus-infected quarters in infections with minor pathogens compared with major pathogens. The influence of individual coagulase-negative Staphylococcus species on new infections was also analyzed. However, numbers of existing infections by Staphylococcus species other than Staphylococcus chromogenes were limited. Therefore, the protective capacity of each coagulase-negative Staphylococcus species was difficult to assess. Overall, a significant restriction of bacterial invasion was observed in quarters with a preexisting infection. These results suggest that quarters harboring a coagulase-negative Staphylococcus infection suppress colonization of the mammary gland by mastitis-causing pathogens.

Isolation of Staphylococcus aureus from sites other than the lactating mammary gland.

The body sites of 163 heifers, bedding and feedstuff samples, flies, and hands and nares of the research personnel were sampled in order to determine the sources of Staphylococcus aureus in a dairy herd other than the lactating mammary gland. Lesions on the udder of lactating animals and the air in the milking parlor were also sampled. Staphylococci isolated from bedding samples were identified as to species. Staphylococcus aureus was isolated from all sources examined except flies. An enrichment procedure was necessary for isolating S. aureus from two bedding samples although other Staphylococcus species were present in high numbers. The designation "environmental staphylococci" is proposed for Staphylococcus species that were apparently free-living in the environment.

Effect of BiTek agar on lysostaphin susceptibility of staphylococci.

Staphylococci which were considered to be lysostaphin susceptible on P agar containing Bacto-Agar showed different degrees of resistance to lysostaphin when tested on P agar made with BiTek agar. As a result, lysostaphin-susceptible strains were misidentified as lysostaphin-resistant strains.

Fluorogenic assay for differentiating Staphylococcus warneri and Staphylococcus hominis strains of bovine origin.

A fluorogenic assay for the detection of beta-glucosidase was developed as part of a simplified conventional method to distinguish Staphylococcus warneri and Staphylococcus hominis isolated from bovine body sites. The assay is based on the fact that strains of S. warneri produce beta-glucosidase, while strains of S. hominis do not.

Antibiotic resistance of gram-negative enteric bacteria from pigs in three herds with different histories of antibiotic exposure.

The antibiotic resistance patterns of gram-negative fecal bacteria from pigs in three herds with different histories of antibiotic exposure were examined. In general, smaller proportions of antibiotic-resistant or multiply resistant fecal isolates (P less than 0.05) were obtained from pigs in a herd not exposed to antimicrobial agents for 154 months than from pigs in a herd continuously exposed to antimicrobial agents at subtherapeutic doses or from pigs in a herd exposed only to therapeutic doses of antimicrobial agents. The proportions of antibiotic-resistant and multiply resistant strains were greater among isolates from pigs in the therapeutic herd than in the non-antibiotic-exposed herd (P less than 0.05). The proportion of antibiotic-resistant isolates in the non-lactose-fermenting population was greater than that in the lactose-fermenting population, regardless of herd. The results suggest that any form of antimicrobial exposure will increase the prevalence of antimicrobial resistance and multiple resistance of fecal bacteria.

Isolation and identification of coagulase-negative Staphylococcus species from bovine body sites and streak canals of nulliparous heifers.

Heifers (n = 103) ranging in age from 1d to 2 yr were sampled to determine the coagulase-negative staphylococcal flora of haircoat, nares, vagina, teat skin, and streak canal. A total of 2706 staphylococal strains were identified from 3612 bacterial isolates. Other genera or groups identified included Bacillus, Micrococcus, Corynebacterium, and coliforms. Staphylococci were identified utilizing a simplified biochemical scheme. Staphylococcus xylosus, S. chromogenes, and S. warneri were the predominant species recovered from anatomic sites and streak canal. Staphylococcal strains identified from specific body sites (expressed as percentage of heifers harboring these species) were: nares 74% S. xylosus and 48% S. warneri; haircoat, 70% S. xylosus and 57% S. chromogenes; vagina, 60% S. chromogenes and 54% S. xylosus; teat skin 62% S. chromogenes and 61% S. warneri; streak canal 53% S. chromogenes, and 43% S. warneri. The prevalent staphylococcal strains identified differed from heifers in confined housing compared with heifers on pasture. Differences observed in distribution of Staphylococcus species among body sites, particularly those between teat skin and streak canal, suggest that establishment of staphylococcal microflora depends on the ability of a species to adapt to and colonize anatomic sites as well as on environmental conditions present.

Comparison of methods for determining DNase and phosphatase activities of staphylococci.

A greater percentage of DNase-positive strains was detected with DNase test agar than with DNase test agar containing 0.005% methyl green or 0.005% toluidine blue (P less than 0.01). No significant differences were obtained in the percentage of phosphatase-positive strains with the four methods compared. On the basis of ease of use, P agar containing para-nitrophenylphosphate disodium (0.495 mg/ml) would be the preferred method for determining phosphatase activity of staphylococci.

Antimicrobial resistance of fecal coliforms from pigs in a herd not exposed to antimicrobial agents for 126 months.

Patterns of antimicrobial resistance were determined for lactose-fermenting fecal coliforms obtained during a 20-month period from pigs in a herd without exposure to antimicrobial drugs for 126 months. Mean percent resistance to cephalothin, chloramphenicol, kanamycin, naladixic acid, streptomycin and tetracycline was lower (P less than 0.01) for isolates obtained during the 20-month period (Group 2) than for isolates obtained during the first 13 months (Group 1) after the withdrawal of antibiotics. Mean tetracycline resistance was 40.5% for Group 2 isolates. Approximately 44% of the Group 2 isolates were resistant to the 10 antimicrobial agents compared with 74.9% for the Group 1 isolates (P less than 0.01). Multiple resistance was lower (P less than 0.01) in the Group 2 isolates than in the Group 1 isolates. Four resistance patterns (tetracycline, tetracycline-streptomycin, tetracycline-sulfisoxazole, tetracycline-streptomycin-sulfisoxazole) accounted for 74% of the resistant isolates.

Use of latex teat dip with germicide during the prepartum period.

The efficacy of an acrylic latex barrier teat dip with germicide on new infections at parturition was tested on 113 cows and heifers during the prepartum period. A split udder design was used in which right quarters were undipped controls and left quarters were dipped with latex dip once daily for approximately 14 d prior to parturition. Distal streak canal swabs were taken from all quarters prior to the beginning of dipping, and all cows were quarter sampled in duplicate at drying off, parturition, and the first 5 consecutive wk of lactation. All quarters received antibiotic therapy at drying off. Excluding infections detected by distal streak canal swabs, the number of new infections at parturition in control and dipped quarters was 32 and 36. New infections at parturition in control and dipped quarters were Staphylococcus aureus, 2 and 2; coagulase-negative staphylococci, 8 and 9; stretptococci, 10 and 12; coliforms, 8 and 8; and others, 4 and 5. Differences were not significant. These results suggest no benefit to use of prepartum latex teat dip with germicide on new infections at parturition.

Effect of age and housing location on antibiotic resistance of fecal coliforms from pigs in a non-antibiotic-exposed herd.

The relationship of age and housing location to single antibiotic resistance, multiple antibiotic resistance, and resistance patterns of fecal coliforms obtained during a 20-month period from pigs in a herd that was not exposed to antibiotics for 126 months was determined. Bacteria resistant to single and multiple antibiotics were isolated more frequently (P less than 0.01) from pigs under 7 months of age. A greater proportion of isolates from pigs over 6 months of age was sensitive to the 13 antimicrobial agents tested (P less than 0.01), while a smaller proportion showed resistance to single (P less than 0.05) and multiple (P less than 0.01) antibiotics. More than 80% of the resistant isolates were resistant to tetracycline, streptomycin, or sulfisoxazole. Resistance was greater (P less than 0.01) for pigs in the finishing unit than for those on pasture. Resistance to ampicillin, carbenicillin, and tetracycline was greater (P less than 0.05) for pigs in the finishing unit than for those in the farrowing house. More isolates from pigs on pasture were sensitive to all antimicrobial agents tested (P less than 0.01). A greater proportion of isolates from pigs in the finishing unit showed resistance to a single antibiotic (P less than 0.01). The data from this study suggest that exposure to antibiotics is not the only factor that influences the prevalence of bacteria that are resistant to single and multiple antibiotics in the feces of domestic animals and that considerable research is needed to define the factors influencing antibiotic resistance in fecal bacteria.

Use of Lysostaphin and Bacitracin Susceptibility for Routine Presumptive Identification of Staphylococci of Bovine Origin 1.

A simple agar plate system based on susceptibility to lysostaphin and bacitracin to differentiate between staphylococci and micrococci is described. The system also uses susceptibility to novobiocin to aid in identification to species level of staphylococci. Growth from the agar plate can be used to prepare inocula for inoculation of rapid identification systems.

A Comparison of Six Selective Media for the Enumeration and Isolation of Staphylococci 1.

The efficacy of Baird-Parker agar, Baird-Parker agar with bacitracin (0.8 µg/ml), Baird-Parker agar supplemented with acriflavin (7 µg/ml), polymyxin (20 units/ml) and sulphonamides (55 µg/ml), KRANEP agar, mannitol salt agar, and Staphylococcus medium 110 agar for the isolation and enumeration of Staphylococcus species was investigated. Bovine blood agar was used as the control medium. Thirty-seven staphylococci strains representing 23 species and 19 non- Staphylococcus species were tested. None of the six selective media supported the growth of all 37 Staphylococcus strains. The number of Staphylococcus species that grew on a specific medium ranged from only the S. aureus strains on Baird-Parker agar supplemented with acriflavin, polymyxin, and sulphonamides, to all but S. warneri BG 647 on Staphylococcus medium 110 agar. Strains of Bacillus , Corynebacterium , and Micrococcus grew on all six selective media.