T.M.A. Santos

Diversity and Succession of Bacterial Communities in the Uterine Fluid of Postpartum Metritic, Endometritic and Healthy Dairy Cows

The diversity of the uterine bacterial composition in dairy cows is still poorly understood, although the emerging picture has shown to be increasingly complex. Understanding the complexity and ecology of microorganisms in the uterus of postpartum dairy cows is critical for developing strategies to block their action in reproductive disorders, such as metritis/endometritis. Here, we used PCR-Denaturing Gradient Gel Electrophoresis (DGGE) and DNA pyrosequencing to provide a comprehensive description of the uterine bacterial diversity and compare its succession in healthy, metritic and endometritic Holstein dairy cows at three intervals following calving. Samples were collected from 16 dairy cows housed in a dairy farm located in upstate New York. PCR-DGGE revealed a complex profile with extensive differences in the community structure. With few exceptions, clustering analysis grouped samples from cows presenting the same health status. Analysis of >65,000 high-quality 16S rRNA gene sequences showed that the uterine bacterial consortia, regardless of the health status, is mainly composed of members of the phyla Bacteroidetes, Fusobacteria, Firmicutes, Proteobacteria, and Tenericutes. In addition to these co-dominant phyla, sequences from Spirochaetes, Synergistetes, and Actinobacteria appear less frequently. It is possible that some sequences detected in the uterine fluid resulted from the presence of fecal or vaginal contaminants. Overall, the bacterial core community was different in uterine fluid of healthy cows, when compared to cows suffering from postpartum diseases, and the phylogenetic diversity in all the combined samples changed gradually over time. Particularly at the 34–36 days postpartum (DPP), the core community seemed to be specific for each health status. Our finding reveals that the uterine microbiota in dairy cows varies according with health status and DPP. Also, it adds further support to the hypothesis that there is uterine contamination with diverse bacterial groups following calving and emphasizes the role of unidentified microorganisms in this context.

Antimicrobial resistance and presence of virulence factor genes in Arcanobacterium pyogenes isolated from the uterus of postpartum dairy cows.

Arcanobacterium pyogenes is considered the most significant bacterium involved in the pathogenesis of metritis in cattle. Infections caused by antimicrobial-resistant bacteria are a great challenge in both human and veterinary medicine. The purpose of this study was to present an overview of antimicrobial resistance in A. pyogenes isolated from the uteruses of postpartum Holstein dairy cows and to identify virulence factors. Seventy-two A. pyogenes isolates were phenotypically characterized for antimicrobial resistance to amoxicillin, ampicillin, ceftiofur, chloramphenicol, florfenicol, oxytetracycline, penicillin, spectinomycin, streptomycin and tetracycline by the broth microdilution method. Presence of virulence factor genes of A. pyogenes was investigated. Isolates exhibited resistance to all antimicrobial agents tested; high levels of resistance were found to amoxicillin (56.9%); ampicillin (86.1%), chloramphenicol (100%), florfenicol (59.7%), oxytetracycline (54.2%), penicillin (86.1%) and tetracycline (50%). Of all isolates, 69 (95.8%) were resistant to at least 2 of the antimicrobial agents tested and multidrug resistance (resistant to at least 3 antimicrobials) was observed in 64 (88.9%) of the A. pyogenes isolates. The major multidrug resistance profile was found for chloramphenicol-ampicillin-penicillin-florfenicol-amoxicillin-tetracycline, which was observed in 21 (29.2%) multidrug resistant isolates. No isolate was resistant to all nine antimicrobial agents tested but four isolates (5.6%) were resistant to eight antimicrobials. The information highlights the need for prudent use of specific antimicrobial agents. All four virulence factor genes occurred in isolates from normal puerperium and clinical metritis; however, the fimA gene was present in significantly higher frequency in isolates from metritis cows.

Subcutaneous Immunization with Inactivated Bacterial Components and Purified Protein of Escherichia coli, Fusobacterium necrophorum and Trueperella pyogenes Prevents Puerperal Metritis in Holstein Dairy Cows

In this study we evaluate the efficacy of five vaccine formulations containing different combinations of proteins (FimH; leukotoxin, LKT; and pyolysin, PLO) and/or inactivated whole cells (Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes) in preventing postpartum uterine diseases. Inactivated whole cells were produced using two genetically distinct strains of each bacterial species (E. coli, F. necrophorum, and T. pyogenes). FimH and PLO subunits were produced using recombinant protein expression, and LKT was recovered from culturing a wild F. necrophorum strain. Three subcutaneous vaccines were formulated: Vaccine 1 was composed of inactivated bacterial whole cells and proteins; Vaccine 2 was composed of proteins only; and Vaccine 3 was composed of inactivated bacterial whole cells only. Two intravaginal vaccines were formulated: Vaccine 4 was composed of inactivated bacterial whole cells and proteins; and Vaccine 5 was composed of PLO and LKT. To evaluate vaccine efficacy, a randomized clinical trial was conducted at a commercial dairy farm; 371 spring heifers were allocated randomly into one of six different treatments groups: control, Vaccine 1, Vaccine 2, Vaccine 3, Vaccine 4 and Vaccine 5. Late pregnant heifers assigned to one of the vaccine groups were each vaccinated twice: at 230 and 260 days of pregnancy. When vaccines were evaluated grouped as subcutaneous and intravaginal, the subcutaneous ones were found to significantly reduce the incidence of puerperal metritis. Additionally, subcutaneous vaccination significantly reduced rectal temperature at 6±1 days in milk. Reproduction was improved for cows that received subcutaneous vaccines. In general, vaccination induced a significant increase in serum IgG titers against all antigens, with subcutaneous vaccination again being more effective. In conclusion, subcutaneous vaccination with inactivated bacterial components and/or protein subunits of E. coli, F. necrophorum and T. pyogenes can prevent puerperal metritis during the first lactation of dairy cows, leading to improved reproduction.

Microbial diversity in bovine papillomatous digital dermatitis in Holstein dairy cows from upstate New York

Papillomatous digital dermatitis (PDD) is one of the most prevalent diseases of cattle, adversely affecting the dairy industry by its negative effect on milk production and reproductive performance. Our objective was to use culture-independent methods to determine the microbial diversity in different strata of PDD lesions of three Holstein dairy cows, analyzing whether major differences exist compared to foot skin of three non-infected cows. Both group-specific 16S rRNA gene PCR-denaturing gradient gel electrophoresis and clone library sequencing of broad-range 16S rRNA gene showed differences between the microbial composition of healthy dairy cows and the different strata of the lesion. The predominant bacterial community in the lesion, regardless of the stratum, consisted of 166 specific phylotypes belonging to seven bacterial phyla. Spirochetes (particularly, treponemes) was the most prominent group detected in PDD deep biopsies and was only found in samples from the lesion. Additionally, one phylotype phylogenetically affiliated with uncultured Euryarchaeota was detected in two strata of the lesion. Sequences from healthy foot skin samples revealed 86 specific phylotypes that were affiliated with Firmicutes and Proteobacteria. Our study corroborates the theory that treponemes are involved in PDD disease etiology and suggests, for the first time, the presence of archaeal members in this particular bovine infection.

Susceptibility of Escherichia coli isolated from uteri of postpartum dairy cows to antibiotic and environmental bacteriophages. Part II: In vitro antimicrobial activity evaluation of a bacteriophage cocktail and several antibiotics.

The use of pathogenic-specific antimicrobials, as proposed by bacteriophage therapy, is expected to reduce the incidence of resistance development. Eighty Escherichia coli isolated from uteri of Holstein dairy cows were phenotypically characterized for antimicrobial resistance to ampicillin, ceftiofur, chloramphenicol, florfenicol, spectinomycin, streptomycin, and tetracycline by broth microdilution method. The lytic activity of a bacteriophage cocktail against all isolates was performed by a similar method. Additionally, the effect of different concentrations of antimicrobials and multiplicities of infections (MOI) of the bacteriophage cocktail on E. coli growth curve was measured. Isolates exhibited resistance to ampicillin (33.7%), ceftiofur (1.2%), chloramphenicol (100%), and florfenicol (100%). All strains were resistant to at least 2 of the antimicrobial agents tested; multidrug resistance (>or=3 of 7 antimicrobials tested) was observed in 35% of E. coli isolates. The major multidrug resistance profile was found for ampicillin-chloramphenicol-florfenicol, which was observed in more than 96.4% of the multidrug-resistant isolates. The bacteriophage cocktail preparation showed strong antimicrobial activity against multidrug-resistant E. coli. Multiplicity of infection as low as 10(-4) affected the growth of the E. coli isolates. The ratio of 10 bacteriophage particles per bacterial cell (MOI=10(1)) was efficient in inhibiting at least 50% of all isolates. Higher MOI should be tested in future in vitro studies to establish ratios that completely inhibit bacterial growth during longer periods. All isolates resistant to florfenicol were resistant to chloramphenicol and, because florfenicol was recently introduced into veterinary clinics, this finding suggests that the selection pressure of chloramphenicol, as well as other antimicrobials, may still play a relevant role in the emergence and dissemination of florfenicol resistance in E. coli. The bacteriophage cocktail had a notable capacity to inhibit the in vitro growth of E. coli isolates, and it may be an attractive alternative to conventional treatment of metritis by reducing E. coli in uteri of postpartum dairy cows.

Susceptibility of Escherichia coli isolated from uteri of postpartum dairy cows to antibiotic and environmental bacteriophages. Part I: Isolation and lytic activity estimation of bacteriophages.

The objective of this study was to isolate bacteriophages from environmental samples of 2 large commercial dairy farms using Escherichia coli isolated from the uteri of postpartum Holstein dairy cows as hosts. A total of 11 bacteriophage preparations were isolated from manure systems of commercial dairy farms and characterized for in vitro antimicrobial activity. In addition, a total of 57 E. coli uterine isolates from 5 dairy cows were phylogenetically grouped by triplex PCR. Each E. coli bacterial host from the uterus was inoculated with their respective bacteriophage preparation at several different multiplicities of infections (MOI) to determine minimum inhibitory MOI. The effect of a single dose (MOI=10(2)) of bacteriophage on the growth curve of all 57 E. coli isolates was assessed using a microplate technique. Furthermore, genetic diversity within and between the different bacteriophage preparations was assessed by bacteriophage purification followed by DNA extraction, restriction, and agarose gel electrophoresis. Phylogenetic grouping based on triplex PCR showed that all isolates of E. coli belonged to phylogroup B1. Bacterial growth was completely inhibited at considerably low MOI, and the effect of a single dose (MOI=10(2)) of bacteriophage preparations on the growth curve of all 57 E. coli isolates showed that all bacteriophage preparations significantly decreased the growth rate of the isolates. Bacteriophage preparation 1230-10 had the greatest antimicrobial activity and completely inhibited the growth of 71.7% (n=57) of the isolates. The combined action of bacteriophage preparations 1230-10, 6375-10, 2540-4, and 6547-2, each at MOI=10(2), had the broadest spectrum of action and completely inhibited the growth (final optical density at 600 nm <or=0.1) of 80% of the E. coli isolates and considerably inhibited the growth (final optical density at 600 nm <or=0.2) of 90% of the E. coli isolates. Restriction profile analysis demonstrated that all 4 phage preparations contained bacteriophages that were genetically distinct from each other according to the banding pattern of the fragments. The combination of several different bacteriophages can improve the spectrum of action, and the results of this study suggest that bacteriophages 1230-10, 6375-10, 2540-4, and 6547-2 should be used in combination as a cocktail.

Antimicrobial resistance and prevalence of virulence factor genes in fecal Escherichia coli of Holstein calves fed milk with and without antimicrobials

Diarrhea in calves has a significant effect on the dairy industry. A common management practice for preventing or decreasing the effects of such disease in preweaned calves is by the use of antimicrobials in milk or milk replacer. In this study, Escherichia coli antimicrobial resistance in fecal samples collected from calves 2 to 8 d of age that had or had not received antimicrobials in the milk and that had or had not signs of diarrhea by inspection of fecal consistency were investigated. Specifically, resistance of E. coli isolates to individual antimicrobials, multiresistance patterns, and presence of virulence factors were analyzed. Escherichia coli isolates were tested for susceptibility to 12 antimicrobials by use of a Kirby-Bauer disk diffusion assay. The study was conducted at 3 farms, 1 administering growth-promoting antimicrobials (GPA) in the milk and 2 not using GPA in the milk (NGPA). All isolates were susceptible to ciprofloxacin and cefepime. From the total isolates tested, 84% (n=251) were resistant to at least 2 antimicrobials and 81% (n=251) were resistant to 3 or more antimicrobials. When antimicrobial resistance was compared between GPA and NGPA, it was observed that the GPA group had higher odds of antimicrobial resistance for most of the individual antimicrobials tested. No significant correlation of virulence factors in GPA or NGPA and diarrheic or non-diarrheic (control) fecal samples was found. Of the 32 virulence factors evaluated, 21 were detected in the study population; the incidence of only 1 virulence factor was statistically significant in each of the diarrheic status (diarrheic or non-diarrheic) and treatment status (NGPA or GPA) groups. Phylogenetic analysis based on the nucleotide sequence of the DNA gyrase gene (gyrB) from 31 fecal E. coli isolates revealed 3 main clades.

Isolation and characterization of two bacteriophages with strong in vitro antimicrobial activity against Pseudomonas aeruginosa isolated from dogs with ocular infections

OBJECTIVE To isolate and characterize bacteriophages with strong in vitro lytic activity against various pathogenic Pseudomonas aeruginosa strains isolated from dogs with ocular infections. SAMPLE 26 genetically distinct P aeruginosa isolates. PROCEDURES P aeruginosa strains were derived from dogs with naturally acquired ulcerative keratitis. From a large-scale screening for bacteriophages with potential therapeutic benefit against canine ocular infections, 2 bacteriophages (P2S2 and P5U5) were selected; host ranges were determined, and phage nucleic acid type and genetic profile were identified via enzymatic digestion. Electron microscopy was used to characterize bacteriophage ultrastructure. Bacteriophage temperature and pH stabilities were assessed by use of double-layer agar overlay titration. A cocultivation assay was used to evaluate the effect of the bacteriophages on bacterial host growth. RESULTS P5U5 was active against all P aeruginosa isolates, whereas P2S2 formed lytic plaques on plates of 21 (80.8%) isolates. For each bacteriophage, the genomic nucleic acid was DNA; each was genetically distinct. Ultrastructurally, P2S2 and P5U5 appeared likely to belong to the Podoviridae and Siphoviridae families, respectively. The bacteriophages were stable within a pH range of 4 to 12; however, titers of both bacteriophages decreased following heating for 10 to 50 minutes at 45° or 60°C. Growth of each P aeruginosa isolate was significantly inhibited in coculture with P2S2 or P5U5; the dose response was related to the plaque-forming unit-to-CFU ratios. CONCLUSIONS AND CLINICAL RELEVANCE Bacteriophages P2S2 and P5U5 appear to be good candidates for phage treatment of infection caused by pathogenic P aeruginosa in dogs.

Evaluation of oral administration of bacteriophages to neonatal calves: Phage survival and impact on fecal Escherichia coli

Abstract Diarrhea is an important cause of morbidity and mortality in neonatal calves. Several enteropathogens are associated with diarrhea in young calves, with Enterotoxigenic Escherichia coli (ETEC) infection being the most common type of colibacillosis. The rise of antibiotic resistance in a number of medically important bacterial pathogens has revived interest in the use of bacteriophages as anti-bacterial therapeutic agents. Here we describe the results of a randomized, placebo-controlled, double-blinded study designed to evaluate the effect of an oral bacteriophage cocktail on fecal E. coli colony-forming units (CFU). Ten calves were enrolled in the study to either the bacteriophage group or the placebo group for 22 feedings. Calves in the bacteriophage treatment group (n=6) received a total of 106 plaque-forming units (PFU) (volume=5ml) of each of four bacteriophages while the placebo group (n=4) received only phosphate-buffered saline (5ml). Fresh fecal samples and blood samples were collected daily from each calf and analyzed for bacterial count and presence of bacteriophage. E. coli-infecting phages were recovered from all phage-treated calves at concentrations of 10² to 10³ PFU per gram of rectal contents, but none was detected in serum. Phage treatment caused a reduction in fecal E. coli when compared to the control group: the mean log CFU for the placebo-treated group was 9.25 (SE=0.42) versus 9.11 (SE=0.34) for the phage-treated group, but the difference was not statistically significant.