Antibacterial Efficacy of Silver Nanoparticles on Endometritis Caused by Prevotella melaninogenica and Arcanobacterum pyogenes in Dairy Cattle.

Bovine postpartum diseases remain one of the most significant and highly prevalent illnesses with negative effects on the productivity, survival, and welfare of dairy cows. Antibiotics are generally considered beneficial in the treatment of endometritis; however, frequent usage of each antibiotic drug is reason for the emergence of multidrug resistance (MDR) of the pathogenic microorganisms, representing a major impediment for the successful diagnosis and management of infectious diseases in both humans and animals. We synthesized silver nanoparticles (AgNPs) with an average size of 10 nm using the novel biomolecule apigenin as a reducing and stabilizing agent, and evaluated the efficacy of the AgNPs on the MDR pathogenic bacteria Prevotella melaninogenica and Arcanobacterium pyogenes isolated from uterine secretion samples. AgNPs inhibited cell viability and biofilm formation in a dose- and time-dependent manner. Moreover, the metabolic toxicity of the AgNPs was assessed through various cellular assays. The major toxic effect of cell death was caused by an increase in oxidative stress, as evidenced by the increased generation of reactive oxygen species (ROS), malondialdehyde, protein carbonyl content, and nitric oxide. The formation of ROS is considered to be the primary mechanism of bacterial death. Therefore, the biomolecule-mediated synthesis of AgNPs shows potential as an alternative antimicrobial therapy for bovine metritis and endometritis.

Antimicrobial Resistance in Corynebacterium spp., Arcanobacterium spp., and Trueperella pyogenes.

There is currently only limited information on the antimicrobial susceptibility and resistance of Corynebacterium spp., Arcanobacterium spp., and Trueperella pyogenes from animals. The comparability of the data is hampered by the use of different antimicrobial susceptibility testing methods and interpretive criteria. To date, standard broth microdilution methods and clinical breakpoints that are approved by the Clinical and Laboratory Standards Institute and are applicable to Corynebacterium spp., Arcanobacterium spp., and T. pyogenes are available. The lack of species-specific clinical breakpoints for the different animal species reduces the explanatory power of the data. Among the isolates of the three genera, elevated MICs for different classes of antimicrobial agents (e.g., ß-lactams, macrolides, lincosamides, tetracyclines, aminoglycosides, phenicols, sulfonamides/diaminopyrimidines, and fluoroquinolones) have been described. The most comprehensive data set is available for T. pyogenes, which also includes information about genes and mutations involved in antimicrobial resistance. In T. pyogenes isolates, the macrolide-lincosamide-streptogramin B resistance genes erm(B) and erm(X) were identified. Tetracycline resistance in T. pyogenes was based on the resistance genes tet(W), tet(Z), and tet(33), whereas the aminoglycoside resistance genes aacC, aadA1, aadA2, aadA5, aadA24, and aadB have been described in T. pyogenes. So far, only single genes conferring either phenicol resistance (cmlA6), trimethoprim resistance (dfrB2a), or ß-lactam resistance (blaP1) are known to occur in T. pyogenes isolates. Various 23S rRNA mutations, including A2058T, A2058G, and G2137C, were identified in macrolide/lincosamide-resistant T. pyogenes.

Properties and antimicrobial susceptibility of Trueperella pyogenes isolated from bovine mastitis in China.

Trueperella (T.) pyogenes is an opportunistic pathogen that causes suppurative diseases in domestic animals. In this work, the properties, pathogenesis and phenotypic diversity of T. pyogenes isolates from bovine mastitis were studied. Both pyolysin (plo) and collagen-binding protein (cbp) virulence factor genes were detected by PCR in all T. pyogenes isolates (n = 50). Using the tissue culture plate method, 90% of T. pyogenes isolates were able to form biofilms. The minimum inhibitory concentrations (MICs) of 13 antimicrobials against T. pyogenes isolates were determined. High susceptibility was observed to rifampin (96%), ampicillin (94%), ciprofloxacin (94%), and penicillin (92%), while low susceptibility was found to trimethoprim-sulphamethoxazole (10%) and bacitracin (2%). The intracellular assay revealed that T. pyogenes isolates had different cytopathogenic effects on cells. The high percentage (28.6%) of T. pyogenes isolates suggests that this bacterium is an important contributor to mastitis. Moreover, the high occurrence of multidrug resistance, biofilm production, intracellular survival, and the temporal dynamics of T. pyogenes interactions are key factors for a better understanding of how immunity acts on infections with these bacteria and how they evade immune surveillance, thus highlighting the need for the prudent use of antimicrobial agents in veterinary medicine.

Brain abscess due to Trueperella bernardiae.

We present the case of a brain abscess caused by a combination of rare organisms (Trueperella bernardiae and Peptoniphilus harei) in a patient with chronic suppurative otitis media that had been complicated by the presence of a cholesteatoma. The authors believe this is the first report published in the literature.

Genotypic characterization and evaluation of an antibiotic resistance of Trueperella pyogenes (Arcanobacterium pyogenes) isolated from milk of dairy cows with clinical mastitis.

Trueperella pyogenes, recently reclassified from the genus Arcanobacterium, is considered the causative agent of acute suppurative mastitis called summer mastitis. T. pyogenes produces a variety of known and putative virulence factors that include pyolysin and factors promoting adhesion to host cells. The objective of this study was to report the presence of virulence genes in T. pyogenes isolates that were identified as etiological agents of clinical mastitis in cows, as well as to determine antimicrobial resistance and distribution of selected determinants that can be associated with phenotypic resistance among these isolates. The presence of genes (plo, nanH, nanP, cbpA, fimA, fimC, fimE, fimG, tet(W), erm(X), erm(B)) was examined by conventional PCRs. Resistance to 10 antimicrobial agents was determined by the broth microdilution method. Among T. pyogenes isolates of bovine mastitis origin the genes encoding all virulence factors occurred. Besides pyolysin gene plo, the fimA was the only gene detected in all isolates, whereas other virulence factor genes were found with different frequencies. Phenotypic antimicrobial resistance was observed to tetracycline (85.5% isolates) and erythromycin (9.1%). Isolates non-susceptible to erythromycin simultaneously exhibited increased MIC of pirlimycin. Beta-lactams were active against isolates. We found the correlation between the presence of tetracycline and macrolide resistance genes and corresponding resistance phenotype. Genotypic characterization of a large number of T. pyogenes isolates from different herds performed in this study may be useful in explanation, which virulence factors play a significant role in the establishment of bovine mammary gland infection.

The effect of intrauterine administration of mannose or bacteriophage on uterine health and fertility of dairy cows with special focus on Escherichia coli and Arcanobacterium pyogenes.

The objective of this study was to evaluate the effects of intrauterine administration of 50 g of ultrapure mannose or a bacteriophage cocktail and the presence of Escherichia coli and Arcanobacterium pyogenes in the uterine lumen on uterine health and reproductive performance of lactating dairy cows. The study was conducted on a commercial dairy farm located near Ithaca, New York, from May 4 to January 20, 2011, and 597 cows were enrolled. The cows were divided randomly into 3 treatment groups, and treatments were administered at 2 ± 1 d in milk (DIM). Treatment 1 consisted of intrauterine administration of 50 g of ultrapure mannose powder divided in 4 pills; treatment 2 consisted of intrauterine administration of a bacteriophage cocktail that included 4 different phages in one pill, with a dose of approximately 107 plaque-forming units; and treatment 3 consisted of intrauterine administration of one empty pill (control). Intrauterine fluid swabs were collected on day of treatment and at 10 ± 3 DIM; uterine lavage samples were collected at 35 ± 3 DIM. Swabs and uterine lavage samples were cultured for E. coli and A. pyogenes. The intrauterine administration of mannose and bacteriophage did not affect uterine health, reproduction performance, or outcome of intrauterine secretion cultures for E. coli and A. pyogenes. Prevalence of intrauterine E. coli at 2 ± 1 DIM and A. pyogenes at 2 ± 1, 10 ± 3, and 35 ± 3 were affected by retained placenta. Additionally, prevalence of intrauterine E. coli at 10 ± 3 and A. pyogenes at 35 ± 3 DIM were associated with metritis, and cows that were diagnosed with clinical endometritis at 35 ± 3 DIM had greater prevalence of intrauterine E. coli at 2 ± 1 DIM and A. pyogenes at 35 ± 3 DIM. Furthermore, cows positive for E. coli at 2 ± 1 and 10 ± 3 DIM and for A. pyogenes at 10 ± 3 DIM had 1.63, 2.34, and 1.54 increased odds of having metritis. Cows positive for A. pyogenes at 35 ± 3 DIM and for E. coli at 2 ± 1 DIM had 19.8 and 2.66 higher odds of being diagnosed with clinical endometritis, respectively. Additionally, cows negative for E. coli at 2 ± 1 DIM were 1.39 times more likely to conceive than cows positive cows for E. coli.

Antimicrobial susceptibility of Arcanobacterium pyogenes isolated from the lungs of white-tailed deer (Odocoileus virginianus) with pneumonia.

In vitro susceptibilities of 29 strains of Arcanobacterium pyogenes isolated from lung lesions of white-tailed deer (Odocoileus virginianus) with pneumonia were determined using the broth microdilution method to ascertain efficacious treatment options for pneumonic white-tailed deer. All 29 A. pyogenes strains tested were susceptible to ceftiofur, spectinomycin, tiamulin, and trimethoprim-sulfamethoxazole but were resistant to both danofloxacin and sulfadimethoxine. Likewise, all 29 isolates were either fully susceptible or intermediately susceptible to gentamicin (25 susceptible; 4 intermediate) and tulathromycin (25 susceptible; 4 intermediate). At least one isolate of A. pyogenes tested was resistant to ampicillin, chlortetracycline, clindamycin, enrofloxacin, florfenicol, oxytetracycline, penicillin, and tilmicosin suggesting their ineffectiveness in treating A. pyogenes-associated lung infections in white-tailed deer. Minimum inhibitory concentration (MIC) data for tylosin and neomycin could not be interpreted due to unavailability of Clinical and Laboratory Standards Institute (CLSI)-approved breakpoints for these 2 agents. In summary, based on MIC values, ceftiofur, spectinomycin, tiamulin, and trimethoprim-sulfamethoxazole are more efficacious than other antimicrobial agents for treating A. pyogenes-related pneumonia in white-tailed deer. However, ceftiofur may be preferred over the other 4 drugs as it is being widely used to treat respiratory disease in cattle and other animal species, as well as is available for single dose parenteral administration.

Detection and characterization of antibiotic-resistance genes in Arcanobacterium pyogenes strains from abscesses of forest musk deer.

Arcanobacterium pyogenes is commonly isolated from ruminant animals as an opportunistic pathogen that co-infects with other bacteria, normally causing surface or internal abscesses. Twenty-eight strains of A. pyogenes isolated from forest musk deer suppurative samples were identified by their 16S rRNA gene sequences, and confirmed by amplification of the pyolysin-encoding gene (plo) in all isolates. The MICs of 14 commonly used antibiotics were determined by an agar dilution method. Class 1 and 2 intI genes were amplified to determine whether integrons were present in the A. pyogenes genome. Class 1 gene cassettes were detected by specific primers and analysed by sequencing. All of the strains were susceptible to most fluoroquinolone antibiotics; however, high resistance rates were observed for ß-lactams and trimethoprim. A total of 18 of the isolates (64.3%) were positive for the class 1 intI gene, and 16 (57.1%) contained class 1 gene cassettes with the aacC, aadA1, aadA2, blaP1 and dfr2a genes. Most were present in the multi-resistant isolates, indicating a general concordance between the presence of gene cassettes and antibiotic resistance, and that the integrons have played an important role in the dissemination of antimicrobial resistance in this species.

Infective endocarditis caused by Arcanobacterium haemolyticum: a case report.

Arcanobacterium haemolyticum is an organism that commonly causes pharyngitis and wound infections. It does not usually cause systemic invasive disease. The organism presents a difficult diagnostic problem because the Clinical Microbiology laboratory has a propensity to view them as diphtheroid organisms of the Corynebacterium species, thus contaminants or normal flora. We describe a case of a 21-year-old female who had endocarditis with cerebral emboli due to Arcanobacterium haemolyticum. This rare condition is associated with significant mortality and to the best of our knowledge; this is the first successfully treated case of A. haemolyticum endocarditis complicated by embolic phenomenon.

Sensitivity to antibiotics of Arcanobacterium pyogenes and Escherichia coli from the uteri of cows with metritis/endometritis.

The aim of the study was to determine the in vitro sensitivity of 161 Arcanobacterium pyogenes strains and 99 Escherichia coli strains isolated from the uteri of 312 cows with metritis/endometritis. Animals with pathological discharges were clinically diagnosed per rectum and per vaginum, and then swabs from the lumen of the uterus were aseptically collected. Bacteriological examination of swabs was according to commonly accepted procedures with the additional use of API tests. Sensitivity to antibiotics was tested by the disk diffusion method and performed in Mueller-Hinton agar. Arc. pyogenes strains were the most sensitive to amoxicillin/clavulanic acid (Amc) (97.3%), bacitracin (96.7%), ceftiofur (95.8%) and cephapirin (77.5%). E. coli strains were the most sensitive to norfloxacin (98.1%), marbofloxacin (95.8%), gentamycin (88%), Amc (80.7%) and ceftiofur (73.1%). Arc. pyogenes and E. coli were most resistant to oxytetracycline (63.7% and 31%, respectively).

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.

Identification, susceptibility, and detection of integron-gene cassettes of Arcanobacterium pyogenes in bovine endometritis.

The present study aimed to identify, determine the susceptibility, and detect gene cassettes of Arcanobacterium (Actinomyces) pyogenes isolates from cows with endometritis. Arcanobacterium pyogenes isolates were identified first by using the API Coryne Vit system test, and further through PCR. Minimum inhibitory concentrations of 23 antimicrobial agents against A. pyogenes were tested using standard broth microdilution assays according to the protocols of the Clinical and Laboratory Standards Institute. The genes of integrons I and II were amplified by PCR using specific primers. Thirty-two A. pyogenes isolates were isolated from 136 endometritic cows in the Hohhot region. Antibiotic susceptibility tests revealed that all isolates were highly sensitive to fluoroquinolones (100%), macrolides (approximately 81.2 to 100%) and florfenicol (90.6%), aminoglycosides (approximately 15.6 to 81.2%), and tetracyclines (approximately 43.7 to 68.7%). However, 53.1% were resistant to clindamycin, approximately 50 to 65.6% were resistant to penicillins, and approximately 37.5 to 71.9% were resistant to cephalosporins. One hundred percent were resistant to sulfonamides and bacitracin zinc. The integrons were further confirmed by sequencing. No class II integrons were detected, whereas 50% (n = 16) of the A. pyogenes isolates were positive for the presence of the intI I gene, but only 13 contained gene cassettes. Sequence analysis of gene cassettes revealed 6 gene cassettes, 4 of which encode resistant determinants of aminoglycosides (aadA1, aadA5, aadA24, and aadB) and 1 of which encodes the resistance gene of chloramphenicol (cmlA6). The function of the sixth identified cassette, designated ORF1, is unknown. The gene cassette arrays aadA24-ORF1, aadA5, and aadA1-addB-cmlA6 were found in 46.13% (6/13), 38.46% (5/13), and 38.46% (5/13) of the isolates, respectively. These cassettes segregated according to a consistent pattern, with aadA5 always alone, ORF1 always with aadA24, and aadA1-aadB and cmlA6 always together. Most of the positive integrons existed in the multiresistant isolates (n = approximately 3 to 7), indicating that the integrons played an important role in the dissemination and spread of antimicrobial resistance. This is the first report of A. pyogenes infections in dairy cows in China and of detection of gene cassettes and integrons in A. pyogenes.

Characterization of tet(32) genes from the oral metagenome.

tet(32) Was identified in three bacterial isolates and in metagenomic DNA from the human oral cavity. The regions immediately flanking the gene were found to have similarities to the mobile elements TnB1230 from Butyrivibrio fibrisolvens, ATE-3 from Arcanobacterium pyogenes, and CTn5 from Clostridium difficile.