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.

Relationship between Escherichia coli virulence factors and postpartum metritis in dairy cows.

The objectives of this study were to report the prevalence of Escherichia coli and Trueperella pyogenes in the uterus of postpartum dairy cows before the onset of postpartum metritis (PPM) and to quantify their association with subsequent occurrence of PPM, to quantify the association between the presence of genes encoding E. coli virulence factors (VF) and PPM, and to determine the accuracy of using early postpartum uterine bacteriology results (bacteria and VF) to identify cows at risk of PPM. A prospective cohort study was conducted on 3 commercial dairy farms. Uterine swabs were collected from 371 Holstein dairy cows (3 commercial herds) at 1 to 7d in milk and submitted to the laboratory for identification of E. coli, T. pyogenes, and E. coli VF. A total of 40 VF were tested using the radioactive probe hybridization method. Postpartum metritis was defined as the presence of a fetid watery red-brown uterine discharge, associated with fever (rectal temperature >39.5°C), and systemic signs of illness (dullness, reduced appetite, and milk production). Surveillance of PPM was done by trained farmers blinded to laboratory results and cows were followed until 21d in milk. Statistical analyses were conducted using 2×2 tables and mixed logistical regression models. Prevalences of E. coli, T. pyogenes, and PPM were 42, 34, and 15%, respectively. A total of 32 VF were found in E. coli isolates. Most prevalent VF were extraintestinal pathogenic genes such as fimH (89%), hlyE (87%), and iss (70%). Cows positive for intrauterine E. coli were 3.2 times more likely to have subsequent PPM compared with bacteriologically negative cows. Cows with VF hra1 in their uterus were 2.7 times more likely to have PPM than cows positive for E. coli and negative for hra1 and 5.9 times more likely than bacteriologically negative cows. Cows with VF kpsMTII in their uterus were 3.2 times more likely to have PPM than cows positive for E. coli and negative for kpsMTII and 6.2 times more likely than bacteriologically negative cows. Using E. coli, hra1, and kpsMTII as predictors for subsequent PPM, positive predictive values were 23, 31, and 42%, respectively, whereas the negative predictive values were 91, 80, and 78%, respectively. Overall, these results showed that E. coli and some VF were associated with PPM.

Phenotypic and genotypic approach to characterize Arcanobacterium pluranimalium isolated from bovine milk samples.

In the present study, three Arcanobacterium pluranimalium strains isolated from bovine milk samples of three cows of three farms (two cows with subclinical mastitis) could successfully be identified by phenotypical investigations, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis and genotypically by sequencing the molecular targets 16S rDNA, 16S-23S rDNA intergenic spacer region (ISR), the ß subunit of bacterial RNA polymerase encoding gene rpoB, the glyceraldehyde 3-phosphate dehydrogenase encoding gene gap, the elongation factor tu encoding gene tuf, and the pluranimaliumlysin encoding gene pla. The latter could also be identified by a loop-mediated isothermal amplification (LAMP) assay. The presented phenotypic and genotypic approaches might support the identification of A. pluranimalium in future and might help to understand the role this species plays in bovine mastitis.

Phenotypic and genotypic characteristics of Arcanobacterium haemolyticum isolated from clinical samples in a Danish hospital.

Six Arcanobacterium haemolyticum strains isolated from six patients of two hospitals in Denmark were identified phenotypically, also including matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and by genotypic methods. The latter were performed by sequencing 16S rDNA and glyceraldehyde 3-phosphate dehydrogenase encoding gene gap and by amplification of an A. haemolyticum specific region of 16S-23S rDNA intergenic spacer region and 23S rDNA. The six A. haemolyticum strains were further investigated for the presence of seven potential virulence genes encoding arcanolysin, phospholipase D, hemolysin A, CAMP factor family protein, collagen binding protein, neuraminidase A and neuraminidase H which appeared to be present in two (seven virulence genes), two (six virulence genes) and two strains (four virulence genes), respectively. The phenotypic and genotypic properties described in the present study might help to reliably identify and further characterize A. haemolyticum isolated from human patients, a species which seems to be of increasing importance.

First description of Trueperella (Arcanobacterium) bernardiae of animal origin.

In the present study a Trueperella (Arcanobacterium) bernardiae strain isolated from an anal swab of a three-day-old piglet could be identified phenotypically, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing the 16S rDNA, the 16S-23S rDNA intergenic spacer region (ISR) and by sequencing the superoxide dismutase A encoding gene sodA. The present study gives the first information about the presence of T. (A.) bernardiae in specimen of animals.

Septicaemia caused by Arcanobacterium haemolyticum smooth type in an immunocompetent patient.

Arcanobacterium haemolyticum is a rarely reported human pathogen but can cause wound infections in elderly patients with immunodeficiency and pharyngotonsillitis in adolescents and young adults. A. haemolyticum septicaemia originating from a wound rarely occurs and mainly affects immunocompromised patients. Here, we report a case of A. haemolyticum septicaemia in an immunocompetent patient with no underlying diseases.

Phenotypic and molecular characterization of Arcanobacterium haemolyticum isolated from clinical samples.

The aim of this study was to assess the phenotypic and genotypic diversity of 56 Arcanobacterium haemolyticum isolates isolated from 51 patients attending primary health care centres and emergency units in the health area of Santander (Cantabria, northern Spain). Phenotypic characterization was based on morphological, biochemical, and antigenic tests. Species identification was confirmed by amplification and sequencing of the 16S rDNA gene. Antimicrobial susceptibility testing was determined by microdilution following the Clinical and Laboratory Standards Institute recommendations for coryneform bacteria. Genetic diversity was evaluated using BOX-PCR and pulsed-field gel electrophoresis. Eighty percent of the isolates had an identical BOX-PCR pattern, suggesting the spread of a single clone. The present report provides extensive information on the phenotypic and genotypic characterization of A. haemolyticum.

Phospholipase D promotes Arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion.

BACKGROUND: Arcanobacterium haemolyticum is an emerging bacterial pathogen, causing pharyngitis and more invasive infections. This organism expresses an unusual phospholipase D (PLD), which we propose promotes bacterial pathogenesis through its action on host cell membranes. The pld gene is found on a genomic region of reduced %G + C, suggesting recent horizontal acquisition. RESULTS: Recombinant PLD rearranged HeLa cell lipid rafts in a dose-dependent manner and this was inhibited by cholesterol sequestration. PLD also promoted host cell adhesion, as a pld mutant had a 60.3% reduction in its ability to adhere to HeLa cells as compared to the wild type. Conversely, the pld mutant appeared to invade HeLa cells approximately two-fold more efficiently as the wild type. This finding was attributable to a significant loss of host cell viability following secretion of PLD from intracellular bacteria. As determined by viability assay, only 15.6% and 82.3% of HeLa cells remained viable following invasion by the wild type or pld mutant, respectively, as compared to untreated HeLa cells. Transmission electron microscopy of HeLa cells inoculated with A. haemolyticum strains revealed that the pld mutant was contained within intracellular vacuoles, as compared to the wild type, which escaped the vacuole. Wild type-infected HeLa cells also displayed the hallmarks of necrosis. Similarly inoculated HeLa cells displayed no signs of apoptosis, as measured by induction of caspase 3/7, 8 or 9 activities. CONCLUSIONS: These data indicate that PLD enhances bacterial adhesion and promotes host cell necrosis following invasion, and therefore, may be important in the disease pathogenesis of A. haemolyticum infections.

Identification of Arcanobacterium haemolyticum isolated from postcastrational complications of a horse.

An Arcanobacterium haemolyticum strain isolated from a postcastrational lesion of a horse was identified phenotypically and genotypically. The latter was performed by sequencing the 16S-23S rDNA intergenic spacer region (ISR), by amplification of the gene encoding A. haemolyticum phospholipase D, by amplification of A. haemolyticum specific parts of ISR-23S rDNA and by amplification of the newly described CAMP factor family protein encoding gene of A. haemolyticum. This indicates (as described previously for seven additional A. haemolyticum strains; Hassan et al. 2009) that A. haemolyticum seems to occur also in infections of horses.

Arcanobacterium abortisuis sp. nov., isolated from a placenta of a sow following an abortion.

A Gram-positive, short diphtheroid-shaped organism was isolated from a sow's placenta of an abortion. This novel isolate, strain Murakami(T), was examined physiologically, chemotaxonomically and phylogenetically. Cells had an irregular V-shaped or palisade arrangement. Colonies appeared translucent on TMVL agar. Cells were strictly anaerobic, negative for catalase and gelatin decomposition and positive for nitrate reduction and soluble starch hydrolysis. Fourteen sugars including glucose were utilized as carbon sources for growth, but 15 sugars including arabinose were not. alpha-Galactosidase, beta-galactosidase, alpha-glucosidase and leucine arylamidase were produced, but beta-glucosidase was not. Fermentation products were lactic, succinic and acetic acids. Sugars of whole cells consisted of rhamnose and ribose. The amino-acid composition of the peptidoglycan was glutamic acid, alanine and lysine in the molar ratio of 1 : 2 : 1. The main fatty acid components of whole cells were C(14 : 0), C(16 : 0), C(16 : 1)omega7 and C(18 : 1)omega9. The bacterial menaquinone was MK-10(H(4)). The polar lipids were phosphatidylethanolamine and two unknown phosphatidylinositol mannosides. The G+C content of the genomic DNA of strain Murakami(T) was 63.8 mol%. Phylogenetic analysis of 16S rRNA gene sequences from strain Murakami(T) and other members of the genus Arcanobacterium supported the phenotypic findings that strain Murakami(T) represents a novel species, for which the name Arcanobacterium abortisuis sp. nov. is proposed. The type strain is Murakami(T) (=ATCC BAA-1522(T) =DSM 19515(T) =JCM 14813(T)).

Synergistic and antagonistic hemolytic activities of bacteria of genus Arcanobacterium and CAMP-like hemolysis of Arcanobacterium phocae and Arcanobacterium haemolyticum with Psychrobacter phenylpyruvicus.

A total of 57 bacteria representing eight species of genus Arcanobacterium (A.) were investigated for hemolytic properties on blood agar containing sheep and rabbit blood and for CAMP-like reactions. An enhanced hemolysis on blood agar containing rabbit blood compared to sheep blood could be observed for A. haemolyticum, less pronounced for A. hippocoleae and A. pluranimalium. A synergistic hemolytic reaction with staphylococcal beta-hemolysin appeared to be constantly visible for A. hippocoleae, A. pluranimalium and A. pyogenes, with Streptococcus agalactiae for A. phocae and A. haemolyticum, with Rhodococcus equi for A. phocae, A. haemolyticum, A. pluranimalium and A. pyogenes and with A. haemolyticum for A. hippocoleae, A. pluranimalium and A. pyogenes, respectively. A reverse CAMP-reaction in the zone of staphylococcal beta-hemolysin could be observed for A.phocae and A.haemolyticum. In addition, a novel CAMP-like reaction could be noted between Psychrobacter phenylpyruvicus, identified by 16S rDNA sequencing, and A. phocae and A. haemolyticum. These synergistic or antagonistic hemolytic properties could possibly be used as additional criteria for identification of bacteria of genus Arcanobacterium.

The roles of PGF(2alpha) and PGE(2) in regression of the corpus luteum after intrauterine infusion of Arcanobacterium pyogenes in cows.

To study the effect of bacteria in the uterus on the fate of the corpus luteum (CL), Arcanobacterium pyogenes was inoculated into the uteri of cows on Day 3 (Day 0=day of spontaneous ovulation). Plasma concentrations of 13,14-dihydro-15-keto-PGF(2alpha) (PGFM), 13,14-dihydro-15-keto-PGE(2) (PGEM) and progesterone (P(4)) were determined. In five cows, the developing CL regressed and first-wave dominant follicles, which normally become atretic, ovulated (Group OV) after bacterial inoculation. In another five cows (Group NOV) and five control cows, the developing CL did not regress and first-wave dominant follicles did not ovulate. In Group OV, PGFM concentrations increased by 126.2pg/mL (from 36.8+/-7.8pg/mL on Day 3 to 163+/-37.2pg/mL on Day 6), with an increase ratio of 5.8-fold. Conversely, in Group NOV, PGFM had a greater increase of 198.4pg/mL (from 128.2+/-27.8pg/mL on Day 3 to 326.6+/-115.1pg/mL on Day 5), but the increase ratio was only 2.3-fold. Although PGEM tended to increase in both groups, raw increases and increase ratios were small. Bacterial inoculation into the uterus stimulated the release of prostaglandins and affected the fate of the CL; in that regard, the CL was affected more by PGF(2alpha) than by PGE(2), and the increase ratio of PGF(2alpha) was more important than the raw increase.