Effects of Luteolin on Biofilm of Trueperella pyogenes and Its Therapeutic Effect on Rat Endometritis.

Trueperella pyogenes is an opportunistic pathogen that causes suppurative infections in animals. The development of new anti-biofilm drugs will improve the current treatment status for controlling T. pyogenes infections in the animal husbandry industry. Luteolin is a naturally derived flavonoid compound with antibacterial properties. In this study, the effects and the mechanism of luteolin on T. pyogenes biofilm were analyzed and explored. The MBIC and MBEC of luteolin on T. pyogenes were 156 µg/mL and 312 µg/mL, respectively. The anti-biofilm effects of luteolin were also observed by a confocal laser microscope and scanning electron microscope. The results indicated that 312 µg/mL of luteolin could disperse large pieces of biofilm into small clusters after 8 h of treatment. According to the real-time quantitative PCR detection results, luteolin could significantly inhibit the relative expression of the biofilm-associated genes luxS, plo, rbsB and lsrB. In addition, the in vivo anti-biofilm activity of luteolin against T. pyogenes was studied using a rat endometritis model established by glacial acetic acid stimulation and T. pyogenes intrauterine infusion. Our study showed that luteolin could significantly reduce the symptoms of rat endometritis. These data may provide new opinions on the clinical treatment of luteolin and other flavonoid compounds on T. pyogenes biofilm-associated infections.

The phylogenetic group, antimicrobial susceptibility, and virulence genes of Escherichia coli from clinical bovine mastitis.

Bovine mastitis is still a central problem on dairy farms despite control programs, and Escherichia coli is a crucial pathogen during the development of bovine mastitis. The virulence genes, antimicrobial susceptibility, and mortality of mice infected with different E. coli isolates from bovine mastitis were determined in this study. According to the presence of the specific genes chuA, yjaA, and TspE4.C2, these isolates mainly belonged to 2 different groups: group A (47/79) and group B1 (22/79). The ompC gene was detected in all the isolates, followed by fimH (89.9%), ECs3703 (88.6%), and ompF (73.4%), whereas most of the virulence genes were not detected in these isolates. The results of the antimicrobial susceptibility tests indicated that the isolates were susceptible to the fluoroquinolones and aminoglycosides. An inverse relationship was shown between the expression level of ompF and antimicrobial resistance; additionally, the isolates that were nonsusceptible to at least 4 classes of antimicrobial agents showed a lower mortality to mice in comparison with the susceptible isolates. This study indicated that antibiotic resistance had emerged in E. coli from bovine mastitis in this area, and appropriate measures should be taken to avoid potential threats to humans and other animals.

Trueperella pyogenes isolated from dairy cows with endometritis in Inner Mongolia, China: Tetracycline susceptibility and tetracycline-resistance gene distribution.

Trueperella pyogenes plays a crucial role in endometritis pathogenesis and is also associated with many infections, including metritis, mastitis, arthritis and liver abscessation, in many domestic animals. In this study, we investigated the prevalence of tetracycline resistance in T. pyogenes isolated from dairy cows with endometritis in Inner Mongolia, China, and we assessed tetracycline-resistance gene distribution among the isolates. Our results indicated that 68.7% and 62.5% of the isolates were resistant to tetracycline and doxycycline, respectively, and the rate of resistance to metacycline was 18.8%. The tetracycline resistance gene tetK was present in all isolates (n = 32), whereas the tetM gene was identified in 12.5% and 9.4% of the isolates, in the chromosome and plasmid, respectively. Strains carrying tetW were also common in the chromosome and plasmid, with abundances of 53.1% and 46.9%, respectively. However, tetO and otrA were absent in all isolates. The resistance phenotype analysis indicated that 6.3% of strains were susceptible to all tetracyclines, while 3.1% showed resistance to all tetracyclines.

Antibacterial and antioxidant properties of various solvents extracts of Abutilon theophrasti Medic. leaves.

This paper described the extraction procedure of six extracts from Abutilon theophrasti Medic. leaves and evaluated antioxidant and antibacterial activity of different extracts by hydroxyl radical, DPPH radical scavenging, broth micro-dilution and agar-well diffusion methods. The six extracts were prepared by the two extraction procedures: (I) water was the extraction solvent; (II) 90% alcohol extract was extracted by petroleum ether, chloroform, ethyl acetate and n-butanol in turn. Extract yields were 7.34%, 7.31%, 0.45%, 0.12%, 2.70% and 5.68% for extract I to VI. It was revealed that the various extracts had effective antibacterial activity against four test strains from Staphylococcus aureus (ATCC 25923), Streptococcus (ATCC 49619), Escherichia coli (ATCC 25922) and Salmonella (ATCC 01303); meanwhile, the six extracts demonstrated potent antioxidant activity, achieved by hydroxyl radical and DPPH radical scavenging assay. Minimum inhibitory concentrations (MICs) for the bacterial species ranged from 2.21 to 539.46 mg/ml, diameter of inhibition zone ranged from 2.08 to 15.05mm. The scavenging •OH and DPPH• rates were 62.37% to 81.86% with the concentration 0.06 to 1.89mg/ml and 37.80% to 81.23% with the concentration 1.07 to 35.52mg/ml. According to the results, these extracts have antioxidant and antibacterial activity. In view of all the facts collectively, the six extracts will become natural and nontoxic antioxidant and antibacterial agent, and be applied in food and pharmaceutical industries for the prevention or treatment caused by microorganisms and free radicals.

Resistance to ß-lactam antibiotic may influence nanH gene expression in Trueperella pyogenes isolated from bovine endometritis.

Virulence could be modulated by many instinctive and environmental factors including oxygen, osmolarity and antimicrobial agents. This study aimed to investigate the correlation between drug resistance and the nanH expression in Trueperella pyogenes (T. pyogenes). Minimum inhibitory concentrations (MICs) of 6 ß-lactam antimicrobial agents (penicillin G, amoxicillin, oxacillin, cefazolin, ceftiofur, and ampicillin) against T. pyogenes were tested by standard broth dilution method according to the protocols of the Clinical and Laboratory Standards Institute (CLSI), and real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) was selected to investigate the mRNA expression levels of the nanH in T. pyogenes. All the isolates were resistant to atleast 2 of antimicrobial agents, and multidrug resistance (resistance to atleast 3 antimicrobials) was observed in 84.38% (27/32) of isolates. The mRNA expression levels of the nanH were significantly higher in comparison with that in ATCC19411, as the resistance profile enlarged, the nanH mRNA expression levels decreased in T. pyogenes. These results indicated that ß-lactam antibiotic resistance in T. pyogenes may alter the expression of the nanH.

In vitro Antibiotic Susceptibility, Virulence Genes Distribution and Biofilm Production of Staphylococcus aureus Isolates from Bovine Mastitis in the Liaoning Province of China.

PURPOSE: The aim of this study was to identify the subtype, characterize the antimicrobial resistance, determine the virulence gene distribution, and analyze the biofilm production of Staphylococcus aureus isolates from bovine mastitis milk samples in the Liaoning Province of China. MATERIALS AND METHODS: In total, 56 Staph. aureus isolates were collected and identified in this study; the isolates were divided into different spa types based on the sequence of the polymorphic X region of the spa gene. Additionally, antimicrobial susceptibility was investigated using the broth microdilution method, and 18 virulence genes were detected using PCR. Biofilm formation was measured by spectrophotometry with crystal violet staining and observed using confocal laser scanning microscopy. RESULTS: There were 12.12% (56/462) milk samples that were positive for Staph. aureus. These isolates were nonsusceptible to sulfamethoxazole (100%), penicillin (76.9%), daptomycin (76.79%), clindamycin (69.64%), and oxacillin (60.71%); however, the majority of the isolates (80.4%) were susceptible to amoxicillin/clavulanate. The predominant virulence genes encoded the cytotoxins, hla (94.64%) and hlb (89.29%), and the adhesion factors clfA (89.29%), clfB (89.29%), and fnbB (80.36%). Comparatively, virulence genes related to other adhesion factors such as cna (8.93%) and enterotoxins, such as seg (26.79%), sea (16.07%), seb (7.14%), and sec (7.14%) were detected at relatively lower rates. The following eight spa types were identified: t267 (35.84%), t730 (22.64%), t518 (15.09%), t1190 (11.32%), t1456 (9.43%), t224 (1.88%), t9129 (1.88%), and t177 (1.88%). The highest biofilm production was observed for t267. Staph. aureus exhibited various patterns of biofilm formation, with the biofilm often being associated with a tower-shaped structure or a thicker biofilm. CONCLUSION: Our results indicated that Staph. aureus isolates from dairy cows with mastitis in the Liaoning Province of China were non-susceptible to sulfamethoxazole, penicillin, daptomycin, oxacillin, and clindamycin. Additionally, the most prevalent subtype was t267, which displayed resistance to multiple antimicrobial agents and harbored several virulence genes, including clfA, clfB, fnbB, hla, and hlb.

Luteolin Showed a Resistance Elimination Effect on Gentamicin by Decreasing MATE mRNA Expression in Trueperella pyogenes.

Trueperella pyogenes is a common inhabitant of mucosal surfaces in animals and causes a variety of infections, including endometritis, mastitis, and liver abscessation, in dairy cows. Many antimicrobial agents are used for treatment of infections caused by T. pyogenes; however, antibiotic resistance has recently become a serious problem. The objective of this study was to characterize the effect of the efflux pump-encoding multidrug and toxic compound extrusion (MATE) gene on antibiotic resistance in T. pyogenes isolates from cows with signs of endometritis. As a compound from plants, luteolin showed antimicrobial activities in Escherichia coli and Staphylococcus aureus; therefore, we also investigated whether luteolin can eliminate antibiotic resistance. We constructed a MATE deletion mutant in BM-H06-3 to identify the function of MATE in antibiotic resistance. MATE mRNA expression was measured to identify the mechanism of luteolin in gentamicin resistance elimination effect in T. pyogenes. The T. pyogenes isolate BM-H06-3 became susceptible to gentamicin, amikacin, streptomycin, erythromycin, and roxithromycin after MATE deletion. No synergistic effect between luteolin and gentamicin was observed in eight isolates, which were randomly selected from 34 T. pyogenes isolates, but the isolates became susceptible to gentamicin after luteolin treatment at a subinhibitory concentration (1/4 minimum inhibitory concentration [MIC]) for 36 hr. Furthermore, luteolin can decrease MATE mRNA expression after luteolin treatment at a subinhibitory concentration (1/4 MIC). We found that the MATE gene was involved in antibiotic resistance and that luteolin induces a resistance elimination effect in T. pyogenes. Therefore, luteolin may be a potential agent to inhibit efflux pumps in multidrug-resistant T. pyogenes.