Luteolin increases susceptibility to macrolides by inhibiting MsrA efflux pump in Trueperella pyogenes.

Trueperella pyogenes (T. pyogenes) is an opportunistic pathogen associated with a variety of diseases in many domestic animals. Therapeutic treatment options for T. pyogenes infections are becoming limited due to antimicrobial resistance, in which efflux pumps play an important role. This study aims to evaluate the inhibitory activity of luteolin, a natural flavonoid, on the MsrA efflux pump and investigate its mechanism. The results of antimicrobial susceptibility testing indicated that the susceptibility of msrA-positive T. pyogenes isolates to six macrolides increased after luteolin treatment, while the susceptibility of msrA-negative isolates showed no change after luteolin treatment. It is suspected that luteolin may increase the susceptibility of T. pyogenes isolates by inhibiting MsrA activity. After 1/2 MIC luteolin treatment for 36 h, the transcription level of the msrA gene and the expression level of the MsrA protein decreased by 55.0-97.7% and 36.5-71.5%, respectively. The results of an affinity test showed that the equilibrium dissociation constant (KD) of luteolin and MsrA was 6.462 × 10-5 M, and hydrogen bonding was predominant in the interaction of luteolin and MsrA. Luteolin may inhibit the ATPase activity of the MsrA protein, resulting in its lack of an energy source. The current study illustrates the effect of luteolin on MsrA in T. pyogenes isolates and provides insight into the development of luteolin as an innovative agent in combating infections caused by antimicrobial-resistant bacteria.

Septic hip abscess due to Fusobacterium nucleatum and Actinomyces turicensis in an immunocompetent SARS-CoV-2 positive patient.

A 42-year-old man was referred to the Department of Orthopedic Surgery with pain over his right greater trochanter and signs of systemic infection. CT showed an enhanced mass in his gluteus maximus as well as gas in the biceps femoris over the underlying hip joint. Tissue biopsy yielded Fusobacterium nucleatum and Actinomyces turicensis. The patient was successfully treated for 6 weeks with amoxicillin/clavulanic acid 875mg/125mg and metronidazole 500mg.

Effect of intramolecular disulfide bond of bovine lactoferricin on its molecular structure and antibacterial activity against Trueperella pyogenes separated from cow milk with mastitis.

BACKGROUND: Lactoferricin (Lfcin) is an antimicrobial activity center of lactoferrin, produced by hydrolysis from the N-terminal of lactoferrin. It was hypothesized that the intramolecular disulfide bond in Lfcin could affect its antibacterial function through influencing its molecular structure. To prove this hypothesis, bovine Lfcin (bLfcin) and its two derivatives, bLfcin with an intramolecular disulfate bond (bLfcin DB) and bLfcin with a mutation C36G (bLfcin C36G), were synthesized, purified, and identified. The circular dichroism spectra of the peptides were detected in solutions with different ionic and hydrophobic strength. The antibacterial activity of the peptides against Trueperella pyogenes, separated from cow milk with mastitis, were determined. RESULTS: The secondary structure of bLfcin DB showed more ß-turn and less random coil than the other peptides in H2O, similar ratios of secondary structures with bLfcin and bLfcin C36G under ionic conditions, and close percentages of secondary structure with bLfcin under hydrophobic conditions. The synthetic peptides exhibited strong antimicrobial activity against T. pyogenes isolates, T. pyogenes ATCC 19,411, and E. coli ATCC 25,922. The antimicrobial activities of the three peptides were greater against T. pyogenes than against E. coli, and bLfcin DB exhibited higher antibacterial activity compared with its derivatives. CONCLUSIONS: The intramolecular disulfide bond could change the molecular structure of bLfcin under alternative ionic strengths and hydrophobic effects, and the formation of the disulfide bond is beneficial to executing the antibacterial function of bLfcin.

New Determinants of Aminoglycoside Resistance and Their Association with the Class 1 Integron Gene Cassettes in Trueperella pyogenes.

Trueperella pyogenes is an important opportunistic animal pathogen. Different antimicrobials, including aminoglycosides, are used to treat T. pyogenes infections. The aim of the present study was to evaluate aminoglycoside susceptibility and to detect aminoglycoside resistance determinants in 86 T. pyogenes isolates of different origin. Minimum inhibitory concentration of gentamicin, streptomycin, and kanamycin was determined using a standard broth microdilution method. Genetic elements associated with aminoglycoside resistance were investigated by PCR and DNA sequencing. All studied isolates were susceptible to gentamicin, but 32.6% and 11.6% of them were classified as resistant to streptomycin and kanamycin, respectively. A total of 30 (34.9%) isolates contained class 1 integrons. Class 1 integron gene cassettes carrying aminoglycoside resistance genes, aadA11 and aadA9, were found in seven and two isolates, respectively. Additionally, the aadA9 gene found in six isolates was not associated with mobile genetic elements. Moreover, other, not carried by gene cassettes, aminoglycoside resistance genes, strA-strB and aph(3')-IIIa, were also detected. Most importantly, this is the first description of all reported genes in T. pyogenes. Nevertheless, the relevance of the resistance phenotype to genotype was not perfectly matched in 14 isolates. Therefore, further investigations are needed to fully explain aminoglycoside resistance mechanisms in T. pyogenes.

Minimum inhibitory concentrations of frequently used antibiotics against Escherichia coli and Trueperella pyogenes isolated from uteri of postpartum dairy cows.

The objective of this study was to determine minimum inhibitory concentrations (MIC) of frequently used antimicrobials for Escherichia coli and Trueperella pyogenes isolated from postpartum bovine uteri of cows with acute puerperal metritis (APM, n = 67), cows suspected to have APM (n = 37), and healthy cows (n = 37) and to evaluate possible differences in MIC according to clinical signs. Cows with APM had reddish-brown, fetid vaginal discharge and rectal temperature (RT) ≥39.5°C within 21 d in milk; cows suspected to have APM had either reddish-brown, fetid vaginal discharge or RT ≥39.5°C within 21 d in milk; and healthy cows had neither fetid discharge nor RT ≥39.5°C. Samples were collected from cows on commercial dairy herds (n = 7) using the cytobrush technique. A total of 37 T. pyogenes isolates and 85 E. coli isolates were tested. Ceftiofur, a third-generation cephalosporin that is often used to treat APM, was the focus of analysis. Trueperella pyogenes and E. coli were isolated more often from samples of cows with APM (46 and 90%, respectively) compared with samples from healthy cows (19 and 54%, respectively). Regarding cows suspected to have APM, T. pyogenes and E. coli were numerically more often isolated (30 and 70%, respectively) than in healthy cows (19 and 54%, respectively). Minimum inhibitory concentrations of ceftiofur were low. For T. pyogenes and E. coli, MIC50 (concentration that inhibited growth of 50% of isolates) were 0.25 and 0.5 µg/mL and MIC90 (concentration that inhibited growth of 90% of isolates) were 0.5 and 1 µg/mL, respectively. Although ceftiofur inhibited all T. pyogenes at the highest concentration tested (64 µg/mL), the growth of 5.9% of E. coli was not impaired. Recently, ampicillin has been suggested as an alternative treatment for APM. Although the T. pyogenes isolates exhibited low MIC in general (MIC50 ≤0.015 µg/mL and MIC90 = 0.06 µg/mL) and 81.1% of all T. pyogenes could be inhibited at the lowest ampicillin concentration tested, 11.8% of the E. coli isolates were not impaired at the highest concentration (64 µg/mL) tested in this study. The MIC50 and MIC90 of E. coli were 4 and ≥128 µg/mL, respectively. We detected no difference in the MIC distributions of ceftiofur or ampicillin among isolates from the 3 APM groups. In summary, E. coli with high MIC against ceftiofur as well as against ampicillin were found in this study.

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.

Clinical and microbiological features of Actinotignum bacteremia: a retrospective observational study of 57 cases.

The purpose of this study was to investigate the incidence, clinical presentation, and prognosis of Actinotignum bacteremia in southern Sweden. Actinotignum isolates in blood cultures were identified retrospectively between 1st January 2012 and 31st March 2016 through searches in the clinical microbiology laboratory database. The population covered by this laboratory is approximately 1.3 million. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for species determination. Etests were used for minimum inhibitory concentration (MIC) determination. The patients' medical charts were reviewed. Fifty-eight episodes in fifty-seven patients with Actinotignum bacteremia were identified (A. schaalii = 53, A. sanguinis = 1, A. urinale = 2, and Actinotignum species = 3), which corresponds to an incidence of 11 cases per million inhabitants. Fifty-one percent of the isolates were in pure culture. The MICs were low for ß-lactam antibiotics, whereas high MICs were recorded for ciprofloxacin and trimethoprim. Patients had a median age of 82 years, 72% were male, and a majority had underlying urological conditions. Thirty-six of the patients were diagnosed with a focus from the urinary tract. Thirty-one patients developed severe sepsis and nine patients died during the hospital stay. Our study is the largest of Actinotignum bacteremia and demonstrates that it is a condition with a significant fatality that affects elderly persons with underlying conditions. ß-Lactams represent a rational treatment option.

Actinobaculum schaalii, a new cause of knee prosthetic joint infection in elderly.

Actinobaculum schaalii is an emerging pathogen particularly involved in urinary tract infection of elderly people and/or patient with urological risk factors of urinary tract infection. This microorganism is a difficult-to-diagnose pathogen and is rarely involved in systemic or deep infections. Here, we report the first case of prosthetic joint infection due to A. schaalii in an 84-year-old man with a benign prostatic hyperplasia associated with chronic retention of urine. This case underlines the importance to optimize the diagnosis of emerging uropathogens as A. schaalii, to prevent systemic infections, particularly in patients with orthopaedic implants.

Minimum inhibitory concentrations of selected antimicrobials against Escherichia coli and Trueperella pyogenes of bovine uterine origin.

Minimum inhibitory concentrations (MIC) of 9 antimicrobials for isolates of 2 common bovine intrauterine bacterial pathogens, Escherichia coli (n=209) and Trueperella pyogenes (n=35), were determined using broth microdilution methodology. The isolates were recovered from dairy cows from 7 herds postpartum using the cytobrush technique. The pathogens were initially identified using phenotypic techniques. Additionally, PCR was used to confirm the identity of T. pyogenes isolates and to categorize the E. coli isolates into phylogenetic groups A, B1, B2, and D. Minimum inhibitory concentrations in excess of published cut-points or bimodal distributions of MIC indicated potential antimicrobial resistance to ampicillin, cefuroxime, cephapirin, and oxytetracycline for E. coli, and to oxytetracycline for T. pyogenes. Of the antimicrobials tested, ticarcillin/clavulanic acid, ceftiofur, and enrofloxacin had the lowest MIC for these 2 pathogens. Differences in MIC of some antimicrobials were found between herds, age, breeds, and E. coli phylogenetic groups. Isolation of E. coli with an MIC ≥8µg/mL of oxytetracycline at 23d postpartum was associated with a lower probability of pregnancy within 6wk of commencement of breeding compared with those isolates with an MIC <8µg/mL (relative risk=0.66). Minimum inhibitory concentrations for uterine pathogens were determined for isolates from New Zealand dairy cows. However, in the absence of either epidemiological or clinical interpretive criteria, the interpretation of these MIC remains unclear. Further studies are required to define interpretative criteria, including determination of pharmacokinetic and pharmacodynamic profiles for antimicrobials.

Erm(X)-mediated resistance to macrolides, lincosamides and streptogramins in Actinobaculum schaalii.

OBJECTIVES: Actinobaculum schaalii is a Gram-positive bacillus increasingly reported as a causative agent of urinary tract infections as well as invasive infections, mainly in the elderly and patients with underlying urological conditions. Since little is known about the molecular basis of antimicrobial resistance in A. schaalii, the aim of this study was to investigate resistance to macrolides, lincosamides and streptogramins (MLS) in this emerging pathogen. METHODS: A total of 32 A. schaalii clinical isolates from France and Switzerland were studied. MICs of erythromycin, spiramycin, lincomycin, clindamycin and quinupristin/dalfopristin were determined by the agar dilution method. Resistance genes erm(A), erm(B), erm(C), erm(F), erm(G), erm(X), msr(A) and mef(A) were screened by PCR. The genetic environment was determined by random cloning and PCR mapping. RESULTS: Out of 32 isolates tested, 21 were highly resistant to erythromycin, spiramycin, lincomycin and clindamycin (MICs >256 mg/L), whereas 11 exhibited low MICs (MICs < 0.12 mg/L). On the other hand, quinupristin/dalfopristin remained active against all the isolates. An inducible MLSB resistance phenotype was noted in all cases. The erm(X) gene was detected among all resistant strains, whereas none was detected in susceptible strains. Analysis of genetic support and environment revealed that erm(X) was probably part of the chromosome of A. schaalii. CONCLUSIONS: This study is the first molecular characterization of MLS resistance in A. schaalii. In all cases, it was due to the presence of erm(X), a methylase gene previously identified in other clinically relevant Gram-positive bacilli.

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.

Anti-biofilm and antibacterial activities of zinc oxide nanoparticles against the oral opportunistic pathogens Rothia dentocariosa and Rothia mucilaginosa.

Species of the genus Rothia that inhabit the oral cavity have recently been implicated in a number of diseases. To minimize their role in oral infections, it is imperative to reduce and/or control the growth and biofilm formation activity of Rothia spp. In this study, two bacterial isolates, Ora-7 and Ora-16, were obtained from the oral cavity of a healthy male subject and identified as Rothia dentocariosa and Rothia mucilaginosa, respectively, using a polyphasic taxonomic approach. Antimicrobial and anti-biofilm formation activities of zinc oxide nanoparticles (ZnO-NPs), of average size 35 nm, were assessed in in vitro assays using Crystal Violet and live and dead staining techniques. The ZnO-NPs exhibited an IC50 value of 53 and 76 µg ml(-1) against R. dentocariosa and R. mucilaginosa, respectively. Biofilm-formation assays, performed on the surfaces of polystyrene plates, artificial teeth, and dental prostheses, revealed the efficacy of ZnO-NPs as a potential antibacterial agent for controlling the growth of Rothia isolates in both planktonic form and biofilm.

Trueperella pyogenes and Escherichia coli as an etiological factor of endometritis in cows and the susceptibility of these bacteria to selected antibiotics.

The aim of this study was to determine the percentage of participation of particular species of microorganisms, isolated from the uterus of cows with endometritis and from cows without inflammatory lesions of the uterus, in the same postpartum period. The aim of the study was also to examine how long after parturition non-treated endometritis persists. Moreover, antibiotic susceptibility tests were carried out of the bacterial isolates dominating in the uterus. Forty cows were included in the study: 20 cows with endometritis (experimental group) and 20 cows without any inflammatory condition of the uterus (control group). The material for cytological and bacteriological tests was collected on the 5th, 26th, 40th and 60th day after parturition, using an intrauterine brush adapted for cows. The total number of collected isolates was 149, including 120 isolates from the uterus of cows with endometritis and 29 isolates from the uterus of cows without endometritis. The following species of microorganisms were isolated from the material collected from cows with endometritis: T. pyogenes (49.2%), E.coli (22.5%), F. necrophorum (11.7%), Staphylococcus sp. (6.7%), B. melaninogenicus (5.8%), and Streptococcus sp. (4.1%). The participation percentage of particular species of bacteria in the material collected from the uterus of cows without endometritis was as follows: T. pyogenes (27.6%), E.coli (24.2%), Staphylococcus sp. (20.7%), Streptococcus sp. (20.7%), B. melaninogenicus (3.4%) and F. necrophorum (3.4%). The highest percentage of T. pyogenes isolates was susceptible to ceftiofur (89.6%); cefoperazone (85.1%) and amoxicillin combined with clavulanic acid (79.1%). E. coli isolates were most susceptible to amoxicillin combined with clavulanic acid (100%), cefoperazone (94.1%) and oxytetracycline (82.3%).

Whole genome-based antimicrobial resistance, virulence, and phylogenetic characteristics of Trueperella pyogenes clinical isolates from humans and animals.

Trueperella pyogenes is an opportunistic zoonotic bacterial pathogen, whose antimicrobial resistance, virulence, and genetic relatedness between strains from animals and humans are barely studied. These characteristics were therefore analyzed for clinical T. pyogenes strains from 31 animals of 11 different species and 8 humans determining their complete circular genome sequence and antimicrobial susceptibility. The MICs of 19 antimicrobials including 3 antiseptics correlated to the resistance genes identified in silico within the genomes revealing a predominance of resistance to streptomycin (aadA9), sulfamethoxazole (sul1), and tetracycline (tet(33), tet(W/N/W)) among strains from humans and cattle. Additional resistance genes (erm(X), erm(56), cmx, drfA1, aadA1, aph(3'')-Ib (strA), aph(6)-Id (strB), aac(3)-IVa, aph(4)-Ia) were found only sporadically. The resistance genes were localized on genetic elements integrated into the chromosome. A cgMLST-based phylogenetic analysis revealed two major clusters each containing genetically diverse strains. The human strains showed the closest relatedness to strains from cattle. Virulence genes coding for fimbriae (fimA, fimC), neuroamidase (nanP, nanH), pyolysin (plo), and collagen binding protein (cbpA) were identified in strains from different hosts, but no correlation was observed between virulence factors and strain origin. The existence of resistance genes typically found in Gram-negative bacteria within the Gram-positive T. pyogenes indicates a wider capacity to adapt to antimicrobial selective pressure. Moreover, the presence of similar antimicrobial resistance profiles found in cattle and human strains as well as their closest relatedness suggests common zoonotic features and cattle as the potential source for human infections.

Polymicrobial bloodstream infection involving Oligella urethralis and Trueperella bernardiae: a case report and literature review.

Oligella urethralis and Trueperella bernardiae are rare causes of bacteremia. A 96-year-old female nursing home resident with diabetes presented with polymicrobial bloodstream infection comprising these organisms and obstructive urinary tract infection. O. urethralis identified from blood cultures was resistant to fluoroquinolones and non-fourth-generation cephalosporins. Cases must be accumulated to define the clinical features of these organisms for infection control.

Antimicrobial efficiency of bromhexine hydrochloride against endometritis-causing Escherichia coli and Trueperella pyogenes in bovines.

In this study, the main agents associated with endometritis in cows in the state of Santa Catarina, Brazil, were identified and the resistance profile and virulence mechanisms of the bacterial isolates were evaluated. Isolates of Escherichia coli and Trueperella pyogenes were tested for their biofilm forming ability and the antimicrobial action of bromhexine hydrochloride in combination with other antimicrobials. A total of 37 uterine lavage samples were collected from cows with endometritis. Of the 55 bacteria isolated, 25.4% were identified as T. pyogenes and 16.3% as E. coli. The bacterial isolates showed greater resistance to sulfamethoxazole + trimethoprim (58.2%) and tetracycline (56.3%). Among the species, E. coli showed the highest resistance rates, with 100% of isolates showing resistance to amoxicillin, streptomycin, and gentamicin. The results of the minimum inhibitory concentration for the T. pyogenes isolates showed that 91.6% of the isolates were resistant to enrofloxacin and tetracycline, and 75% were resistant to ceftiofur and sulfamethoxazole + trimethoprim. All E. coli and T. pyogenes isolates showed biofilm forming ability. The plo, fimA, and nanH genes were identified in 100% of T. pyogenes isolates. In parallel, 100% of E. coli isolates had the fimH gene, and 11.1% had the csgD gene. Bromhexine hydrochloride showed antimicrobial activity against 100% of E. coli isolates and 66.6% of T. pyogenes isolates. Furthermore, when associated with antimicrobials, bromhexine hydrochloride has a synergistic and additive effect, proving to be an option in the treatment of endometritis in cows and an alternative for reducing the use of antimicrobials.

Virulence determinants and biofilm production among Trueperella pyogenes recovered from abscesses of captive forest musk deer.

Trueperella pyogenes (formerly Arcanobacterium) is commonly isolated from domesticated or wild ruminants as an opportunistic pathogen. To investigate the role of virulence determinants (VDs) and biofilm production in T. pyogenes isolates, a total of 36 T. pyogenes were collected from abscesses of forest musk deer in Miyaluo Farm (Sichuan Province, China). The prevalence of VDs and associations with clonal types, antibiotic resistance and biofilm production were analyzed by PCR and bioassay. Finally, T. pyogenes isolates were separated into three clonal types based on the DNA fingerprinting of BOX-PCR. Isolates with less VDs obtained from sick forest musk deer were mainly belonged to Type 1, and the isolates with robust VD repertoire obtained from dead forest musk deer were included in Type 3. Accordingly, resistant isolates exhibited significant lower virulence than susceptible ones. Majority of T. pyogenes isolates of this study were capable of producing a biofilm. However, no VDs presence and antibiotic resistance were statistically associated with biofilm production. In conclusion, the current study demonstrated that T. pyogenes was probably the primary pathogen of abscesses in the forest musk deer. Moreover, as an animal origin pathogen, the increasing resistance of T. pyogenes isolates could also associate with a decreased virulence.

An in vitro comparison of antimicrobial efficacy and cytotoxicity between povidone-iodine and chlorhexidine for treating clinical endometritis in dairy cows.

This study aimed to assess the in vitro antimicrobial effects of chlorhexidine (CHX) and povidone-iodine (PI) on clinical isolates of Escherichia coli (E. coli) and Trueperella pyogenes (T. pyogenes) from the vaginal discharge of dairy cows, as well as to compare the cytotoxicity effects of CHX and PI on bovine endometrial epithelial cells (BEnEpC). In Experiment 1, 12 E. coli and 10 T. pyogenes were isolated from the vaginal discharge of cows with a uterine infection. The MIC and MBC against CHX and PI were analyzed in vitro. In Experiment 2, the cytotoxicity effects of CHX and PI on BEnEpC were analyzed using a Viability/Cytotoxicity Kit, wound scratch healing assay, and the expression of pro-inflammatory cytokine genes (IL-6, IL-8, and TNF-α). In Experiment 1, the MIC and MBC values of CHX against E. coli were 0.0002% and 0.0002 to 0.00025%, respectively. The MIC and MBC values of PI were 1.25 to 2.5% and 1.25 to 5%, respectively. For T. pyogenes, the MIC and MBC values of CHX were 0.00002%. The MIC and MBC values of PI were 1.25%. In Experiment 2, the cell viability significantly decreased, and wound closures were significantly inhibited after treatment with ≥ 0.002% CHX and ≥ 0.025% PI. The expression of IL-6, IL-8, and TNF-α significantly increased after treatment with PI. Only IL-6 showed a significant increase after cells were treated with 0.00002% and 0.0002% CHX. The results suggested that both CHX and PI had high antibacterial effects. However, veterinarians and farmers should be aware of their cytotoxicity, which decrease viability of endometrial epithelial cells and inhibit wound healing in vitro.