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LC-MS/MS quantitative analysis of the bacterial secondary messenger molecule, cyclic di-GMP, from uterine infections to detect P. aeruginosa biofilms. Elevated cyclic di-GMP levels were detected in a majority of tissue-adherent bacterial samples, except for those from samples obtained from horse 5, which were not elevated compared to those of control samples. Four samples of intraluminal fluid and tissue-adherent bacteria were collected at random locations from each infected uterus (n = 6). Four control samples were collected by uterine biopsy procedure from two uninfected mares. Amounts are represented as picomoles of cyclic di-GMP per gram of sample. The calculated limit of detection (LOD) is 0.64 pmol, and the calculated limit of quantification (LOQ) is 2.14 pmol. Experimental samples range from 3.1 pmol/g to 2,033 pmol/g cyclic di-GMP. Samples from which no cyclic di-GMP was detected are represented under the LOD line.
Source publication
Bacteria in a biofilm community have increased tolerance to antimicrobial therapy. To characterize the role of biofilms in equine endometritis, six mares were inoculated with lux -engineered Pseudomonas aeruginosa strains isolated from equine uterine infections. Following establishment of infection, the horses were euthanized and the endometrial su...
Citations
... In 2017, Ryan A. Ferris and colleagues declared that the clinical isolates of P. aeruginosa from the equine uterus can produce a biofilm [29]. In other words, P. aeruginosa can lead to chronic endometritis that resists treatment. ...
... Some E. coli strains can replicate intracellularly (17) but it is not known whether this occurs in the equine endometrium. The healthy uterus harbors a core uterine microbiome of which bacteria of the Escherichia genus are a part, together with Lactobacillus, Shigella, Streptococcus, Blautia, Staphylococcus, Klebsiella, Acinetobacter, and Peptoanaerobacter (18). Persistent or recurrent infections are likely associated with mare-dependent factors, such as altered uterine immunity, or anatomical or functional defects (2). ...
... Mare age at biopsy (years) 14.5 ± 1.1 (10)(11)(12)(13)(14)(15)(16)(17)(18)(19) 14.2 ± 2.0 (9-20) 13.8 ± 2.1 (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) Mare status at biopsy 2 maiden Frontiers in Veterinary Science 05 frontiersin.org urinary tract and endometrium, differences between host species, or the selection and low number of cases. ...
... Mare age at biopsy (years) 14.5 ± 1.1 (10)(11)(12)(13)(14)(15)(16)(17)(18)(19) 14.2 ± 2.0 (9-20) 13.8 ± 2.1 (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) Mare status at biopsy 2 maiden Frontiers in Veterinary Science 05 frontiersin.org urinary tract and endometrium, differences between host species, or the selection and low number of cases. ...
Endometritis is one of the major causes of infertility in mares. Escherichia coli and β-haemolytic streptococci are among the bacterial species most frequently isolated from the equine uterus. Some bacteria such as β-hemolytic streptococci, can persist in dormant forms and cause prolonged, latent or recurrent infections. Dormant bacteria may be present despite negative bacterial cultures, and they are resistant to antimicrobial treatment due to their resting metabolic state. The purpose of this study was to study formalin-fixed paraffin-embedded equine endometrial biopsies for the presence and localization of E. coli—bacteria, with a chromogenic RNAscope®-method for detection of E. coli-related 16S ribosomal RNA. Hematoxylin-eosin—stained endometrial biopsies were evaluated to determine the level of inflammation and degeneration. During estrus, samples were taken for endometrial culture and cytology with a double-guarded uterine swab. The samples included eight samples with moderate to severe endometrial inflammation detected in endometrial histopathology, and growth of E. coli in bacterial culture, six samples with moderate to severe endometrial inflammation but negative bacterial culture, and five samples with no endometrial pathology (grade I endometrial biopsy, negative endometrial culture and cytology) serving as controls. Positive and negative control probes were included in the RNA in situ hybridization, and results were confirmed with a fluorescence detection method (fluorescence in situ hybridization). Only unspecific signals of limited size and frequency of occurrence were detected in all samples, with random localization in the endometrium. No samples contained rod-shaped signals corresponding to bacterial findings. In conclusion, there was no evidence of bacterial invasion in the endometrium regardless of the inflammatory status of the biopsy or previous bacterial culture results. According to these findings on a small number of samples, invasion of E. coli is not a common finding in the lamina propria of mares, but these bacteria may also evade detection due to localized foci of infections, or supra-epithelial localization under the cover of biofilm. These bacteria and biofilm covering the epithelium may also be lost during formalin-fixation and processing.
... When opening the uterus, the presence of a biofilm was indicated by observations of a luminescent, strongly adherent material, which was positive for P. aeruginosa and was observed on the endometrial surface. The model was further used to study the spatial intrauterine localization of metabolically active bacteria in six mares [64]. The biofilm matrix component Pel was observed in endometrial samples with tissue-adherent bacteria ( Figure 5). ...
... The scale bar is 4 μm. Reprinted from: Ferris et al. 2017[64]. ...
... The scale bar is 4 µm. Reprinted from: Ferris et al. 2017[64]. ...
Biofilms are bacterial aggregates embedded in a self-produced, protective matrix. The biofilm lifestyle offers resilience to external threats such as the immune system, antimicrobials, and other treatments. It is therefore not surprising that biofilms have been observed to be present in a number of bacterial infections. This review describes biofilm-associated bacterial infections in most body systems of husbandry animals, including fish, as well as in sport and companion animals. The biofilms have been observed in the auditory, cardiovascular, central nervous, digestive, integumentary, reproductive, respiratory, urinary, and visual system. A number of potential roles that biofilms can play in disease pathogenesis are also described. Biofilms can induce or regulate local inflammation. For some bacterial species, biofilms appear to facilitate intracellular invasion. Biofilms can also obstruct the healing process by acting as a physical barrier. The long-term protection of bacteria in biofilms can contribute to chronic subclinical infections, Furthermore, a biofilm already present may be used by other pathogens to avoid elimination by the immune system. This review shows the importance of acknowledging the role of biofilms in animal bacterial infections, as this influences both diagnostic procedures and treatment.
... Therefore, rabbits were selected as experimental animals to establish the pathological model of equine endometritis and detect the relevant pathological indicators. Existing studies have shown that IL-1β, IL-6, TNF-a, and other factors play a crucial role in the occurrence and development of inflammation, which may lead to inflammation by activating the NF-kB signaling pathway (18)(19)(20)(21)(22). ...
Pathogenic bacteria were isolated from the uterine lavage fluid of a mare with endometritis. After identification and purification, the pathogenic bacteria were injected into the uterus of rabbits to induce endometritis. Then, anatomical, blood routine, chemical examination, and histopathological examinations were performed on the rabbits. Rabbit uterus was collected, and quantitative polymerase chain reaction (qPCR) was used to detect the mRNA expression of inflammatory factors including IL-1β, IL-6, and TNF-α in the rabbit uterus. In addition, enzyme-linked immunosorbent assay (ELISA) was used to detect the uterine concentrations of the inflammatory factors IL-1β, IL-6, and TNF-α. Western Blot was used to detect the protein expressions of NF-kB, IkBα, and TNF-α in the NF-kB pathway. An antibiotic treatment group was also set up to verify the accuracy of the results. The clinical examination results showed that there was a significant increase of leukocytes in the blood of the rabbits in the model group (P < 0.01). The uterus was congested, enlarged, and purulent. The integrity of the uterine lining was destroyed, and there was a significant increase of lymphocytes in the uterus (P < 0.01). The qPCR and ELISA results showed that the expressions of the inflammatory factors IL-1β, IL-6, and TNF-α in the uterus of rabbits were significantly increased (P < 0.01). Western blot results showed that the inflammatory factors IL-1β, IL-6, and TNF-α play a role in promoting inflammation through the NF-kB pathway. The results of the test provide a simple, economical, and reliable means of studying the occurrence, development, prevention, and treatment of equine endometritis.
... The clinical literature continues to describe the uterus of a normal (healthy) mare as a site that does not host a resident microbial community 11 . However, previous studies based on in vitro culturing indicate that, a variety of microorganisms may be present in the uterus of a clinically healthy mare 12 . ...
The goal of this study was to understand the composition and existence of the resident uterine microbiome in healthy mares and to establish the presence of a core microbiome for the healthy equine uterus. We analyzed the microbiomes of 35 healthy mares that are long-time residents of three farms in Oklahoma, Louisiana, and Australia as well as that of 19 mares purchased from scattered owners in the Southern Mid-Western states of the United States. Over 6 million paired-end reads of the V4 region of the 16S rRNA gene were obtained resulting in 19,542 unique Amplicon Sequence Variants (ASVs). ASVs were assigned to 17 known phyla and 213 known genera. Most abundant genera across all animals were Pseudomonas (27%) followed by Lonsdalea (8%), Lactobacillus (7.5%), Escherichia/Shigella (4.5%), and Prevotella (3%). Oklahoma and Louisiana samples were dominated by Pseudomonas (75%). Lonsdalea (28%) was the most abundant genus in the Australian samples but was not found in any other region. Microbial diversity, richness, and evenness of the equine uterine microbiome is largely dependent on the geographical location of the animal. However, we observed a core uterine microbiome consisting of Lactobacillus, Escherichia/Shigella, Streptococcus, Blautia, Staphylococcus, Klebsiella, Acinetobacter, and Peptoanaerobacter.
... In previous studies, most of the bioluminescent reporter genes, such as lux, luc, ruc, and gfp, were expressed in the recipient hosts through plasmid transformation methods [31,[59][60][61]. The reporter genes were, therefore, not integrated into the host chromosome and were easily lost, even without selection pressure [62]. ...
Some prevention strategies, including vaccines and antibiotic alternatives, have been developed to reduce enterotoxigenic Escherichia coli proliferation in animal production. In this study, a wild-type strain of BE311 with a virulent heat-stable enterotoxin gene identical to E. coli K99 was isolated for its high potential for gene expression ability. The whole genome of E. coli BE311 was sequenced for gene analyses and editing. Subsequently, the fluorescent gene mCherry was successfully knocked into the genome of E. coli BE311 by CRISPR/Cas9. The E. coli BE311–mCherry strain was precisely quantified through the fluorescence intensity and red colony counting. The inflammatory factors in different intestinal tissues all increased significantly after an E. coli BE311–mCherry challenge in Sprague–Dawley rats (p < 0.05). The heat-stable enterotoxin gene of E. coli BE311 was knocked out, and an attenuated vaccine host E. coli BE311-STKO was constructed. Flow cytometry showed apoptotic cell numbers were lower following a challenge of IPEC-J2 cells with E. coli BE311-STKO than with E. coli BE311. Therefore, the E. coli BE311–mCherry and E. coli BE311-STKO strains that were successfully constructed based on the gene knock-in and knock-out technology could be used as ideal candidates in ETEC challenge models and for the development of attenuated vaccines.
... In 2017, Ryan A. Ferris and colleagues declared that the clinical isolates of P. aeruginosa from the equine uterus can produce a biofilm [29]. In other words, P. aeruginosa can lead to chronic endometritis that resists treatment. ...
Bacterial infections of the uterus are known to be an important cause of infertility in the mare. The objective of this study was to determine the species of bacteria isolated from the uterus of infertile Arabic mares, and investigate how identified bacteria are related to parity. A total of 18 Arabic mares with a history of long-term infertility were evaluated. The age range of mares was 4-23 years. For statistical analysis, logistic regression and Chi-square test were used by Proc Logistic of SAS. In this study, low-volume uterine flush and culture techniques were used. P. aeruginosa was the most prevalent bacterium isolated from 26.32% of mares as pure or in conjunction with E. coli, K. pneumoniae, or Citrobacter spp. Furthermore, 23.68% of bacterial infertility cases were related to E. coli. Pure growth of E. coli was observed only in one case. However, mixed growth with P. aeruginosa, S.zooepidemicus, and S. aureus was very prevalent. The present study revealed that the most prevalent bacteria isolated from chronic endometritis in Arabic mares were gram-negative bacteria (p < 0.05), while in some cases may be accompanied by gram-positive bacteria. C. albicans was isolated in only 8% of mares with chronic endometritis. Moreover, older age and higher parity number of the mares were not related to the presence of intrauterine fluid or the species of bacteria (p > 0.05). It can be concluded that the most prevalent bacteria isolated from infertile Arabic mares with chronic endometritis are gram-negative bacteria.
key words: mare, uterus, endometritis, bacteria, parity
... In veterinary medicine, researches involving biofilm formation, treatments and preventions have been limited; however, it is still possible to find few studies demonstrating biofilm-forming bacteria associated with different comorbidities such as otitis, wound infections, UTIs, and endometritis [5,6,14,17]. These studies generally select dogs with chronic and recurrent infections, which could be an important factor in antibiotic resistance. ...
Background: Biofilms have been reported as important virulent markers associated with drug resistance in urinary tract infections (UTIs) in humans and dogs. However, in veterinary medicine, researches involving biofilm formation, treatments and preventions have been limited; yet, it is still possible to find few studies demonstrating biofilm-forming bacteria associated with different comorbidities such as otitis, wound infections, UTIs, and endometritis. These studies generally select dogs with chronic and recurrent infections, which could be an important factor in antibiotic resistance. We aimed to evaluate biofilms in sporadic cystitis regarding prevalence and drug resistance.Materials, Methods & Results: Urine samples were collected by cystocentesis from 36 client-owned dogs under clinical and laboratory suspicion of non-recurrent urinary bladder infection (cystitis). Urine was aseptically plated onto blood agar, MacConkey, and CLED, followed by incubation for 24 to 48 h. Definitive identification of a potential pathogen was made by subculture collected from an isolated colony to obtain a pure culture. The gram staining method and specific biochemical tests (phenol red fermentation, lysine, phenylalanine, citrate, sulfide-indole-motility, and urease) were used to distinguish and classify the bacteria. After identification, the bacteria were tested for antimicrobial susceptibility by a standard disk diffusion method, using the following antimicrobials: amoxicillin with clavulanic acid, ampicillin, ceftriaxone, ciprofloxacin, clindamycin, cefazolin, cephalothin, erythromycin, gentamicin, norfloxacin, and sulfamethoxazole-trimethoprim. The biofilm-forming ability was determined based on a culture in Congo red agar (CRA), where biofilm producer strains formed black colonies with a dry crystalline surface, while non-biofilm producer strains formed red colonies with a smooth surface. A crystal violet dye assay was used to confirm the CRA results. Of the 36 urine samples collected from dogs with suspected cystitis, a total of 37 isolates were obtained, from mixed or pure cultures. The most prevalent bacteria were Escherichia coli (11/37), followed by Staphylococcus spp. (8/37), Proteus spp. (7/37), and Enterococcus spp. (5/37). Other less prevalent bacteria were Klebsiella spp., Streptococcus spp., and Enterobacter spp. As for biofilm-forming ability, 67.6% (25/37) of the 37 bacterial isolates had biofilm formation in CRA and 54.05% (20/37) on the microplates containing crystal violet dye. There was no statistical difference in antimicrobial susceptibility between biofilm producer and non-biofilm producer bacteria.Discussion: We found a high proportion (> 54%) of in vitro biofilm-forming ability by different bacteria, which may indicate that biofilms may also be formed in vivo, in simple cystitis. Antimicrobial resistance was not noticed in bacteria capable of forming a biofilm; however, in a future study it is important to evaluate bacterial resistance in vivo, considering the possibility of having a different response than in vitro. In addition, the problem of the presence of a biofilm in vivo is that it can nullify the antimicrobial efficacy of therapeutic agents even with in vitro susceptibility. Besides the possibility of slow or incomplete diffusion of antibiotics through the extracellular matrix of the biofilm, aspects like hydration level, pCO2, pO2, pH, pyrimidine, and divalent cation concentration that negatively influence antimicrobial activity in vitro can also cause undesirable effects at the profound layers of the biofilm. In conclusion, all of the genera of bacteria isolated from dog’s sporadic cystitis in this study were able to form a biofilm in vitro. The pathogenicity and antibiotic resistance of bacteria appears unrelated to biofilm formation in vitro.Keywords: sessile bacteria, urine, simple cystitis, antibiotic resistance.
... Some strains of Klebsiella can produce virulence factors such as lipopolysaccharide (LPS), which acts on Toll-like receptor 4 (TLR4) to induce fibrosis and inflammation [43]. Pseudomonas aeruginosa can produce a biofilm in the uterus, and the host immune response is modulated focally around areas with biofilms [44]. Whether the microorganisms isolated in the present study affected or were one of the major causative factors of CSD niche formation requires further investigation. ...
Background
Delayed childbearing has been noted in a high percentage of women with a previous Caesarean section (CS). Many women with CS scar defects (CSDs) present with clinical symptoms of irregular vaginal bleeding. The present study aimed to investigate bacterial colonies at CSDs in women suffering from secondary infertility.
Methods
This observational study included 363 women with secondary infertility who visited the Assisted Reproduction Unit between 2008 and 2013. Among them, 172 women with a previous CS and 191 women with no previous CS were approached. The women with a previous CS had their CS operations in the past 1 to 14 years, with a mean of 3.5 years. The presence of CSDs was detected by vaginal ultrasonography. Bacteriology cultures of specimens taken from the uterine niches in those with CSDs were collected during Day 7 to Day 10 of the follicular phase. Specimens were obtained from the endocervical canal for bacterial culture in those without CSDs. The main outcome measure was the detection of the growth of bacterial colonies.
Results
CSDs were found in 60.4% (96 of 159) of women with a previous CS. In women with a previous CS, bacterial colonies were identified in 89.6% (86 of 96) and 69.8% (44 of 63) of women with and without CSDs, respectively. In women with no previous CS, 49.7% (88 out of 177) of bacterial cultures of endocervical samples showed bacterial colony growth. Gram-positive cocci (P = 0.0017, odds ratio (OR) = 1.576, 95% confidence intervals (CI) -22.5 to − 5.4) and Gram-negative rods (P = 0.0016, OR = 1.74, CI − 20.8 to − 5.0) were the most commonly isolated bacteria and contributed to approximately 90% of all microorganisms found in those with a previous CS. In women with a previous CS, more Gram-negative rods were isolated (P = 0.01, OR = 1.765, CI − 27.2 to − 3.8), especially Pseudomonas species (P = 0.02, OR = 1.97, CI − 16.7 to − 1.0), in those with visible CSDs than in those without CSDs.
Conclusions
Bacterial colonization at CSDs was found in a high percentage of women with secondary infertility.
... [17,24]. E. coli, along with P. aeruginosa and K. pneumoniae, have been regarded as biofilm-producing microorganisms in equine endometritis [33][34][35]. All these species showed high prevalence in our study compared with previous studies reported in mares [16,17], implying that biofilm may be a potentially serious problem in endometritis in donkeys. ...
Background:
Endometritis is a common reproductive disease in equine animals. No investigation about the bacterial characteristics and antimicrobial susceptibility pattern of donkeys with endometritis has thus far been reported.
Objectives:
To determine the common uterine bacterial isolates from donkeys with endometritis and to evaluate their susceptibility to antimicrobials used for the treatment thereof.
Study design:
Retrospective case-series.
Methods:
Medical records at an equine clinical diagnostic center were retrospectively reviewed to identify submissions from donkeys with bacterial endometritis between 2018 and 2021. Data were extracted and analyzed descriptively in terms of the frequency of bacterial species, susceptibility to antimicrobials and multidrug resistance.
Results:
A total of 73 isolates were identified from 30 donkeys, of which 92% of the isolates were Gram-negative bacteria. Mixed cultures were found in 90% of the donkeys. The most common isolates were Escherichiacoli (31.5%) and Acinetobacter spp. (21.9%). Susceptibility testing revealed that amikacin (98%), cefoxitin (95%), trimethoprim-sulfamethoxazole (78%) and gentamicin (74%) were the most efficient agents for donkeys. Multidrug resistance (MDR) was found in 20% of all bacterial isolates, of which all Pseudomonas aeruginosa isolates showed a multidrug resistance profile. Main limitations: The sample size was relatively small, which means a bias of selection may exist. The antimicrobial resistance and MDR of agents without break points were not calculated, which means the relative results may be underestimated in our study.
Conclusions:
Severe infections were detected in donkeys with endometritis. Antimicrobial resistance and MDR bacteria are not rare in our study. This study demonstrated that bacteria identification and antimicrobial susceptibility testing are highly recommended before the treatment of uterine infections in donkeys. Further studies, including the epidemiological investigation of bacterial endometritis of donkeys, should be conducted to provide a better understanding of this critical problem.
