RESUMO
Until 2010, our knowledge of the uterine microbiome in cows that developed uterine disease relied almost exclusively on culture-dependent studies and mostly included cows with clinical endometritis (i.e., with purulent uterine discharge). Those studies consistently found a strong positive correlation between Trueperella pyogenes and clinical endometritis, whereas other pathogens such as Escherichia coli, Fusobacterium necrophorum, Prevotella melaninogenica, and Bacteroides spp. were also commonly cocultured. In contrast, Streptococcus spp., Staphylococcus spp., and Bacillus spp. were usually isolated from healthy cows. Starting in 2010, culture-independent studies using PCR explored the microbiome of cows with metritis and clinical endometritis, and observed that E. coli was a pioneer pathogen that predisposed cows to infection with F. necrophorum, which was strongly associated with metritis, and to infection with T. pyogenes, which was strongly associated with clinical endometritis. Starting in 2011, culture-independent studies using metagenomic sequencing expanded our knowledge of the uterine microbiome. It has been shown that cows have bacteria in the uterus even before calving, they have an established uterine microbiome within 20 min of calving, and that the microbiome structure is identical between cows that develop metritis and healthy cows until 2 d postpartum, after which the bacterial structure of cows that developed metritis deviates in favor of greater relative abundance of Bacteroidetes and Fusobacteria and lesser relative abundance of Proteobacteria and Tenericutes. The shift in the uterine microbiome in cows that develop metritis is characterized by a loss of heterogeneity and a decrease in bacterial richness. At the genus level, Bacteroides, Porphyromonas, and Fusobacterium have the strongest association with metritis. At the species level, we observed that Bacteroides pyogenes, Porphyromonas levii, and Helcococcus ovis were potential emerging uterine pathogens. Finally, we have shown that the hematogenous route is a viable route of uterine infection with uterine pathogens. Herein, we propose that metritis is associated with a dysbiosis of the uterine microbiota characterized by decreased richness, and an increase in Bacteroidetes and Fusobacteria, particularly Bacteroides, Porphyromonas, and Fusobacterium.
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Bactérias/isolamento & purificação , Doenças dos Bovinos/microbiologia , Endometrite/veterinária , Microbiota , Doenças Uterinas/veterinária , Animais , Bactérias/genética , Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Bovinos , Doenças dos Bovinos/patologia , Disbiose/microbiologia , Disbiose/veterinária , Endometrite/microbiologia , Feminino , Fusobactérias/genética , Fusobactérias/isolamento & purificação , Reação em Cadeia da Polimerase , Período Pós-Parto , Doenças Uterinas/microbiologia , Doenças Uterinas/patologia , Útero/microbiologiaRESUMO
The objective of this study was to evaluate the progression of the uterine microbiota from calving until establishment of metritis. Uterine swabs (n = 72) collected at 0, 2, and 6 ± 2 days postpartum (dpp) from 12 metritic and 12 healthy cows were used for metagenomic sequencing of the 16S rRNA gene on the Illumina MiSeq platform. A heat map showed that uterine microbiota was established at calving. The microbiota changed rapidly from 0 to 6 ± 2 dpp, with a decrease in the abundance of Proteobacteria and an increase in the abundance of Bacteroidetes and Fusobacteria, which were dominant in metritic cows. Uterine microbiota composition was shared; however, metritic and healthy cows could be discriminated using relative abundance of bacterial genera at 0, 2, and 6 ± 2 dpp. Bacteroides was the main genus associated with metritis because it was the only genus that showed significantly greater abundance in cows with metritis. As the abundance of Bacteroides organisms increased, the uterine discharge score, a measure of uterine health, worsened. Fusobacterium was also an important genus associated with metritis because Fusobacterium abundance increased as Bacteroides abundance increased and the uterine discharge score worsened as the abundance increased. The correlation with uterine discharge score and the correlation with Bacteroides or Fusobacterium showed that other bacteria, such as Helcoccocus, Filifactor, and Porphyromonas, were also associated with metritis. There were also bacteria associated with uterine health, such as "Candidatus Blochmannia," Escherichia, Sneathia, and Pedobacter.
Assuntos
Doenças dos Bovinos/microbiologia , Bovinos/microbiologia , Endometrite/veterinária , Microbiota , Período Pós-Parto , Útero/microbiologia , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Bacteroidetes/isolamento & purificação , Endometrite/microbiologia , Feminino , Fusobactérias/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Interações Microbianas , Proteobactérias/isolamento & purificação , RNA Ribossômico 16S/genética , Fatores de TempoRESUMO
TLR4 is a membrane sensor for lipopolysaccharide (LPS), a major cell wall component of gram-negative bacteria. In this study, we investigated the role of TLR4 on innate immune responses in immune cells against Acinetobacter baumannii. Bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) were isolated from WT and TLR4-deficient mice and infected with A. baumannii ATCC 15150. ELISA assay revealed that the production of IL-6 and TNF-α by A. baumannii was impaired in TLR4-deficient macrophages. However, absence of TLR2 did not affect A. baumannii-induced cytokines production in BMDMs. In addition, TLR4 was required for the optimal production of IL-6, TNF-α, and IL-12 in BMDCs in response to A. baumannii. Western blot analysis showed that A. baumannii leads to the activation of NF-κB and MAPKs (p38, ERK, and JNK) in macrophages via TLR4-dependent pathway. mRNA expression of iNOS and NO production was elicited in WT BMDMs in response to A. baumannii, which was abolished in TLR4-deficienct cells. Bacterial killing ability against A. baumannii was impaired in TLR4-deficient BMDMs. In addition, A. baumannii induced apoptosis in BMDMs via TLR4-independent pathway. Our results demonstrate that TLR4 is essential for initiating innate immune response of macrophages against A. baumannii infection.
Assuntos
Acinetobacter baumannii/imunologia , Células Dendríticas/imunologia , Macrófagos/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Apoptose , Western Blotting , Células Cultivadas , Células Dendríticas/microbiologia , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno/biossíntese , NF-kappa B/biossíntese , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Receptor 4 Toll-Like/deficiência , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Epizootic Shell Disease (ESD) has posed a great threat, both ecologically and economically, to the American lobster population of Long Island Sound since its emergence in the late 1990s. Because of the polymicrobial nature of carapace infections, causative agents for ESD remain unclear. In this study, we aimed to identify carapace microbiota associated with ESD and its potential impact on the microbiota of internal organs (green gland, hepatopancreas, intestine, and testis) using high-throughput 16S rRNA gene sequencing. We found that lobsters with ESD harbored specific carapace microbiota characterized by high abundance of Aquimarina, which was significantly different from healthy lobsters. PICRUSt analysis showed that metabolic pathways such as amino acid metabolism were enriched in the carapace microbiota of lobsters with ESD. Aquimarina, Halocynthiibacter, and Tenacibaculum were identified as core carapace bacteria associated with ESD. Particularly, Aquimarina and Halocynthiibacter were detected in the green gland, hepatopancreas, and testis of lobsters with ESD, but were absent from all internal organs tested in healthy lobsters. Hierarchical clustering analysis revealed that the carapace microbiota of lobsters with ESD was closely related to the green gland microbiota, whereas the carapace microbiota of healthy lobsters was more similar to the testis microbiota. Taken together, our findings suggest that ESD is associated with alterations in the structure and function of carapace microbiota, which may facilitate the invasion of bacteria into the green gland.
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The source and route of bacterial colonization of the uterus are still not established. The objective was to investigate the source and route of bacterial colonization of the uterus by exploring the genetic relationship among E. coli strains isolated from the gastrointestinal and the reproductive tract of dairy cows pre- and postpartum. Secondarily, uterine health status (metritis vs. healthy) was evaluated. Cows (n = 34) had the rectoanal junction (RAJ), vulva, and vagina swabbed every three days starting six days before expected calving until nine days postpartum. The uterus was swabbed postpartum. A blood sample was collected at all time points, but cultures were negative. Whole-genome sequencing was performed on 44 isolates recovered from eight cows (four metritic and four healthy) with growth on selective E. coli media from the RAJ, vulva and/or vagina and uterus. Clonal isolates were found in the RAJ or the vulva prepartum and in the vulva, vagina or uterus postpartum. Clonal isolates were also found in the RAJ, the vulva, the vagina and the uterus postpartum. Clonal isolates were found in individual cows and different cows. Absence of clustering based on virulence factor genes and all genes indicate no strain specificity to body site or uterine health status. These findings indicate that the gastrointestinal tract is the likely source of bacteria that colonize the reproductive tract via ascending colonization of the uterus through the lower genital tract. Additionally, cow to cow transmission occurs, and strains are not specific to body site or to health status.
Assuntos
Doenças dos Bovinos , Endometrite , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Endometrite/veterinária , Escherichia coli/genética , Feminino , Trato Gastrointestinal , Período Pós-Parto , Útero/microbiologiaRESUMO
Striped bass Morone saxatilis were studied in order to characterize their immune responses over the short term following challenge with Mycobacterium marinum. The expression of immunity-related genes (IL-1beta, TNF-alpha, Nramp and TGF-beta) quickly increased following infection with M. marinum, but these genes were subsequently down-regulated despite the fact that bacterial counts remained high. The number of monocytes and neutrophils also initially increased at 1 d postinfection. This confirms the importance of these types of cells in initial inflammation and mycobacterial infection in striped bass. The phagocytic index of splenic leukocytes over these same time frames did not change significantly following infection. The discrete window in which inflammatory mechanisms were stimulated in striped bass may be related to the intracellular nature of this pathogen.
Assuntos
Bass , Doenças dos Peixes/microbiologia , Infecções por Mycobacterium não Tuberculosas/veterinária , Mycobacterium marinum , Animais , Infecções por Mycobacterium não Tuberculosas/microbiologia , Fatores de TempoRESUMO
BACKGROUND: Metritis is an inflammatory uterine disease found in ~ 20% of dairy cows after parturition and associated with uterine microbiota with high abundance of Fusobacterium, Bacteroides, and Porphyromonas. Ceftiofur is a common treatment, but the effect on uterine microbiota is poorly understood. Herein, we investigated the short-term impact of ceftiofur on uterine microbiota structure and function in cows with metritis. Eight cows received ceftiofur (CEF) and 10 remained untreated (CON). Uterine swabs were collected for PCR and metagenomic analysis at diagnosis before treatment (5 ± 1 DPP) and 2 days after diagnosis/treatment (7 ± 1 DPP) from the same individuals. Seven CEF and 9 CON passed quality control and were used for 16S rRNA gene sequencing. RESULTS: Ceftiofur treatment resulted in uterine microbiota alteration, which was attributed to a decrease in relative abundance of Fusobacterium and in gene contents involved in lipopolysaccharide biosynthesis, whereas uterine microbiota diversity and genes involved in pantothenate and coenzyme A biosynthesis increased. Ceftiofur treatment also reduced rectal temperature and tended to reduce total bacteria in the uterus. However, other uterine pathogens such as Bacteroides and Porphyromonas remained unchanged in CEF. The blaCTX-M gene was detected in 37.5% of metritic cows tested but was not affected by CEF. We found that ß-hydroxybutyric acid, pyruvic acid, and L-glutamine were preferentially utilized by Fusobacterium necrophorum according to metabolic activity with 95 carbon sources. CONCLUSIONS: Ceftiofur treatment leads to alterations in the uterine microbiota that were mainly characterized by reductions in Fusobacterium and genes involved in LPS biosynthesis, which may be associated with a decrease in rectal temperature. The increase in pantothenate and coenzyme A biosynthesis indicates microbial response to metabolic stress caused by ceftiofur. Preference of Fusobacterium for ß-hydroxybutyric acid may help to explain why this strain becomes dominant in the uterine microbiota of cows with metritis, and it also may provide a means for development of new therapies for the control of metritis in dairy cows.
RESUMO
Bacteroides pyogenes is found in the human and animal gut and is implicated in the pathogenesis of metritis in cows. We report the draft genome sequences of four Bacteroides pyogenes isolates obtained from the uterus of metritic cows. This will increase the understanding of its pathogenicity, antimicrobial resistance, and differentiation across hosts.
RESUMO
A commensal in the gastrointestinal tract, Fusobacterium necrophorum is involved in the pathogenicity of abscesses, foot rot, and metritis in cattle. Here, we present the draft genomes of two Fusobacterium necrophorum isolates from the uterus of dairy cows with metritis to allow for future comparative genome studies.
RESUMO
Helcococcus ovis is an emerging pathogen implicated in the pathogenesis of metritis in dairy cows. Herein, we report the first draft genome sequences of four Helcococcus ovis isolates from the uterus of dairy cows with metritis. This information will enable a better understanding of the bacterium's pathogenicity and antimicrobial resistance.
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In this study, we have cloned a cDNA encoding for cathepsin X (PoCtX) from the olive flounder, Paralichthys olivaceus. The presence of an HIP motif, which is conserved in the unique cathepsin X family, PoCtX, clearly shows its relation to the cathepsin X group, apart from the cathepsin L or B subfamily. The results of RT-PCR and real-time PCR analyses revealed ubiquitous PoCtX expression in normal and LPS-stimulated tissues. The cDNA encoding for the proenzyme of PoCtX (proPoCtX) was expressed in Escherichia coli as a 57 kDa fusion protein with glutathione S-transferase. Its activity was quantified via the cleavage of the synthetic fluorogenic peptide substrate Z-Phe-Arg-AMC, and the optimal pH for the protease activity was 5. The recombinant proPoCtX was inhibited by antipain and leupeptin. The PoCtX protein from P. olivaceus muscle extracts was purified 9.48-fold via a one-step purification process using a DEAE-Sephagel high performance liquid chromatography (HPLC) column. Western blotting and ELISA were conducted in order to evaluate the reaction ability and detection-specificity of the anti-proPoCtX polyclonal antibody to native PoCtX and recombinant proPoCtX proteins. Our findings indicate that the P. olivaceus cathepsin X is highly conserved within the cathepsin X subfamily in terms of its amino acid sequence, tissue expression, and biochemical activity.
Assuntos
Catepsinas/genética , Catepsinas/metabolismo , Linguado/genética , Linguado/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Catepsina K , Catepsinas/química , Clonagem Molecular , Sequência Conservada , Primers do DNA/genética , DNA Complementar/genética , Expressão Gênica , Cinética , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Metritis, the inflammation of the uterus caused by polymicrobial infections, is a prevalent and costly disease to the dairy industry as it decreases milk yield, survival, and the welfare of dairy cows. Although the antibiotic ceftiofur is widely used for the treatment of metritis, endometrium and ovary function is compromised, resulting in subfertility and infertility. According to culture-dependent studies, uterine pathogens include Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum, and Prevotella melaninogenica. Recent studies using high-throughput sequencing claimed that metritis is a microbiota-associated disease. Herein, we propose that metritis is associated with uterine microbiota with high abundance of Bacteroides, Porphyromonas, and Fusobacterium, but rare bacteria such as Escherichia coli and Helcococcus ovis cannot be ignored.
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Metritis is caused by polymicrobial infection; however, recent metagenomic work challenges the importance of known pathogens such as Escherichia coli and Trueperella pyogenes while identifying potential new pathogens such as Bacteroides pyogenes, Porphyromonas levii and Helcococcus ovis. This study aims to quantify known and emerging uterine pathogens, and to evaluate their association with metritis and fever in dairy cows. Metritis was diagnosed at 6⯱â¯2 days postpartum, a uterine swab was collected and rectal temperature was measured. 39 cows were classified into three groups: Healthy (nâ¯=â¯14), Metritis without fever (MNoFever; nâ¯=â¯12), and Metritis with fever (MFever; nâ¯=â¯13). Absolute copy number was determined for total bacteria and for 8 potentially pathogenic bacteria using droplet digital PCR. Both MNoFever and MFever cows had higher copy number of total bacteria, Fusobacterium necrophorum, Prevotella melaninogenica, Bacteroides pyogenes, Porphyromonas levii, and Helcococcus ovis than Healthy cows. MNoFever and MFever groups were similar. There was no difference among groups in copy number of Escherichia coli, Trueperella pyogenes, and Bacteroides heparinolyticus, and they all had low copy numbers. Our work confirms the importance of some bacteria identified by culture-based studies in the pathogenesis of metritis such as Fusobacterium necrophorum and Prevotella melaninogenica; however, it challenges the importance of others such as Escherichia coli and Trueperella pyogenes at the time of metritis diagnosis. Additionally, Bacteroides pyogenes, Porphyromonas levii, and Helcococcus ovis were recognized as emerging pathogens involved in the etiology of metritis. Furthermore, fever was not associated with the total bacterial load or specific bacteria.
Assuntos
Infecções Bacterianas/veterinária , Doenças dos Bovinos/microbiologia , Endometrite/veterinária , Febre/veterinária , Útero/microbiologia , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Infecções Bacterianas/microbiologia , Infecções Bacterianas/patologia , Bovinos , Doenças dos Bovinos/patologia , Endometrite/microbiologia , Feminino , Febre/microbiologiaRESUMO
Broad-spectrum antibiotics such as ceftiofur and ampicillin are recommended for the treatment of metritis in dairy cows. Nonetheless, little is known about the impacts of antibiotics on the uterine microbiota. Here, we evaluated the shift in uterine microbiota after treating metritic cows with ceftiofur or ampicillin, and also gained insight into the uterine microbiota associated with cure of metritis. Uterine swabs from ceftiofur-treated, ampicillin-treated, and untreated metritic Holstein cows were collected on the day of metritis diagnosis (D1) and on D6 and then used for genomic DNA extraction and sequencing of the V4 hypervariable region of the 16S rRNA gene on the Illumina MiSeq platform. The uterine microbiota consolidated over time by decreasing species richness and increasing evenness; therefore, becoming more homogeneous. The uterine microbial community showed distinct clustering patterns on D6 according to antibiotic treatment, which could be attributed to more dynamic changes in the microbial structure from D1 to D6 in ceftiofur-treated cows. Ceftiofur led to significant changes at the community level, phylum level, and genus level, whereas the changes in ampicillin and untreated cows, although following the same pattern, were mostly non-significant. Bacteroidetes was significantly increased in ceftiofur-treated cows but was not changed after ampicillin and no treatment. Different responses to antibiotics were observed in Porphyromonas, which increased in relative abundance with ceftiofur and decreased with ampicillin. Regardless of treatment group, failure to cure metritis was associated with a decrease in diversity of uterine microbiota and an increase in the relative abundance of Bacteroides, Porphyromonas, and Fusobacterium.
Assuntos
Antibacterianos/uso terapêutico , Bactérias/isolamento & purificação , Endometrite/veterinária , Microbiota/efeitos dos fármacos , Útero/microbiologia , Ampicilina/administração & dosagem , Ampicilina/uso terapêutico , Animais , Antibacterianos/administração & dosagem , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bacteroidetes/efeitos dos fármacos , Bacteroidetes/isolamento & purificação , Bovinos , Cefalosporinas/administração & dosagem , Cefalosporinas/uso terapêutico , Endometrite/tratamento farmacológico , Endometrite/microbiologia , Feminino , Porphyromonas/efeitos dos fármacos , Porphyromonas/isolamento & purificação , Útero/efeitos dos fármacosRESUMO
A cysteine protease gene (ScCtL) homologous to the cathepsin L genes was isolated from a cDNA library of the scuticociliate parasite (Uronema marinum). To express the ScCtL recombinant protein in heterologous system, 17 codons were redesigned to conform to the standard eukaryotic genetic code using PCR-based site-directed mutagenesis. The synthetic U. marinum procathepsin L (proScCtL) was expressed at high levels in E. coli BL21 (DE3) with pGEX-4T-1 vector, and successfully refolded and purified into a functional and enzymatically active form. The optimal pH for protease activity was found to be 4.5. Like any typical cysteine protease, the enzyme was inhibited by E-64 and leupeptin. A dot-blot immunoassay was conducted in an attempt to determine the reaction abilities and sensitivity of the anti-proScCtL polyclonal antibody to the cytosol and to the membrane fraction from the scuticociliate. Our results suggest that the biochemical characteristics of the recombinant ciliate proScCtL protein are similar to that of the cathepsin L-like cysteine protease, and that the PCR-based site-direct mutated ciliate gene was successfully expressed in a biochemically active form.
Assuntos
Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Oligoimenóforos/enzimologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/isolamento & purificação , Inibidores de Cisteína Proteinase/farmacologia , DNA de Protozoário/química , DNA de Protozoário/genética , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Biblioteca Gênica , Concentração de Íons de Hidrogênio , Leucina/análogos & derivados , Leucina/farmacologia , Leupeptinas/farmacologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligoimenóforos/genética , Dobramento de Proteína , Proteínas de Protozoários/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Escherichia coli is involved in the pathogenicity of metritis in cows. We report here the genome sequences of E. coli strains isolated at calving from the uterus, vagina, vulva, and rectoanal junction of a dairy cow that later developed metritis. The genomic similarities will give an insight into phylogenetic relationships among strains.
RESUMO
BACKGROUND: Metritis is an inflammatory disease of the uterus caused by bacterial infection, particularly Bacteroides, Porphyromonas, and Fusobacterium. Bacteria from the environment, feces, or vagina are believed to be the only sources of uterine contamination. Blood seeps into the uterus after calving; therefore, we hypothesized that blood could also be a seeding source of uterine bacteria. Herein, we compared bacterial communities from blood, feces, and uterine samples from the same cows at 0 and 2 days postpartum using deep sequencing and qPCR. The vaginal microbiome 7 days before calving was also compared. RESULTS: There was a unique structure of bacterial communities by sample type. Principal coordinate analysis revealed two distinct clusters for blood and feces, whereas vaginal and uterine bacterial communities were more scattered, indicating greater variability. Cluster analysis indicated that uterine bacterial communities were more similar to fecal bacterial communities than vaginal and blood bacterial communities. Nonetheless, there were core genera shared by all blood, feces, vaginal, and uterine samples. Major uterine pathogens such as Bacteroides, Porphyromonas, and Fusobacterium were part of the core genera in blood, feces, and vagina. Other uterine pathogens such as Prevotella and Helcococcus were not part of the core genera in vaginal samples. In addition, uterine pathogens showed a strong and significant interaction with each other in the network of blood microbiota, but not in feces or vagina. These microbial interactions in blood may be an important component of disease etiology. The copy number of total bacteria in blood and uterus was correlated; the same did not occur in other sites. Bacteroides heparinolyticus was more abundant in the uterus on day 0, and both B. heparinolyticus and Fusobacterium necrophorum were more abundant in the uterus than in the blood and feces on day 2. This indicates that B. heparinolyticus has a tropism for the uterus, whereas both pathogens thrive in the uterine environment early postpartum. CONCLUSIONS: Blood harbored a unique microbiome that contained the main uterine pathogens such as Bacteroides, Porphyromonas, and Fusobacterium. The presence of these pathogens in blood shortly after calving shows the feasibility of hematogenous spread of uterine pathogens in cows.
Assuntos
Bactérias/genética , Bactérias/isolamento & purificação , Sangue/microbiologia , Bovinos/microbiologia , Fezes/microbiologia , Microbiota , Período Pós-Parto , Útero/microbiologia , Animais , Bactérias/classificação , Bactérias/patogenicidade , Bacteroides/fisiologia , Feminino , Redes Reguladoras de Genes , Metagenômica , Interações Microbianas , Reação em Cadeia da Polimerase , Porphyromonas/fisiologia , Vagina/microbiologiaRESUMO
Uterine disease such as metritis is associated with multiple bacterial infections in the uteri after parturition. However, treatment of metritis is challenging due to considerably high antibiotic treatment failure rate with unknown reason. Recently, chitosan microparticles (CM) have been developed to exert broad spectrum antimicrobial activity against bacterial pathogens, including multi-drug resistant bacteria, without raising CM resistant mutants. In this study, we tested, using metagenomics analysis, if CM maintain strong antimicrobial activity against pathogenic bacteria such as Fusobacteriaceae and Bacteroidaceae in cow uteri and evaluated CM's potency as an alternative antimicrobial agent to cure metritis in cows. Here, we report that efficacy of CM treatment for metritis was comparable to the antibiotic ceftiofur, and CM greatly altered uterine microflora of sick animals to healthy uterine microflora. Among uterine bacteria, CM significantly decreased Fusobacterium necrophorum, which is known pathogenic bacteria within the uterus. Taken together, we observed the broad spectrum antimicrobial activity of CM in vivo with an animal model, and further evaluated treatment efficacy in cows with metritis, providing insights into promising use of CM as an alternative antimicrobial agent for controlling uterine disease.
Assuntos
Anti-Infecciosos/uso terapêutico , Infecções por Bacteroidaceae/tratamento farmacológico , Quitosana/uso terapêutico , Infecções por Fusobacteriaceae/tratamento farmacológico , Doenças Uterinas/tratamento farmacológico , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bovinos , Cefalosporinas/farmacologia , Cefalosporinas/uso terapêutico , Quitosana/farmacologia , Modelos Animais de Doenças , Feminino , Fusobacterium necrophorum/efeitos dos fármacos , Humanos , Resultado do Tratamento , Doenças Uterinas/microbiologia , Útero/microbiologiaRESUMO
OBJECTIVE: This study aimed to evaluate bacterial and host factors causing a fever in cows with metritis. For that, we investigated uterine microbiota using a metagenomic sequencing of the 16S rRNA gene (Study 1), and immune response parameters (Study 2) in metritic cows with and without a fever. PRINCIPAL FINDINGS (STUDY1): Bacterial communities were similar between the MNoFever and MFever groups based on distance metrics of relative abundance of bacteria. Metritic cows showed a greater prevalence of Bacteroidetes, and Bacteroides and Porphyromonas were the largest contributors to that difference. A comparison of relative abundance at the species level pointed to Bacteroides pyogenes as a fever-related species which was significantly abundant in the MFever than the MNoFever and Healthy groups; however, absolute abundance of Bacteroides pyogenes determined by droplet digital PCR (ddPCR) was similar between MFever and MNoFever groups, but higher than the Healthy group. The same trend was observed in the total number of bacteria. PRINCIPAL FINDINGS (STUDY2): The activity of polymorphonuclear leukocyte (PMN) and the production of TNFα, PGE2 metabolite, and PGE2 were evaluated in serum, before disease onset, at 0 and 3 DPP. Cows in the MNoFever had decreased proportion of PMN undergoing phagocytosis and oxidative burst compared with the MFever. The low PMN activity in the MNoFever was coupled with the low production of TNFα, but similar PGE2 metabolite and circulating PGE2. CONCLUSION/SIGNIFICANCE: Our study is the first to show a similar microbiome between metritic cows with and without a fever, which indicates that the host response may be more important for fever development than the microbiome. Bacteroides pyogenes was identified as an important pathogen for the development of metritis but not fever. The decreased inflammatory response may explain the lack of a febrile response in the MNoFever group.
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Doenças dos Bovinos , Indústria de Laticínios , Febre/imunologia , Febre/microbiologia , Microbiota , Útero/imunologia , Útero/microbiologia , Animais , Bovinos , Feminino , Febre/complicações , Explosão RespiratóriaRESUMO
The emergence of antibiotic resistant microorganisms is a great public health concern and has triggered an urgent need to develop alternative antibiotics. Chitosan microparticles (CM), derived from chitosan, have been shown to reduce E. coli O157:H7 shedding in a cattle model, indicating potential use as an alternative antimicrobial agent. However, the underlying mechanism of CM on reducing the shedding of this pathogen remains unclear. To understand the mode of action, we studied molecular mechanisms of antimicrobial activity of CM using in vitro and in vivo methods. We report that CM are an effective bactericidal agent with capability to disrupt cell membranes. Binding assays and genetic studies with an ompA mutant strain demonstrated that outer membrane protein OmpA of E. coli O157:H7 is critical for CM binding, and this binding activity is coupled with a bactericidal effect of CM. This activity was also demonstrated in an animal model using cows with uterine diseases. CM treatment effectively reduced shedding of intrauterine pathogenic E. coli (IUPEC) in the uterus compared to antibiotic treatment. Since Shiga-toxins encoded in the genome of bacteriophage is often overexpressed during antibiotic treatment, antibiotic therapy is generally not recommended because of high risk of hemolytic uremic syndrome. However, CM treatment did not induce bacteriophage or Shiga-toxins in E. coli O157:H7; suggesting that CM can be a potential candidate to treat infections caused by this pathogen. This work establishes an underlying mechanism whereby CM exert antimicrobial activity in vitro and in vivo, providing significant insight for the treatment of diseases caused by a broad spectrum of pathogens including antibiotic resistant microorganisms.