This study found that lysates from Gardnerella vaginalis, a bacteria commonly associated with bacterial vaginosis, significantly stimulated HIV expression in monocytoid cells and certain T cell lines. G. vaginalis lysates activated HIV long terminal repeat transcription and increased NF-kB binding activity in HIV-infected cells, indicating an effect on HIV transcription. The activation of HIV production by G. vaginalis suggests that G. vaginalis infection in the genital tract may increase the risk of HIV transmission by enhancing HIV expression levels in the genital tract. This could help explain the link between bacterial vaginosis and increased sexual transmission of HIV.
Incidence rate of multidrug-resistant organisms in a tertiary care hospital, ...Apollo Hospitals
Antimicrobial resistance to microorganisms is a growing public health concern globally, especially in developing countries. This study was conducted to study the incidence rate of multidrug-resistant organisms with their antibiotic sensitivity pattern.
Incidence rate of multidrug-resistant organisms in a tertiary care hospital, ...Apollo Hospitals
Antimicrobial resistance to microorganisms is a growing public health concern globally, especially in developing countries. This study was conducted to study the incidence rate of multidrug-resistant organisms with their antibiotic sensitivity pattern.
Candidemia in HIV-positive patients in Dschang District Hospital (West Region...Claude Nangwat
Candidemia has been identified as a public health problem in HIV-infected patients. The evaluation of CD4 count, transaminases and blood glucose, are being used as a means to monitor the health of HIV-infected patients, without excluding the diagnosis of candidemia and other opportunistic infections. In order to contribute in improving the care of HIV-infected patients attending Dschang District Hospital and later on, in other hospitals in Cameroon, we conducted from June to September 2014 a cross-sectional study, with general objective; to determine the association between candidemia and selected biochemical and haematological parameter changes in HIV-infected patients, as a possible indicator in monitoring HIV disease progression.
To do this, blood samples were collected from HIV-infected patients assigned to the UPEC of Dschang District Hospital for follow up, and haemogram report, CD4 counts, ALAT level, ASAT level, and glucose level in blood were evaluated by cytometric and spectrophotometric assays. Candida species were isolated from some blood samples, and then identified using CHROMagar Candida culture medium. The broth microdilution method was afterwards used to test the susceptibility of the fungal isolates vis-a-vis three conventional antifungal agents.
Mycological analysis of blood samples showed that eight (08) patients had candidemia, a prevalence of 6.11%. Eight (08) isolates were obtained from these eight (08) candidemic HIV-infected patients; this consisted of 4(50%) Candida albicans, 3(37.5%) Candida parapsilosis and 1(12.5%) Candida glabrata. All these isolates were resistant (MICs ranged from 2 to >256 µg/mL) to the antifungals used, that is, ketoconazole, amphotericin B and nystatin.
A significant correlation was found between candidemia and white blood cell count, with a correlation coefficient of r = 0.240 (p < 0.05). Based on the results obtained, the systematic diagnosis of candidemia should be performed in patients infected with HIV in Cameroon in order to improve on their care.
Key words: Candidemia, HIV, biochemical parameters, hematological parameters, Antifungals activities.
Multidrug Resistance Pattern of Staphylococcus Aureus Isolates in Maiduguri M...Scientific Review
Multi drug-resistant (MDR) isolates of Staphylococcus aureus are on rise and are becoming a challenge for timely and appropriate treatment. The present study was carried out with an objective to isolate Staphylococcus aureus from clinical samples and determine their sensitivity. Out of 110 samples collected, 44 were shown to contained S. aureus. The isolates were subjected to antibiotic sensitivity tests using 10 different and commonly used antibiotics by modified Kirby- Bauer disc diffusion technique. Out of the total isolates (42) tested, only 7.1% were susceptible to all the antibiotics. Multiple resistance was eminent in over 92% with highest occurrence in 4.8% where the entire antibiotics were resisted. Multiple antibiotic resistance indixes (MAR index) indicated that 0.6 index occurred most (23.8%) followed by 0.5 (19.0%). On the other hand, 0.1 and 0.8 indexes were the lowest with 0.0% and 1.0% occurrence respectively. Ciprofloxacin was resisted by most of the organisms (64.3%) while amoxicillin (64.3%) and streptomycin (61.9%) were most efficacious. With over 90% isolate having MAR index ≥ 0.2, the multiple drug resistance by the S. aureus is quite alarming and might suggest inappropriate antibiotic usage by the sampled population. Therefore, the need to strategize the nature of antibiotic treatment against S. aureus and massive campaign on indiscriminate antibiotic use is urgent.
Antimicrobial Susceptibility, Biochemical Characterization and Molecular Typi...wilhelm mendel
Pathogenic isolates of Klebsiella pneumoniae (K. pneumoniae), particularly the extended-spectrum β-lactamase (ESBL) producing strains, are mostly associated with the failure of antibiotic therapy in nosocomial infections. The present work was designed to evaluate the impact of Mr. Trivedi’s biofield energy treatment on phenotypic and genotypic characteristics of K. pneumoniae. The strain of K. pneumoniae bearing ATCC 15380 (American Type Culture Collection) was procured from the Bangalore Genei, in sealed pack and divided into control and treated groups. Treated group was subjected to Mr. Trivedi’s biofield energy treatment and analyzed for the antimicrobial susceptibility, minimum inhibitory concentration (MIC), biochemical reactions, and biotyping using automated MicroScan Walk-Away® system. Further, the effect of biofield treatment was also evaluated using Random Amplified Polymorphic DNA (RAPD) in order to determine their epidemiological relatedness and genetic characteristics of biofield treated K. pneumoniae samples. The antimicrobial susceptibility results showed an improve sensitivity (i.e. from intermediate to susceptible) of ampicillin/sulbactam and chloramphenicol, while altered sensitivity of cephalothin (i.e. from susceptible to intermediate) was also reported as compared to the control sample. The MIC value showed two-fold decrease in MIC value of ampicillin/sulbactam (i.e. 16/8 to ≤8/4 μg/mL) and chloramphenicol (i.e. 16 to ≤ 8 μg/mL) as compared to the control. The cephalothin showed two-folds change (i.e. ≤ 8 to 16 μg/mL) in the MIC value as compared with the control. Biofield treatment showed 9.09% alterations in biochemical reactions followed by a change in biotype number (7774 4272) in the treated group with respect to the control (7774 4274). Genetic fingerprinting was performed on control and treated samples using RAPD-PCR biomarkers, which showed an average range of 11 to 15% of polymorphism among the treated samples with respect to the control. These results suggested that Mr. Trivedi’s biofield energy treatment has a significant impact on K. pneumoniae.
Effect of the Gayatri Mantra Playing on Microbial Load in Room AirBhoj Raj Singh
In the study, the effect of the Gayatri Mantra playing for 24 hr on microbial quality of air was examined in 12 rooms of scientists of the ICAR-Indian Veterinary Research Institute, Izatnagar, India willing to participate in the study and in 7 empty classroom/ examination hall lying vacant since March 2020 due to COVID-19. Of the empty rooms, 6 were used as the control for the first two days, then after a week, all rooms were also used to detect the effect of playing the Gayatri Mantra. A total of 31 bacterial species of medical importance were isolated and identified in the air of the rooms. After playing the Gayatri Mantra in Raag Bhairvi for 24 h bacterial count significantly decreased in rooms inhabited by vegetarian (OR 24, CI 95: 0.00-0.88; p, 0.036) than in rooms occupied by non-vegetarians. Rooms occupied by vegetarians. The effect of Gayatri Mantra in the empty room had no significant difference but bacterial count reduced. Bacterial counts of rooms occupied by non-vegetarian increased significantly post Mantra playing. Bacterial counts of rooms occupied by non-vegetarians and empty rooms varied significantly (OR 18, p, 0.02, CI 95: 0.00 – 0.79). Empty rooms with and without mantra not varied significantly for the reduction of the bacterial count. Paenibacillus spp. was not detected in any of the 12 rooms occupied by the scientists/ staff but in 3 of the 7 empty rooms (p, 0.01) it was detected even without playing any mantra. It indicated that daily disturbance in the environment may be detrimental to the survival of Paenibacillus spp. After playing Mantra P. alvei, P. cookie, P. lautus were detected in 5 of the seven rooms occupied by Non-vegetarians, while P. pantothenicus continued to be present in three of the vacant rooms even after playing the Mantra. Paenibacillus spp. bacteria are known for their probiotic potential and its significance in the study is not clear. It seems that the Gayatri mantra has some enrichment effect on Paenibacillus spp. but a detrimental effect on other microbes.
Profiling Hospital-Acquired Pathogens and Antibiotic Resistance Genes WebinarQIAGEN
Hospital-acquired infections (HAIs) are caused by bacterial, viral and fungal pathogens that easily spread through the body. The most common HAIs include urinary tract infections, bloodstream infections and pneumonia. HAIs are becoming more virulent and more resistant to the antibiotics typically used to fight them, making antibiotic resistance a serious public health concern. In this webinar, we will provide an overview of hospital-acquired pathogens and antibiotic resistance. We will also present tools to help you identify and characterize hospital-acquired bacterial species and antibiotic resistance genes in your research samples.
Inducible Clindamycin Resistance in Staphylococcus Aureus: A Study from Weste...paperpublications3
Abstract: The resistance to antimicrobial agents among Staphylococci is an increasing problem. The resistance to macrolide can be mediated by msr a gene coding for efflux mechanism or via erm gene encoding for enzymes that confer inducible or constitutive resistance to macrolide, lincosamide and Type B streptogramin. The present study was aimed to find out the percentage of Staphylococcus aureus having inducible clindamycin resistance (iMLS B) in our geographic area using D-test.
In vitro experiments of prokaryotic and eukaryotic antimicrobial peptide cyto...AI Publications
These proteinaceous molecules, called antimicrobial peptides (AMPs), are a varied collection of antimicrobial peptides. The ability of AMPs to combat gut infections necessitates further study of the AMP-GI tract interaction. These peptides need to be tested in vitro for cytotoxicity before they may be considered for use in clinical infections. Using the MTT conversion assay, neutral red dye absorption assay, and a comparison to vancomycin, researchers examined the cytotoxicity of gallidermin, nisin A, natural magainin peptides, and melittin in two gastrointestinal cell types (HT29 and Caco-2). Sheep erythrocyte hemolytic activity was also studied, and the influence of AMPs on paracellular permeability was assessed using transepithelial resistance (TEER) and TEM. Gallidermin, nisin A, magainin I, magainin II, and melittin were the least cytotoxic AMPs. To our knowledge, only Melittin and NIS caused considerable hemolysis. There are two distinct ways that melittin and nisin differ in their ability to kill bacteria. It was the only AMP that had an effect on the permeability of the paracellular space. Intestinal tight junctions and cell–cell adhesion were destroyed by long-term melittin therapy, as were microvilli, cell debris, and cell–cell adhesion. Antimicrobial activity and low cytotoxicity make Gallidermin a promising therapeutic drug. The antibacterial properties of Melittin are limited, but its ability to transport poorly bioavailable medicines may be useful.
A study of antibiotic resistance of Extended-Spectrum Beta-Lactamases produci...Premier Publishers
Background: Extended-Spectrum Beta-Lactamases - producing Enterobacteriaceae are common in hospitals. This study aims to describe the antibiotic resistance of these bacteria and their associated demographic and clinical factors. Methods: It was a prospective study of 73 isolates of Extended-Spectrum Beta-Lactamases - producing Enterobacteriaceae for a period of six months from July to December 2019 in the laboratory of Befelatanana. Results: This study showed 73 (6.3%) isolates of Extended-Spectrum Beta-Lactamases- producing Enterobacteriaceae, represented by 25 (34.2%) isolates of Klebsiella spp, 24 (32.9%) isolates of Escherichia coli, 22 (30.1%) isolates of Enterobacter spp and 2 (2.7%) isolates of Proteus spp. The antibiotic resistance of these bacteria varied from 0% to 100% for all of the antibiotics tested. Resistance to aminoglycosides ranged from 0% (amikacin) to 69.9% (gentamycin). Resistance to quinolones ranged from 43.8% (levofloxacin) to 76.7% (nalidixic acid). Similarly, 60 (82.2%) isolates were resistant to cotrimoxazole and 25 (34.2%) isolates to chloramphenicol. Patients under 20 years (57.1%) (p=0.03), men (52.2%)(p=0.11; NS), patients with respiratory samples (83.3%)(p=0.004), with pus (61.9%)(p=0.02) and hospitalized in surgery and intensive care units (68.4%)(p=0.0009) were the most affected by these enterobacteria. Conclusion: Extended-Spectrum Beta-Lactamases - producing Enterobacteriaceae are responsible for severe infections and the majorities are multi-resistant bacteria.
Keywords: Beta-lactamase, Enterobacteriaceae, antibiotic resistance, amikacin, imipenem.
Protein was extracted from muscles of Channa striatus and attempts were
made to evaluate in vitro antibacterial activity against clinical bacterial isolates. The
higher concentration of protein (100μg/ml) extracts exhibited a pronounced activity
against Pseudomonas aeruginosa (21 mm), Proteus vulgaris (19 mm), Citrobacter sp
(19 mm), Klebsiella pneumoniae (18 mm), Micrococcus sp (17 mm), Bacillus subtilis (16
mm), Staphylococcus aureus (15 mm), E. coli (14 mm) and Serratia marcescens (5
mm). The minimum inhibitory concentration and minimum bactericidal concentration
were found to be 20-40 μg/ml and 80-100 μg/ml respectively for the extracts of
Channa striatus protein against test organisms. This study confirms that C. striatus fish
protein extracts possess antibacterial activity against a wide range of microbes and
justified that it could be used in the traditional medicine as a remedy for the
treatment of bacterial diseases.
This poster was presented at the 2015 Georgia Bio Conference in Atlanta, GA.
Abstract:
Alarming trends in the spread of antibiotic resistance among top pathogens, including Staphylococcus aureus, have pushed mankind toward what has been coined as the “post-antibiotic era.” Therefore, an indirect attack on bacteria through interfering with their means of communication, quorum sensing, is proposed. An underappreciated source for modern anti-infectives is natural products from terrestrial plants. A rich history of medical traditions developed under the influence of diverse cultures in the Mediterranean and many of these are still practiced by local people. Investigation of botanical folk medicines used in the treatment of skin and soft tissue infections identified Castanea sativa (European Chestnut) for its potential antibacterial activity.
This work demonstrates the quorum sensing inhibitory activity of oleanene and ursene derivatives from a C. sativa leaf extract against all S. aureus accessory gene regulator (agr) alleles. Multiple layers of evidence for agr blocking activity (IC50 1.56-25 µg mL-1) are reported: toxin outputs, reporter assays, hemolytic activity, cytotoxicity studies, and an in vivo abscess model. The C. sativa extract is neither cytotoxic to human keratinocytes, nor murine skin; it neither inhibits S. aureus growth, nor skin commensal growth. Serial passaging experiments with the extract did not result in the development of resistance. In conclusion, the disruption of quorum sensing in the absence of growth inhibition demonstrated by this natural product derived non-biocidal inhibitor of virulence shows potential for future antibiotic therapies.
Candidemia in HIV-positive patients in Dschang District Hospital (West Region...Claude Nangwat
Candidemia has been identified as a public health problem in HIV-infected patients. The evaluation of CD4 count, transaminases and blood glucose, are being used as a means to monitor the health of HIV-infected patients, without excluding the diagnosis of candidemia and other opportunistic infections. In order to contribute in improving the care of HIV-infected patients attending Dschang District Hospital and later on, in other hospitals in Cameroon, we conducted from June to September 2014 a cross-sectional study, with general objective; to determine the association between candidemia and selected biochemical and haematological parameter changes in HIV-infected patients, as a possible indicator in monitoring HIV disease progression.
To do this, blood samples were collected from HIV-infected patients assigned to the UPEC of Dschang District Hospital for follow up, and haemogram report, CD4 counts, ALAT level, ASAT level, and glucose level in blood were evaluated by cytometric and spectrophotometric assays. Candida species were isolated from some blood samples, and then identified using CHROMagar Candida culture medium. The broth microdilution method was afterwards used to test the susceptibility of the fungal isolates vis-a-vis three conventional antifungal agents.
Mycological analysis of blood samples showed that eight (08) patients had candidemia, a prevalence of 6.11%. Eight (08) isolates were obtained from these eight (08) candidemic HIV-infected patients; this consisted of 4(50%) Candida albicans, 3(37.5%) Candida parapsilosis and 1(12.5%) Candida glabrata. All these isolates were resistant (MICs ranged from 2 to >256 µg/mL) to the antifungals used, that is, ketoconazole, amphotericin B and nystatin.
A significant correlation was found between candidemia and white blood cell count, with a correlation coefficient of r = 0.240 (p < 0.05). Based on the results obtained, the systematic diagnosis of candidemia should be performed in patients infected with HIV in Cameroon in order to improve on their care.
Key words: Candidemia, HIV, biochemical parameters, hematological parameters, Antifungals activities.
Multidrug Resistance Pattern of Staphylococcus Aureus Isolates in Maiduguri M...Scientific Review
Multi drug-resistant (MDR) isolates of Staphylococcus aureus are on rise and are becoming a challenge for timely and appropriate treatment. The present study was carried out with an objective to isolate Staphylococcus aureus from clinical samples and determine their sensitivity. Out of 110 samples collected, 44 were shown to contained S. aureus. The isolates were subjected to antibiotic sensitivity tests using 10 different and commonly used antibiotics by modified Kirby- Bauer disc diffusion technique. Out of the total isolates (42) tested, only 7.1% were susceptible to all the antibiotics. Multiple resistance was eminent in over 92% with highest occurrence in 4.8% where the entire antibiotics were resisted. Multiple antibiotic resistance indixes (MAR index) indicated that 0.6 index occurred most (23.8%) followed by 0.5 (19.0%). On the other hand, 0.1 and 0.8 indexes were the lowest with 0.0% and 1.0% occurrence respectively. Ciprofloxacin was resisted by most of the organisms (64.3%) while amoxicillin (64.3%) and streptomycin (61.9%) were most efficacious. With over 90% isolate having MAR index ≥ 0.2, the multiple drug resistance by the S. aureus is quite alarming and might suggest inappropriate antibiotic usage by the sampled population. Therefore, the need to strategize the nature of antibiotic treatment against S. aureus and massive campaign on indiscriminate antibiotic use is urgent.
Antimicrobial Susceptibility, Biochemical Characterization and Molecular Typi...wilhelm mendel
Pathogenic isolates of Klebsiella pneumoniae (K. pneumoniae), particularly the extended-spectrum β-lactamase (ESBL) producing strains, are mostly associated with the failure of antibiotic therapy in nosocomial infections. The present work was designed to evaluate the impact of Mr. Trivedi’s biofield energy treatment on phenotypic and genotypic characteristics of K. pneumoniae. The strain of K. pneumoniae bearing ATCC 15380 (American Type Culture Collection) was procured from the Bangalore Genei, in sealed pack and divided into control and treated groups. Treated group was subjected to Mr. Trivedi’s biofield energy treatment and analyzed for the antimicrobial susceptibility, minimum inhibitory concentration (MIC), biochemical reactions, and biotyping using automated MicroScan Walk-Away® system. Further, the effect of biofield treatment was also evaluated using Random Amplified Polymorphic DNA (RAPD) in order to determine their epidemiological relatedness and genetic characteristics of biofield treated K. pneumoniae samples. The antimicrobial susceptibility results showed an improve sensitivity (i.e. from intermediate to susceptible) of ampicillin/sulbactam and chloramphenicol, while altered sensitivity of cephalothin (i.e. from susceptible to intermediate) was also reported as compared to the control sample. The MIC value showed two-fold decrease in MIC value of ampicillin/sulbactam (i.e. 16/8 to ≤8/4 μg/mL) and chloramphenicol (i.e. 16 to ≤ 8 μg/mL) as compared to the control. The cephalothin showed two-folds change (i.e. ≤ 8 to 16 μg/mL) in the MIC value as compared with the control. Biofield treatment showed 9.09% alterations in biochemical reactions followed by a change in biotype number (7774 4272) in the treated group with respect to the control (7774 4274). Genetic fingerprinting was performed on control and treated samples using RAPD-PCR biomarkers, which showed an average range of 11 to 15% of polymorphism among the treated samples with respect to the control. These results suggested that Mr. Trivedi’s biofield energy treatment has a significant impact on K. pneumoniae.
Effect of the Gayatri Mantra Playing on Microbial Load in Room AirBhoj Raj Singh
In the study, the effect of the Gayatri Mantra playing for 24 hr on microbial quality of air was examined in 12 rooms of scientists of the ICAR-Indian Veterinary Research Institute, Izatnagar, India willing to participate in the study and in 7 empty classroom/ examination hall lying vacant since March 2020 due to COVID-19. Of the empty rooms, 6 were used as the control for the first two days, then after a week, all rooms were also used to detect the effect of playing the Gayatri Mantra. A total of 31 bacterial species of medical importance were isolated and identified in the air of the rooms. After playing the Gayatri Mantra in Raag Bhairvi for 24 h bacterial count significantly decreased in rooms inhabited by vegetarian (OR 24, CI 95: 0.00-0.88; p, 0.036) than in rooms occupied by non-vegetarians. Rooms occupied by vegetarians. The effect of Gayatri Mantra in the empty room had no significant difference but bacterial count reduced. Bacterial counts of rooms occupied by non-vegetarian increased significantly post Mantra playing. Bacterial counts of rooms occupied by non-vegetarians and empty rooms varied significantly (OR 18, p, 0.02, CI 95: 0.00 – 0.79). Empty rooms with and without mantra not varied significantly for the reduction of the bacterial count. Paenibacillus spp. was not detected in any of the 12 rooms occupied by the scientists/ staff but in 3 of the 7 empty rooms (p, 0.01) it was detected even without playing any mantra. It indicated that daily disturbance in the environment may be detrimental to the survival of Paenibacillus spp. After playing Mantra P. alvei, P. cookie, P. lautus were detected in 5 of the seven rooms occupied by Non-vegetarians, while P. pantothenicus continued to be present in three of the vacant rooms even after playing the Mantra. Paenibacillus spp. bacteria are known for their probiotic potential and its significance in the study is not clear. It seems that the Gayatri mantra has some enrichment effect on Paenibacillus spp. but a detrimental effect on other microbes.
Profiling Hospital-Acquired Pathogens and Antibiotic Resistance Genes WebinarQIAGEN
Hospital-acquired infections (HAIs) are caused by bacterial, viral and fungal pathogens that easily spread through the body. The most common HAIs include urinary tract infections, bloodstream infections and pneumonia. HAIs are becoming more virulent and more resistant to the antibiotics typically used to fight them, making antibiotic resistance a serious public health concern. In this webinar, we will provide an overview of hospital-acquired pathogens and antibiotic resistance. We will also present tools to help you identify and characterize hospital-acquired bacterial species and antibiotic resistance genes in your research samples.
Inducible Clindamycin Resistance in Staphylococcus Aureus: A Study from Weste...paperpublications3
Abstract: The resistance to antimicrobial agents among Staphylococci is an increasing problem. The resistance to macrolide can be mediated by msr a gene coding for efflux mechanism or via erm gene encoding for enzymes that confer inducible or constitutive resistance to macrolide, lincosamide and Type B streptogramin. The present study was aimed to find out the percentage of Staphylococcus aureus having inducible clindamycin resistance (iMLS B) in our geographic area using D-test.
In vitro experiments of prokaryotic and eukaryotic antimicrobial peptide cyto...AI Publications
These proteinaceous molecules, called antimicrobial peptides (AMPs), are a varied collection of antimicrobial peptides. The ability of AMPs to combat gut infections necessitates further study of the AMP-GI tract interaction. These peptides need to be tested in vitro for cytotoxicity before they may be considered for use in clinical infections. Using the MTT conversion assay, neutral red dye absorption assay, and a comparison to vancomycin, researchers examined the cytotoxicity of gallidermin, nisin A, natural magainin peptides, and melittin in two gastrointestinal cell types (HT29 and Caco-2). Sheep erythrocyte hemolytic activity was also studied, and the influence of AMPs on paracellular permeability was assessed using transepithelial resistance (TEER) and TEM. Gallidermin, nisin A, magainin I, magainin II, and melittin were the least cytotoxic AMPs. To our knowledge, only Melittin and NIS caused considerable hemolysis. There are two distinct ways that melittin and nisin differ in their ability to kill bacteria. It was the only AMP that had an effect on the permeability of the paracellular space. Intestinal tight junctions and cell–cell adhesion were destroyed by long-term melittin therapy, as were microvilli, cell debris, and cell–cell adhesion. Antimicrobial activity and low cytotoxicity make Gallidermin a promising therapeutic drug. The antibacterial properties of Melittin are limited, but its ability to transport poorly bioavailable medicines may be useful.
A study of antibiotic resistance of Extended-Spectrum Beta-Lactamases produci...Premier Publishers
Background: Extended-Spectrum Beta-Lactamases - producing Enterobacteriaceae are common in hospitals. This study aims to describe the antibiotic resistance of these bacteria and their associated demographic and clinical factors. Methods: It was a prospective study of 73 isolates of Extended-Spectrum Beta-Lactamases - producing Enterobacteriaceae for a period of six months from July to December 2019 in the laboratory of Befelatanana. Results: This study showed 73 (6.3%) isolates of Extended-Spectrum Beta-Lactamases- producing Enterobacteriaceae, represented by 25 (34.2%) isolates of Klebsiella spp, 24 (32.9%) isolates of Escherichia coli, 22 (30.1%) isolates of Enterobacter spp and 2 (2.7%) isolates of Proteus spp. The antibiotic resistance of these bacteria varied from 0% to 100% for all of the antibiotics tested. Resistance to aminoglycosides ranged from 0% (amikacin) to 69.9% (gentamycin). Resistance to quinolones ranged from 43.8% (levofloxacin) to 76.7% (nalidixic acid). Similarly, 60 (82.2%) isolates were resistant to cotrimoxazole and 25 (34.2%) isolates to chloramphenicol. Patients under 20 years (57.1%) (p=0.03), men (52.2%)(p=0.11; NS), patients with respiratory samples (83.3%)(p=0.004), with pus (61.9%)(p=0.02) and hospitalized in surgery and intensive care units (68.4%)(p=0.0009) were the most affected by these enterobacteria. Conclusion: Extended-Spectrum Beta-Lactamases - producing Enterobacteriaceae are responsible for severe infections and the majorities are multi-resistant bacteria.
Keywords: Beta-lactamase, Enterobacteriaceae, antibiotic resistance, amikacin, imipenem.
Protein was extracted from muscles of Channa striatus and attempts were
made to evaluate in vitro antibacterial activity against clinical bacterial isolates. The
higher concentration of protein (100μg/ml) extracts exhibited a pronounced activity
against Pseudomonas aeruginosa (21 mm), Proteus vulgaris (19 mm), Citrobacter sp
(19 mm), Klebsiella pneumoniae (18 mm), Micrococcus sp (17 mm), Bacillus subtilis (16
mm), Staphylococcus aureus (15 mm), E. coli (14 mm) and Serratia marcescens (5
mm). The minimum inhibitory concentration and minimum bactericidal concentration
were found to be 20-40 μg/ml and 80-100 μg/ml respectively for the extracts of
Channa striatus protein against test organisms. This study confirms that C. striatus fish
protein extracts possess antibacterial activity against a wide range of microbes and
justified that it could be used in the traditional medicine as a remedy for the
treatment of bacterial diseases.
This poster was presented at the 2015 Georgia Bio Conference in Atlanta, GA.
Abstract:
Alarming trends in the spread of antibiotic resistance among top pathogens, including Staphylococcus aureus, have pushed mankind toward what has been coined as the “post-antibiotic era.” Therefore, an indirect attack on bacteria through interfering with their means of communication, quorum sensing, is proposed. An underappreciated source for modern anti-infectives is natural products from terrestrial plants. A rich history of medical traditions developed under the influence of diverse cultures in the Mediterranean and many of these are still practiced by local people. Investigation of botanical folk medicines used in the treatment of skin and soft tissue infections identified Castanea sativa (European Chestnut) for its potential antibacterial activity.
This work demonstrates the quorum sensing inhibitory activity of oleanene and ursene derivatives from a C. sativa leaf extract against all S. aureus accessory gene regulator (agr) alleles. Multiple layers of evidence for agr blocking activity (IC50 1.56-25 µg mL-1) are reported: toxin outputs, reporter assays, hemolytic activity, cytotoxicity studies, and an in vivo abscess model. The C. sativa extract is neither cytotoxic to human keratinocytes, nor murine skin; it neither inhibits S. aureus growth, nor skin commensal growth. Serial passaging experiments with the extract did not result in the development of resistance. In conclusion, the disruption of quorum sensing in the absence of growth inhibition demonstrated by this natural product derived non-biocidal inhibitor of virulence shows potential for future antibiotic therapies.
The objective of this study was to examine the antiviral activity of native lactoferrin against Potato virus x, the most important virus that severely affects potato crop and productivity in Egypt, using tissue culture technique and spraying the plants in greenhouse by the aqueous solution of lactoferrin.
Interleukin16 and Interleukin 28B Genes Polymorphism in HBV Infected Saudi pa...iosrjce
The course of hepatitis B virus (HBV) infection is variable depending on many factors. In this study,
we investigated single nucleotide polymorphisms of interleukin-28B and interleukin-16 as possible host factors
which may determine the occurrence of hepatocellular carcinoma (HCC) in Saudi patients. Chronic hepatitis B
(CHB) patients (75), HCC patients (42), and healthy controls (70) were analyzed for polymorphisms of the IL16
and IL28B genes using PCR and restriction fragment length polymorphism (RFLP). Results showed that HCC
and chronic HBV patients had higher prevalence of rs11556218TG genotype than controls. The rs11556218GG
genotype was higher among HCC (14.4%) compared to chronic HBV (2.7%) patients. The IL-16 genotype
rs4072111CT was higher among HCC (47.6%) and chronic HBV (46.7%) patients than controls (28.6%). The
rs4072111TT genotype was higher among HCC patients compared to the other two groups. The T allele
frequency was higher among HCC patients than controls. The CT and TT of the IL-28B rs12979860 genotype
were significantly less frequent in chronic HBV and HCC patients. The IL-28B rs8099917 TG genotype was
more frequent among HCC (19%) compared to chronic HBV (8%) patients. However, no significant difference
was detected in the allele distribution.
Comparative Study of the Prevalence and Antibiogram of Bacterial Isolates fro...iosrjce
The study compared the prevalence and antibiogram of bacterial isolates from the urinary and
genital tracts of pregnant women attending ante-natal clinics in Imo State. Urine and High vaginal swab (HVS)
samples were collected from across the three geopolitical zones of Imo State (Owerri, Orlu and Okigwe).
Federal Medical Centre (FMC) Owerri, Imo State University Teaching Hospital (IMSUTH) Orlu and General
Hospital Okigwe (GHO) were used as focal points. A total of 1197 samples were obtained from women and
used. Infection was significantly more with the urine samples than the HVS samples (P < 0.05) while
polymicrobial growth was more observed with the HVS samples. Escherichia coli was the predominantly
isolated organism (38.3%) from the urine samples while Staphylococcus aureus (29.1%) was the predominant
bacterial isolates in HVS. Other commonly isolated bacterial species include; Enterococcus faecalis and
Staphylococcus epidermidis, Klebsiella pneumoniae, Proteus mirabilis and Bacteriodes were solely isolated
from urine while Lactobacillus was solely isolated from HVS. Overall antibiogram showed ciprofloxacin to be
the most effective antibiotic followed by nalidixic acid and pefloxac in for both specimens. Generally, multidrug
resistance was more in urine isolates (55.7%) than vaginal isolates (53.6%) with many showing the same
resistance patterns. The rate of multi/drug resistance in both samples is high (>50%) and worrisome. These call
for routine HVS as well as urine culture to be carried out on all antenatal women to ensure holistic antenatal care/ management.
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Similar to Activation-of-human-immunodeficiency-virus-type-1-expression-by-Gardnerella-vaginalis_1999_Journal-of-Infectious-Diseases_1 (20)
1. 924
Activation of Human Immunodeficiency Virus Type 1 Expression
by Gardnerella vaginalis
Farhad B. Hashemi, Mahmood Ghassemi,
Kenneth A. Roebuck, and Gregory T. Spear
Department of Immunology/Microbiology, Rush University,
and Department of Medicine, Section of Infectious Diseases,
University of Illinois at Chicago
Bacterial vaginosis (BV) is associated with an increased rate of sexual transmission of human
immunodeficiency virus (HIV) type 1, and Gardnerella vaginalis is frequently isolated from
the genital tracts of women with BV. G. vaginalis lysates were found to significantly stimulate
HIV expression in monocytoid cells. Stimulation was significantly higher when lysates were
heated at 100ЊC for 5 min but was reduced by treatment with lysozyme or protease. G. vaginalis
lysates also activated HIV expression in certain T cell lines. G. vaginalis lysates activated HIV
long-terminal repeat transcription in HIV-infected cells and increased NF-kB binding activity,
indicating an effect by G. vaginalis on HIV transcription. The activation of HIV production
by G. vaginalis suggests that genital tract infection with G. vaginalis increases the risk of HIV
transmission by increasing HIV expression in the genital tract. This may explain, at least in
part, the increased rate of HIV transmission in women with BV.
Sexual transmission of human immunodeficiency virus (HIV)
continues to be the major route of the infection, accounting
for 70%–80% of cases worldwide [1–3]. In the United States,
women are a growing demographic group of newly diagnosed
AIDS cases [4, 5]. Retrospective population studies [6–9] and
a recent prospective investigation [10] show that vaginal infec-
tions, such as bacterial vaginosis (BV) and sexually transmitted
diseases, are associated with an increased risk of HIV trans-
mission. BV is a common disorder characterized by changes in
the vaginal flora in which the normally predominant lactobacilli
are replaced by potential pathogens such as Gardnerella vagin-
alis, genital Mycoplasma infections, and eventually anaerobic
bacteria, accompanied by an increase in vaginal pH [11].
BV-associated microorganisms may increase the incidence of
sexual transmission of HIV by several mechanisms. For ex-
ample, cell-associated or secreted products of microorganisms
may affect the differentiation or proliferation state of HIV tar-
get cells in the genital tract, leading to increased susceptibility
to HIV infection [12]. Alternatively, infected cells, such as mac-
rophages, T cells, and dendritic cells in the genital tract may
be stimulated by these organisms or their products, leading to
Received 24 July 1998; revised 9 December 1998.
The subjects in this study gave informed consent as a part of the Women’s
Interagency HIV Study at Rush-Presbyterian St. Luke’s Medical Center.
Human investigation guidelines of the US Department of Health and Hu-
man Services and of Rush-Presbyterian St. Luke’s Medical Center were
observed.
Grant support: NIH (AI-34993, AI-31812).
Reprints or correspondence: Dr. Farhad B. Hashemi, Dept. of Immu-
nology/Microbiology, Rush University, 1653 W. Congress Pkwy., Chicago,
IL 60612 (fhashem2@rush.edu).
The Journal of Infectious Diseases 1999;179:924–30
᭧ 1999 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/99/7904-0020$02.00
increased HIV expression. Focusing on the latter mechanism,
we hypothesized that G. vaginalis can increase HIV expression.
G. vaginalis, a gram-variable, rod-shaped bacteria, is com-
monly isolated from the female genital tract [13]. Since G. va-
ginalis is among the bacteria frequently (р95%) associated with
BV [14], we studied whether G. vaginalis can modulate HIV
expression in infected cells.
Materials and Methods
Organisms and cells. G. vaginalis cultures were grown on choc-
olate agar plates (Remel Microbiology Products, Lenexa, KS) at
37ЊC for 48 h in a humidified atmosphere with 5% CO2. In this
study, we used G. vaginalis ATCC strain 14018 (American Type
Culture Collection, Rockville, MD), referred to as isolate B, and
3 genital isolates from patients. A genital tract yeast isolate (Can-
dida albicans) and coagulase (Ϫ) staphylococci (referred to as
Staphylococcus species—not aureus or SSNA) was isolated from 1
of the clinical samples and cultured as above. Lactobacillus aci-
dophilus was isolated from a clinical specimen from the genital tract
and grown in MRS broth (Remel) under anaerobic conditions.
Microorganisms were identified by standard procedures recom-
mended by the American Society for Microbiology [15].
We obtained the U1, U38, J1.1, and HeLa cervix epitheloid
carcinoma cell lines from the AIDS Research and Reference Re-
agent Program (Division of AIDS, National Institute of Allergy
and Infectious Diseases, NIH, Rockville, MD; U1 and J1.1 cells
contributed by T. Folks; U38 cells by B. Felber and G. Pavlaskis;
and HeLa cells by R. Axel) [16–18]. Both U1 and U38 are trans-
formed monocytoid cells derived from the U937 cell line. The for-
mer contains a stable copy of the complete HIV-1IIIB genome and
expresses very low amounts of CD4 [16]. The latter has the HIV-1
long-terminal repeat (LTR) promoter linked to the chloramphen-
icol acetyl transferase (CAT) gene and is a very sensitive indicator
of HIV-1 transactivating (Tat) protein [17]. The J1.1 cells are la-
tently HIV-1–infected Jurkat T cells capable of being induced with
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2. JID 1999;179 (April) Effect of G. vaginalis on HIV Replication 925
PMA or tumor necrosis factor (TNF)-a [18]. The HIV-1MN and
HIV-1RF preparations were also obtained from the AIDS Research
and Reference Reagent Program [19, 20]. The human peripheral
blood–acute lymphocytic leukemia (HPB-ALL) cell line is a trans-
formed T lymphocyte cell line [21].
All cell lines were maintained in RPMI 1640 medium supple-
mented with 10 mM HEPES, 2 mM glutamine, and 10% fetal
bovine serum (FBS; BioWhittaker, Walkersville, MD). This culture
medium is referred to as complete medium. The cells were fed twice
weekly using complete medium. Prior to each experiment, cell vi-
ability was routinely checked by trypan blue dye exclusion. All cells
were negative for mycoplasma by polymerase chain reaction [22].
Preparation of lysates from G. vaginalis, L. acidophilus, coagulase
(Ϫ) staphylococci, and yeast. Culture plates growing confluent
lawns of G. vaginalis, SSNA, or C. albicans were harvested, washed
with PBS, centrifuged (700 g) for 10 min at 24ЊC, and then resus-
pended in 3 mL of PBS. For L. acidophilus, broth cultures were
centrifuged for 10 min at 700 g after 24 h of incubation. The
bacterial pellet was then resuspended in 5 mL of PBS. Lysates were
prepared using bacterial pellets as previously described [23]. In
brief, suspensions were sonicated on ice for 3 min using a flat-tip
probe sonifier (model 250; Branson Ultrasonics, Danbury, CT) at
setting 4. Prior to sonication, harvested cultures were tested for
purity by culturing a sample on chocolate agar plates. The protein
concentration of lysates was determined by BSA protein assay
(Pierce, Rockford, IL). Lysates were stored at Ϫ20ЊC until use.
Prior to bioassay, lysates were diluted in complete medium to a
final concentration of 5000, 500, or 50 ng/mL lysate protein.
Stimulation of U1, J1.1, HPB-ALL, and HeLa cells by G. va-
ginalis lysates. To test the HIV-inducing activity, U1 or4
4 ϫ 10
J1.1 cells were incubated with lysates in microtiter plate wells at
37ЊC. After 3 days, culture supernatant fluids were collected, mixed
with Triton X-100 (final concentration 0.1%), and p24 concentra-
tions were measured by ELISA (National Cancer Institute, Rock-
ville, MD). Similar procedures were used for the HPB-ALL cul-
tures, except that before treatment with lysates, HPB-ALL cells
were infected with HIV-1 as described previously [24]. In brief,
HPB-ALL cells were incubated with HIV-1MN (6 4
2 ϫ 10 3 ϫ 10
TCID/mL) at 37ЊC for 2 h. Cells were then washed once and re-
suspended in complete medium at cells/mL and treated with5
5 ϫ 10
lysates followed by culture for 3 days at 37ЊC. Where appropriate,
complete medium, TNF-a (50 U; R&D Systems, Minneapolis), or
PMA (100 ng/mL) were included as controls.
HeLa cells ( total) were incubated with 5000 ng/mL pro-5
2 ϫ 10
tein of heated lysate in 24-well plates (Corning, New York, NY)
at 37ЊC. After 3 days, culture supernatant fluids were collected,
and cytokine concentrations were determined.
Cytokine measurements by ELISA. Cytokine levels in super-
natant fluids of Hela cell cultures were determined by ELISA. To
test interleukin (IL)-8 concentrations, we used an ELISA (Cyto-
Screen; Biosource International, Camarillo, CA). For TNF-a, IL-
1a, and IL-6 measurements, we used CYTELISA kits (CYT-
immune, College Park, MD). All determinations were made ac-
cording to manufacturers’ recommendations.
Heat, lysozyme, and protease treatments of G. vaginalis lysates.
For heat treatment, G. vaginalis lysates were heated at 100ЊC in a
water bath for 5 min. For protease treatment, proteinase K (Sigma,
St. Louis) was added to heated G. vaginalis lysates at 10 mg/mL
final concentration. For lysozyme treatment, egg white lysozyme
(Sigma) was added to heated lysates at 250 mg/mL final concen-
tration. The mixture of enzyme plus lysate were incubated for 30
min at 37ЊC and then heated again at 100ЊC for 5 min. Samples
were diluted in complete medium and added to culture wells. As
controls, the G. vaginalis lysates and enzymes were incubated for
30 min at 37ЊC in separate tubes. Samples were then heated and
added to the cells.
Stimulation of HIV-LTR activity by G. vaginalis lysates. HIV
LTR activity was measured as previously described [17]. Briefly,
U38 cells were infected with HIV-1RF (∼100 pg of p246
3 ϫ 10
content) in the presence or absence of G. vaginalis–heated lysates
(5000 ng/mL protein). After 48 h, cells were harvested and washed.
Protein extracts were prepared by 4 freeze-thaw cycles of cell pellets.
Pellets were resuspended in 100 mL of 0.25 M Tris (pH 7.8), frozen
at Ϫ70ЊC for 10 min, and thawed at 37ЊC. After the fourth thaw,
samples were centrifuged for 5 min, and protein concentrations of
supernatants were determined by protein assay (Bio-Rad Labo-
ratories, Hercules, CA).
Equal amounts of protein extract were analyzed by CAT assay
as described [25] with minor modifications. In brief, 50 mL of cell
extract, adjusted to contain 100 mg of protein, was incubated for
5 h at 37ЊC with 50 mL of CAT assay mix (containing 20 mL of
0.01 Ci/mL [3
H]chloramphenicol [DuPont, Boston], 5 mL of 5 mg/
mL butyl CoA [Sigma], 5 mL of 2 M Tris-Cl, pH 8, and 20 mL of
distilled water). The acetylated chloramphenicol was then extracted
with the addition of 200 mL of 2:1 (vol/vol) tetramethylpentade-
cane/xylenes (Sigma) by vigorous shaking. After the tubes were
centrifuged, 85% of the organic phase was transferred to scintil-
lation vials and counts per minute were determined.
Electrophoretic mobility shift assays (EMSA). U1 cells were
washed in serum-free RPMI 1640 and resuspended in RPMI sup-
plemented with 0.5% FBS. After overnight incubation at 37ЊC, cells
were adjusted to /mL and treated with heated G. vaginalis6
5 ϫ 10
lysate (5000 ng/mL protein) for 2 h at 37ЊC. Cells were then washed
in PBS and centrifuged (500 g) for 5 min. Cell pellets were used
to prepare nuclear protein extracts for the EMSA by the method
of Osborn et al. [26] with the addition of proteinase inhibitors.
Extracted proteins (5 mg) were incubated with AP-1 or NF-kB 32
P-
labeled oligonucleotide (AP-1, 5-CGCTTGATGAGTCAGCCG-
GAA-3; NFk-B, 5-AGTTGAGGGGACTTTCCCAGGC-3; Pro-
mega, Madison, WI) for 30 min at 24ЊC. Positive controls included
extracts from cells stimulated with PMA or TNF-a. Incubated
mixtures were separated on a 5% nondenaturing polyacrylamide
gel. Scanning and densitometry were done by personal scanner SI
(Molecular Dynamics, Sunnyville, CA). To generate stimulation
index ratios, the density of an empty lane on the scanned film was
selected as the background value for band comparison.
Results
Stimulation of HIV production in U1 monocytic cells by G.
vaginalis lysates. In order to determine the effect of G. va-
ginalis on HIV expression, lysates from 4 G. vaginalis isolates
were prepared by sonication. The resulting lysates were then
added to cultures of the chronically HIV-infected U1 cell line.
These cells have been used as a model to test the effects of
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3. 926 Hashemi et al. JID 1999;179 (April)
Figure 1. Dose-dependent activation of HIV-1 expression in U1 cells
by 4 heated or unheated G. vaginalis lysates. Isolates were incubated
with U1 cells at 50, 500, or 5000 ng/mL G. vaginalis lysate protein;
heated and unheated lysates of coagulase (Ϫ) staphylococci (SSNA)
and Lactobacillus acidophilus at 50, 500, or 5000 ng/mL lysate protein
were also used. Control media (dashed line). After 3 days, culture
supernatant fluids were collected, and p24 concentrations were mea-
sured by ELISA. Values are mean of triplicate culture wells. Data are
representative of 2 separate experiments.
Figure 2. Effects of G. vaginalis lysates on HIV expression in J1.1
T cells. Heated or unheated G. vaginalis lysates were incubated with
J1.1 or HIV-infected HPB-ALL cells at 50, 500, or 5000 ng/mL lysate
protein. After 3 days, p24 concentrations in culture supernatant fluids
were measured by ELISA. Controls included TNF-a (50 U/well) and
media (dashed line). Data are mean of triplicate culture wells from 1
experiment.
various substances, including cytokines and bacterial products
[27, 28] on HIV replication in monocytoid cells.
Although untreated U1 cells produced low levels of HIV as
measured by release of p24 into culture medium, addition of
5000 ng/mL G. vaginalis lysate protein significantly increased
HIV replication compared with that in cells cultured in medium
alone ( , one-group t test; figure 1). The increase wasP ϭ .003
13- to 77-fold higher than in untreated cells. For cells that
received 500 ng/mL G. vaginalis lysate protein, the increase in
p24 production was 6- to 11-fold above control cultures (figure
1). Lower amounts of lysate (50 ng/mL; figure 1) did not sig-
nificantly increase HIV production. As shown in figure 1, in
contrast to G. vaginalis lysates, lysates of genital tract–derived
coagulase (Ϫ) staphylococci (SSNA), L. acidophilus, and C.
albicans (not shown), at lysate protein levels of 50–5000 ng/
mL, did not increase HIV production in U1 cells.
To assess the heat stability of the stimulatory factor in G.
vaginalis, the lysates were heated at 100ЊC for 5 min. The heated
lysates at 5000 and 500 ng/mL stimulated higher p24 produc-
tion from U1 cells than unheated lysates ( and .04,P ϭ .002
respectively, paired t test; figure 1). Addition of heated lysates
from isolates C and D at 50 ng/mL also increased p24 pro-
duction 18- to 20-fold higher than control, whereas isolates A
and B at 50 ng/mL isolates did not affect p24 production. These
results show that the stimulatory activity from G. vaginalis is
not only heat-stable, but that 100ЊC treatment for 5 min also
increases its activity.
Stimulation of HIV production in T lymphocyte lines by G.
vaginalis lysates. The ability of G. vaginalis lysates to stim-
ulate HIV replication in 2 T lymphocytic cell lines was also
evaluated. Although the effect of G. vaginalis in J1.1 cells was
less remarkable than in U1 cells, as a group, G. vaginalis lysates
(5000 ng/mL lysate protein) significantly increased HIV ex-
pression in J1.1 than in cells cultured in medium alone (P ϭ
, one-group t test; figure 2). Isolate B did not stimulate HIV.017
expression at any concentration; however, for isolates A, C,
and D, the increase in p24 production was 1.2- to 2.6-fold above
control cultures. For cells that received 500 ng/mL G. vaginalis
lysate protein of isolates C and D, the increase in p24 produc-
tion was 1.7- and 2.0-fold over control cultures, respectively
(figure 2). Isolates A and B did not stimulate p24 production
in J1.1 cells at 500 ng/mL lysate protein (figure 2). Overall, the
group of J1.1 cultures treated with 500 ng/mL lysates did not
show any significant increase over control cultures ( ,P ϭ .13
one-group t test; figure 2). Similar to U1 cells, neither 50 ng/
mL lysate protein (figure 2) nor C. albicans lysates (not shown)
affected p24 production by J1.1 cells.
Heating significantly increased stimulatory activity of the lys-
ates at 5000 ng/mL compared with unheated samples (P ϭ
, paired t test; figure 2). Although less marked, heat also.006
enhanced the stimulatory activity of lysates at the 500 ng/mL
protein lysate level ( , paired t test; figure 2). The J1.1P ϭ .09
cells responded to TNF-a (50 U) treatment by production of
pg/mL p24 protein (not shown).5
4 ϫ 10
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4. JID 1999;179 (April) Effect of G. vaginalis on HIV Replication 927
Figure 3. Physical characterization of HIV-activating factor(s) from
G. vaginalis lysates. Heated lysates (5000 ng/mL lysate protein) from
4 G. vaginalis isolates were incubated with lysozyme (250 mg/mL) or
proteinase K (10 mg/mL) and heated at 100ЊC for 5 min to inactivate
enzymes then incubated with U1 cells. Culture supernatant fluids were
collected after 3 days, and p24 levels were measured by ELISA. Data
represent 2 separate experiments run in triplicate culture wells.
Figure 4. Effect of G. vaginalis lysates on HIV long-terminal repeat
activation in U38 cells. Cells were treated with heated G. vaginalis
lysates (5000 ng/mL lysate protein) in presence or absence of exogenous
HIVRF. After 48 h, cells were harvested, and chloramphenicol acetyl
transferase (CAT) activity of cellular extract was determined. Data are
representative of 3 independent experiments.
Lysates from G. vaginalis isolates A and C were also tested
for stimulatory activity on the HPB-ALL T cell line (not
shown). In contrast to U1 and J1.1 cells, heated or unheated
lysates from these isolates did not stimulate any increase in
HIV production by HPB-ALL cells, whereas TNF-a (50 U)
induced HIV production by these cells (not shown).
Physical properties of the G. vaginalis stimulatory substance.
Although the above experiments showed that the stimulatory
activity in G. vaginalis lysates was heat-stable, further char-
acteristics of the stimulatory activity were determined. Lysates
from isolates were heated at 100ЊC and then treated with either
lysozyme or proteinase K. Both lysozyme and proteinase K
treatment significantly reduced the ability of the lysates to stim-
ulate HIV production by U1 cells (figure 3), suggesting that
the stimulatory substance(s) contained a protein, lipoprotein,
or peptidoglycan-like component that was required for full ac-
tivity. Control samples containing only enzymes had no effect
on p24 production by U1 cells (not shown).
In further experiments, G. vaginalis lysates were separated
into soluble and particulate material by ultracentrifugation and
then added separately to U1 cells. All of the recoverable stim-
ulatory activity for all 4 G. vaginalis isolates was associated
with the particulate fraction (not shown), suggesting that the
stimulatory component is associated with the G. vaginalis cell
wall or membrane. Taken together, these results provide evi-
dence that the HIV activating factor(s) may be a protein de-
rivative associated with the G. vaginalis cell wall or membrane.
Effect of G. vaginalis on HIV transcription through activation
of HIV-LTR. In order to determine the effect of G. vaginalis
on HIV-1 LTR promoter activity, U38 cells were infected with
HIV in the presence or absence of heated G. vaginalis lysates
followed by measurement of HIV-LTR activity by CAT assay.
Since the U38 cells have the HIV-1 LTR promoter linked to
the CAT gene, they are a sensitive indicator of HIV-1 transac-
tivating (Tat) protein [17].
As expected, HIV infection of U38 cells enhanced HIV-LTR
CAT transcription. When cells were infected in the presence of
lysates, the enhancement of HIV-LTR CAT expression was sig-
nificantly higher than in untreated HIV-infected cells (P ϭ
, one-group t test; figure 4). The increases over cells that.018
were HIV-infected but not treated with lysates ranged from 1.6-
to 3.4-fold for isolates A and D, respectively. Incubation with
G. vaginalis lysates did not increase CAT expression in unin-
fected U38 cells (not shown).
Effect of G. vaginalis lysates on transcription factors NF-kB
and AP-1. The mechanism of stimulation of HIV production
by G. vaginalis lysates was further investigated by assessing
induction of transcription factors NF-kB and AP-1 in U1 cells.
Lysates from G. vaginalis isolates A and B induced increases
in NF-kB binding activity (figure 5A) that corresponded to
about a 15-fold induction of NF-kB binding over untreated U1
cells. In contrast, G. vaginalis lysates did not affect AP-1 binding
in these cells (figure 5B).
Effect of G. vaginalis lysates on cytokine production by HeLa
epithelial cells. Another possible mechanism of increasing
HIV replication in the genital tract by G. vaginalis is to induce
proinflammatory cytokine production by epithelial cells. To de-
termine whether G. vaginalis stimulates these cells, Hela cells
were incubated with heated G. vaginalis lysates (5000 ng/mL
lysate protein) for 72 h, and proinflammatory cytokines levels
in culture supernatant fluids were measured. Heated G. vaginalis
lysates from isolates A and B did not increase secretion of IL-
6, IL-1a, TNF-a, or IL-8 by Hela cells (table 1). In contrast,
the positive controls increased secretion of these cytokines (ta-
ble 1).
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5. 928 Hashemi et al. JID 1999;179 (April)
Figure 5. NF-kB and AP-1 binding activity in U1 cells treated with G. vaginalis lysates. U1 cells were treated with heated lysates of G. vaginalis
(5000 ng/mL lysate protein) for 2 h at 37ЊC. Complete medium, tumor necrosis factor (TNF)-a, and PMA served as controls. Nuclear protein
extracts were analyzed by electrophoretic mobility shift assay using consensus NFk-B (A) or AP-1 (B) probes. Autoradiographs were scanned
and optical density was measured by densitometry. Density of empty lane on scanned film was selected as background (index ) forratio ϭ 1
comparison of bands. Bar graph compares stimulatory ratio of lysate-treated U1 cells with cells treated with PMA, TNF-a, and complete medium
alone.
Discussion
BV is associated with a higher incidence of sexual transmis-
sion of HIV. The process by which microbial or host-related
factors in the genital tract leads to increased risk of HIV trans-
mission is not well understood. We hypothesized that G. va-
ginalis, which is frequently associated with BV, activates HIV
expression in cells in the genital tract. This activation could
then contribute to the higher risk of HIV sexual transmission
by increasing the amount of virus in the genital tract. To test
this hypothesis, we evaluated the effect of G. vaginalis on HIV
replication.
We found that G. vaginalis activated HIV expression in
monocytoid and in certain T cells. The HIV-inducing effect was
specific to G. vaginalis, since common nonpathogenic bacteria
often isolated in the vaginal tract (e.g., coagulase [Ϫ] staphy-
lococci and L. acidophilus) did not induce HIV expression. All
4 G. vaginalis isolates used in this study stimulated HIV pro-
duction by U1 cells, although the stimulatory activity of some
isolates was modest unless the lysates were heated. These results
suggest that some strains of G. vaginalis may be more effective
at increasing HIV expression in the genital tract than others.
Although we have not studied the mechanism by which heat
treatment releases the HIV-inducing activity by G. vaginalis
lysates, it is possible that in the vaginal tract, factors such as
degradative enzymes or the low pH of vaginal secretions release
the activity of G. vaginalis similar to how heat acts on G. va-
ginalis components in vitro. Thus, our data support the hy-
pothesis that increased HIV production by the cells in the gen-
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6. JID 1999;179 (April) Effect of G. vaginalis on HIV Replication 929
Table 1. The effect of G. vaginalis lysates on cytokine production by
HeLa cells.
Cytokine concentration (pg/mL)
IL-6 IL-1 TNF-a IL-8
G. vaginalis A !16 59 !16 96
G. vaginalis B 744 9 109 78
Culture medium 907 16 161 214
TNF-a (50 U) 11,844 55 NA 2301
NOTE. Cells ( ) were incubated with G. vaginalis (5000 ng/mL lysate5
2 ϫ 10
protein) at 37ЊC. Lysates were heated (100ЊC for 5 min) before incubation. After
3 days, culture supernatant fluids were collected and cytokine levels were deter-
mined by ELISA. Values are mean of duplicate cultures; data are representative
of 3 separate experiments. IL, interleukin; TNF, tumor necrosis factor; NA, not
applicable.
ital tract may affect the risk of HIV transmission by increasing
virus load in the genital tract. It will be of interest to investigate
whether other organisms associated with BV also activate HIV
expression independent or in combination with G. vaginalis.
We hypothesized that G. vaginalis could also activate HIV
replication in the genital tract indirectly by inducing secretion
of proinflammatory cytokines by epithelial cells. In fact, other
bacteria from the genital tract, such as Chlamydia species, have
increased inflammatory cytokine production [29], and some
studies have shown increased IL-1a, IL-8, IL-1b, IL-6, and
TNF-a in vaginal secretions of pregnant women with BV [30,
31]. However, G. vaginalis did not stimulate production of IL-
1a, IL-6, TNF-a, or IL-8 in epithelial cells. Perhaps the lack
of inflammatory cytokine secretion by epithelial cells in re-
sponse to G. vaginalis is one way this organism evades the
immune response. For instance, Darveau et al. [32] recently
reported that Porphromonas gingivalis not only does not induce
IL-8 secretion by epithelial cells but that it also degrades IL-8
and inhibits its production.
Although we have not identified the HIV-inducing factor(s)
in G. vaginalis lysates, the evidence of its heat stability and its
sensitivity to protease suggest that the active component is at
least in part a heat-stable lipoprotein or proteoglycan. The HIV
activation seems to be specific to G. vaginalis components, since
other vaginal gram-positive bacteria with generally similar cell
wall structure, such as Staphylococcus species (not aureus) and
L. acidophilus, did not induce HIV expression. The reduction
of activity after lysozyme treatment combined with the recovery
of HIV-inducing activity from only the particulate part of the
lysate indicate that the activating factor(s) is a cell wall or
membrane-associated component of G. vaginalis, such as the
proteogylcans. In fact, ultrastructure studies reveal that G. va-
ginalis has no outer membrane and the thin peptidoglycan layer
of cell wall is exposed on the surface [33]. That the enzymes
had various degrees of effect on the G. vaginalis lysates from
different isolates suggests a qualitative or quantitativedifference
in isolates. For example, the side chains on the active protein
or proteoglycan could vary or the concentration of the active
factor(s) may vary in different isolates.
The up-regulation of HIV by G. vaginalis was probably not
due to lipopolysaccharide (LPS) for the following reasons.
First, G. vaginalis does not possess LPS [33]. Second, flow cy-
tometric analysis of U1 cells by our laboratory confirmed re-
ports by other investigators [34] that U1 cells do not express
the LPS receptor (CD14 molecule) unless they are stimulated
with granulocyte-macrophage colony-stimulating factor. In ad-
dition, in our hands, Escherichia coli LPS at 1 mg/mL (Sigma)
had no effect on HIV replication in U1 cells (unpublished data).
Thus, the activation of HIV by G. vaginalis was probably not
due to LPS.
The induction of HIV expression by G. vaginalis in chroni-
cally infected U1 cells indicates an effect on the expression of
HIV after integration of the virus rather than infection by HIV.
This is consistent with the observation that the G. vaginalis
lysate also activated HIV replication in chronically infected J1.1
cells. Increased HIV-driven CAT activity induced by G. vagin-
alis indicates that the mechanism by which G. vaginalis lysates
stimulated increased production of virus is due to up-regulation
of HIV transcription. Since the presence of exogenous HIV was
required for the HIV-LTR activation by the G. vaginalis lysates
in U38 cells, we conclude that the G. vaginalis–mediated en-
hancement of HIV expression involves a pathway that requires
HIV infection.
We believe this to be the first report to demonstrate that G.
vaginalis activates HIV expression. These findings warrant fur-
ther studies to evaluate the in vivo significance of G. vaginalis
activation of HIV-1. Our results suggest that strategies aimed
at prevention or eliminating genital infections with G. vaginalis
may decrease the frequency of sexual transmission of HIV.
Acknowledgments
We thank Beverly Sha, Department of Internal Medicine, Section
for Infectious Diseases, Rush University, for helping with collection of
patient samples, and Mary Hayden, director, and members of the Rush-
Presbyterian St. Luke’s Medical Center clinical microbiology labora-
tory for assistance in the isolation and identification of the microor-
ganisms used in this study.
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