Antibacterial Activities of Methanolic Seeds Extract of Black pepper (Piper nigrum L.) against Gram Positive Staphylococcus aureus & Gram-Negative Escherichia coli.

Evaluation of the in vitro antibacterial activity of Methanolic extracts isolated from Black pepper seeds (Piper nigrum L.) against two infection causing pathogens, Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Between July 2022 and June 2023, this experimental study was conducted at the Mymensingh Medical College's Department of Pharmacology and Therapeutics in conjunction with the Department of Microbiology. Using the disc diffusion and broth dilution methods, the antibacterial activity of methanolic extract of black pepper seeds (MBPE) was evaluated at various doses. The solvents Methanol and 10.0% Di Methyl Sulfoxide (DMSO) were used to make the extract. Using the broth dilution procedure, the conventional antibiotic Ciprofloxacin was utilized and the outcome was contrasted with that of Methanol extracts. Methanolic extract of black pepper seeds (MBPE) at seven distinct concentrations (100, 80, 60, 40, 20, 10 and 5 mg/ml) were utilized, then later in chosen concentrations as needed to confirm the extracts' more precise margin of antimicrobial sensitivity. At 80 mg/ml and above doses of the MBPE, it had an inhibitory impact against the aforementioned microorganisms. For Staphylococcus aureus and Escherichia coli the MIC were 60 and 75 mg/ml in MBPE respectively. As of the MIC of Ciprofloxacin was 1µg/ml against Staphylococcus aureus and Escherichia coli. In comparison to MICs of MBPE for the test organisms, the MIC of Ciprofloxacin was the lowest. This study clearly shows that Staphylococcus aureus and Escherichia coli are sensitive to the methanolic extract of black pepper seeds' antibacterial properties.

Comparative Efficacy and Safety of Amlodipine and Losartan Potassium in Essential Hypertension in a Tertiary Hospital of Bangladesh.

Hypertension is a common disorder of major clinical, public health and economic importance. It affects men and women of all ages, and the prevalence is increasing in most countries. Maintenance of blood pressure below 140/90 mm of Hg is recommended by most of the guideline available around the world. Various classes of drugs are being used in the treatment of hypertension. Losartan potassium and amlodipine are two different antihypertensive agents belonging to two different groups used commonly around the world in treating essential hypertension. Losartan potassium is non-peptide Angiotensin-II receptor antagonist. Amlodipine which is the third generation dihydropyridine group of calcium channel blocker. The aim of the study was to compare the efficacy and safety of amlodipine and losartan for the treatment of essential hypertensive patients (18-75 years). A non-randomized comparative observational study was conducted in the Department of Pharmacology and Therapeutics in collaboration with Department of Medicine, Sylhet, MAG Osmani Medical College, Sylhet, Bangladesh from July 2021 to June 2022. In this study non-randomization was in two groups. Group A received amlodipine 5mg daily at morning and Group B received losartan potassium 50mg daily at night. The study parameters were systolic blood pressure (SBP), diastolic blood pressure (DBP), ankle oedema, serum K+ level. The result of treatment outcome was compared between two groups. After treatment the reduction of SBP was 5.19±2.93mm of Hg versus 3.27±1.34mm of Hg (p<0.001); reduction of DBP was 1.7±0.70 mm of Hg versus 0.68 mm of Hg (p<0.001) and serum K+ level 4.22±0.27mmol/L versus 4.21±0.16mmol/L (p<0.719) in amlodipine and losartan group respectively. Amlodipine is more effective than losartan potassium in respect to treatment of essential hypertension. Regarding adverse events losartan potassium causes angioedema, hyperkalemia, headache, dizziness etc. The study concluded that amlodipine is superior to losartan potassium in treating essential hypertension.

Current Trends of Using Antimicrobials in Acute Watery Diarrhoea in Children Below 5 Years of Age at Paediatrics Outpatient Department of a Tertiary Care Hospital in Bangladesh.

Childhood diarrhoea is a major public health problem in developing countries like Bangladesh which is commonly caused by intestinal infection, mainly viral. Diarrhoea is causing second leading mortality in children below 5 years, where about 2 billion cases occur globally in each year. This study was proposed to evaluate the antimicrobials utilization pattern prescribed for AWD in children below 5 years of age, in a tertiary care hospital in Bangladesh. This record based, cross-sectional, descriptive type of observational study carried out at pharmacology department of Mymensingh Medical College. A total of 205 diarrhoeal patient's up to 5 years of age, attending the paediatric OPD from January 2021 to December 2021, were enrolled in the study. Out of 205 patients 182(88.8%) were prescribed antimicrobials. Azithromycin was the most frequently prescribed antimicrobial (60.0%) and among the antiprotozoals metronidazole was the prescribed most (24.9%). ORS and Zinc were prescribed in all patients (100%). Empirical excessive use of antibiotics was observed in this study. Emphasis on educational and training programs may help in a better and judicious use of drugs in children.

Antibacterial Effects of Methanol Henna (Lawsone inermis) Leaf Extracts against Two Food Borne Infection Causing Pathogens: Gram-Positive Staphylococcus aureus and Gram Negative Klebsiella pneumoniae.

Evaluation of the in vitro antibacterial activity of Methanol extracts isolated from Henna (Lawsonia inermis) leaf against two food born infection causing pathogens, gram-positive Staphylococcus aureus and gram-negative Klebsiella pneumoniae. This interventional study was carried out in the Department of Pharmacology and Therapeutics in collaboration with the Department of Microbiology, Mymensingh Medical College, Bangladesh from January 2021 to December 2021. The antibacterial activity was tested at different concentrations of Methanol Henna leaf extracts by using disc diffusion and broth dilution method. The extract was prepared by using solvents Methanol and 0.1% dimethyl sulfoxide (DMSO). The test microorganisms were also tested for their activity against a standard antibiotic Ciprofloxacin by broth dilution method and the result was compared with that of Methanol extracts. Methanol Henna Extracts (MHE) were used initially in nine different concentrations (2.5, 5, 10, 20, 50, 100, 200, 500 and 1000mg/ml) and later in selected concentrations as needed to confirm the more precise margin of antimicrobial sensitivity of the extracts. Among different concentrations of the MHE, 100mg/ml and above concentrations showed inhibitory effect against afore said bacteria. The MIC for Staphylococcus aureus and Klebsiella pneumoniae were 100mg/ml in MHE. The MIC of Ciprofloxacin was 1µg/ml against Staphylococcus aureus and 1.5µg/ml for Klebsiella pneumoniae. The MIC of Ciprofloxacin was the lowest in comparison to MICs of MHE for the test organisms. This study showed that Methanol Henna extracts demonstrated antibacterial effects against pathogens. From this study, it is clearly observed that there is definite antibacterial effect of the methanolic extract of Henna leaves (Lawsonia inermis) against Staphylococcus aureus and Klebsiella pneumoniae.

Antibacterial Effects of Chloroform Henna (Lawsonia inermis) Leaf Extracts against Two Nosocomial Infection Causing Pathogens: Gram-positive Staphylococcus aureus and Gram-negative Klebsiella pneumoniae: A Comparative Study.

Evaluation of the in vitro antibacterial activity of Chloroform extracts isolated from Henna (Lawsonia inermis) leaf against two nosocomial infection causing pathogens, gram-positive Staphylococcus aureus and gram-negative Klebsiella pneumoniae. This interventional study was carried out for the period of January 2021 to December 2021 in the Department of Pharmacology and Therapeutics in collaboration with the Department of Microbiology, Mymensingh Medical College, Bangladesh. The antibacterial activity was tested at different concentrations of Chloroform Henna leaf extracts by using disc diffusion and broth dilution method. The extract was prepared by using solvents chloroform and 0.1% Dimethyl sulfoxide (DMSO). The test microorganisms were also tested for their activity against a standard antibiotic Ciprofloxacin by broth dilution method and the result was compared with that of Chloroform extracts. Chloroform Henna Extracts (CHE) were used initially in nine different concentrations (2.5, 5, 10, 20, 50, 100, 200, 500 and 1000 mg/ml). Among different concentrations of the CHE, 100mg/ml and above concentrations showed inhibitory effect against Staphylococcus aureus and Klebsiella pneumoniae. The MIC for Staphylococcus aureus and Klebsiella pneumoniae were 100 and 200mg/ml in CHE respectively. The MIC of Ciprofloxacin was 1µg/ml against Staphylococcus aureus and 1.5µg/ml against Klebsiella pneumoniae. The MIC of Ciprofloxacin was the lowest in comparison to MICs of CHE for the test organisms. This study showed that Chloroform Henna extracts demonstrated antibacterial effects against food borne pathogens. It is clearly observed that there is definite antibacterial effect of the Chloroform extract of Henna leaves (Lawsonia inermis) against Staphylococcus aureus and Klebsiella pneumoniae.

Status of Serum Calcium and Magnesium in Hospital Admitted Chronic Kidney Disease Patients in Mymensingh Locality of Bangladesh.

The study was designed to evaluate the serum calcium and magnesium levels in hospitalized patients with Chronic Kidney Disease (CKD). This cross-sectional study was carried out in the Department of Biochemistry, Mymensingh Medical College (MMC), Bangladesh with the help of the Department of Nephrology, Mymensingh Medical College Hospital, Bangladesh from the period of January 2021 to December 2021. The subjects were selected on the basis of inclusion and exclusion criteria by purposive and convenient sampling method. A total of 110 subjects were included in this study. Among them, 55 were diagnosed CKD patients denoted as Group I and 55 were normal healthy individuals denoted as Group II. The subjects were briefed and written consents were taken. Under aseptic precaution 5.0ml of venous blood was collected from median cubital vein. Analyses were carried out in the Department of Biochemistry, Mymensingh Medical College and serum calcium and magnesium levels were measured. All values were expressed as Mean±SD. All statistical analysis was done by using SPSS (statistical package for social science) windows package version 21.0. Statistical significance of difference between Group I and Group II were evaluated by using Student's unpaired 't' test and the significance was defined as p<0.05. Correlation was done by using Pearson's correlation coefficient test. The mean±SD values of serum calcium were 8.15±0.54mg/dl and 9.80±0.50mg/dl and serum magnesium were 2.25±0.17mg/dl and 1.95±0.50mg/dl in Group I and Group II respectively. We found mean serum calcium was highly significantly (<0.001) decreased and serum magnesium was highly significantly increased (<0.001) in CKD patients in comparison to that of healthy individual.

Pattern of Prescribing Drugs in Chronic Obstructive Pulmonary Disease in a Tertiary Care Hospital of Bangladesh.

Chronic obstructive pulmonary disease (COPD) is a chronic obstructive disease of the airways. It is one of the most common and important chronic respiratory conditions in terms of years lived with disability. Incidence is increasing in Bangladesh like other developing countries. To evaluate drug prescription pattern for COPD, this cross-sectional, observational study was conducted from January to December in 2020 at the Department of Pharmacology in collaboration with the Department of Medicine in Mymensingh Medical College, Bangladesh. A total of 168 patients were selected for the study by non-random purposive sampling technique. Age distribution indicates that 31.5% of patients were in the 50-59 years age group and males were 93.5%. The majority (82.1%) of the participants were smokers. In this study, majority (34.12%) of the drugs were used as oral form and second most common dosage form was nebulization (26.75%). Bronchodilators were mostly prescribed 652(57.19%), followed by corticosteroids 222(19.47%) and antibiotics 165(14.47%) among drugs used for COPD. Beta sympathomimetics 322(45.49%) were mostly prescribed, followed by anticholinergics 186(28.52%) and methylxanthines 144(22.08%) as bronchodilators. Out of 1140 drugs for COPD, 53.06% and 34.12% were delivered as inhalation and oral forms, respectively. Inhalation route was the most (60.37%) preferred one over oral route (37.63%) for steroid use. The most of the patients [152 (90.48%)] were treated with combination therapy. Mostly (39.6%) used Fixed Dose Combination (FDC) therapy was salbutamol and ipratropium bromide followed by salmeteroal and Fluticasone (30.83%). Both FDC were prescribed in 57.7% of study population. Considering nomenclature, trade name was used in 24.4% of prescription.

Antibacterial Activities of Mint (Mentha piperita) Leaf Extracts (Aqueous) Against Two Food Borne infection causing pathogens: Staphylococcus aureus and Escherichia coli.

Evaluation of the in vitro antibacterial activity of Aqueous extracts isolated from Mint (Mentha piperita) leaf against two food born infection causing pathogens, gram-positive Staphylococcus aureus and gram-negative Escherichia coli. This interventional study was carried out in the Department of Pharmacology and Therapeutics in collaboration with the Department of Microbiology, Mymensingh Medical College, Bangladesh from January 2021 to December 2021. The antibacterial activity was tested at different concentrations of Aqueous Mint leaf extracts by using disc diffusion & broth dilution method. The extract was prepared by using solvents Aqueous. The test microorganisms were also tested for their activity against a standard antibiotic Gentamicin by broth dilution method and the result was compared with that of Aqueous extracts. Aqueous extract of Mint leaves (AMLE) were used initially in eight different concentrations (25, 50, 100, 200, 400, 600, 800 and 1000µg/ml) and later in selected concentrations as needed to confirm the more precise margin of antimicrobial sensitivity of the extracts. Among different concentrations of the AMLE, 200µg/ml and above concentrations showed inhibitory effect against Staphylococcus aureus and 400µg/ml and above concentrations showed inhibitory effect against Escherichia coli. Minimum inhibitory concentration (MIC) for Staphylococcus aureus and Escherichia coli were 200 and 400µg/ml in AMLE respectively. The MIC of Gentamicin was 1µg/ml against Staphylococcus aureus and 1.5µg/ml against Escherichia coli. The MIC of Gentamicin was the lowest in comparison to MICs of AMLE for the test organisms. This study showed that Aqueous Mint extracts demonstrated antibacterial effects against food borne pathogens. It is clearly observed that there is definite antibacterial effect of the aqueous extract of Mint leaves against Staphylococcus aureus and Escherichia coli.

Neisseria gonorrhoeae MtrCDE Efflux Pump During In Vivo Experimental Genital Tract Infection in Men and Mice Reveals the Presence of Within-Host Colonization Bottleneck.

The MtrCDE efflux pump of Neisseria gonorrhoeae exports a wide range of antimicrobial compounds that the gonococcus encounters at mucosal surfaces during colonization and infection. Here, we evaluate the role of this efflux pump system in strain FA1090 in human male urethral infection with a Controlled Human Infection Model. Using the strategy of competitive multi-strain infection with wild-type FA1090 and an isogenic mutant strain that does not contain a functional MtrCDE pump, we found that the presence of the efflux pump during human experimental infection did not confer a competitive advantage. This finding is in contrast to previous findings in female mice, which demonstrated that gonococci of strain FA19 lacking a functional MtrCDE pump had a significantly reduced fitness compared to the wild type strain in the lower genital tract of female mice. We conducted competitive infections in female mice with FA19 and FA1090 strains, including mutants that do not assemble a functional Mtr efflux pump, demonstrating the fitness advantage provided byt the MtrCDE efflux pump during infection of mice is strain dependent. Our data indicate that new gonorrhea treatment strategies targeting the MtrCDE efflux pump functions may not be universally efficacious in naturally occurring infections. Owing to the equal fitness of FA1090 strains in men, our experiments unexpectedly demonstrated the likely presence of an early colonization bottleneck of N. gonorrhoeae in the human male urethra. TRIAL REGISTRATION: Clinicaltrials.gov NCT03840811 .

Antibacterial Effects of Methanolic Leaf Extracts of Henna (Lawsonia inermis) Against Two Most Common Pathogenic Organisms: Gram Positive Staphylococcus aureus and Gram-Negative Escherichia coli.

Evaluation of the in vitro antibacterial activity of Methanolic extracts isolated from Henna (Lawsonia inermis) leaf against two nosocomial infection causing pathogens, gram-positive Staphylococcus aureus and gram-negative Escherichia coli. This interventional study was carried out during the period of January 2021 to December 2021 in the Department of Pharmacology and Therapeutics in collaboration with the Department of Microbiology, Mymensingh Medical College, Mymensingh, Bangladesh. The antibacterial activity was tested at different concentrations of Methanolic Henna leaf extracts by using disc diffusion and broth dilution method. The extract was prepared by using solvents Methanol and 0.1% DMSO (Dimethyl sulfoxide). The test microorganisms were also tested for their activity against a standard antibiotic Ciprofloxacin by broth dilution method and the result was compared with that of Methanolic leaf extracts. Methanolic Henna leaf Extracts (MHE) were used initially in nine different concentrations (2.5, 5, 10, 20, 50, 100, 200, 500 and 1000 mg/ml) and later in selected concentrations as needed to confirm the more precise margin of antimicrobial sensitivity of the extracts. Among different concentrations of the MHE, 100mg/ml and above concentrations showed inhibitory effect against aforesaid bacteria. The MIC for Staphylococcus aureus and Escherichia coli were 100 and 200 mg/ml in MHE respectively. The MIC of Ciprofloxacin was 1µg/ml against both Staphylococcus aureus and Escherichia coli. The MIC of Ciprofloxacin was the lowest in comparison to MICs of MHE for the test organisms. The present study showed that Methanol Henna extracts demonstrated antibacterial effects against nosocomial infection pathogens. From this study, it is clearly observed that there are definite antibacterial effects of the methanolic extract of Henna leaves (Lawsonia inermis) against Staphylococcus aureus and Escherichia coli.

Serum Albumin and C-reactive protein in Hospital Admitted Patients with Chronic Kidney Disease.

Adequate knowledge about the causes of chronic kidney disease and their potential prevention can improve poor clinical outcome in chronic kidney disease (CKD) patients. The study was designed to evaluate the serum albumin and C-reactive protein (CRP) levels in hospitalized patients with Chronic Kidney Disease. This cross-sectional study was carried out in the Department of Biochemistry, Mymensingh Medical College, Mymensingh, Bangladesh with the collaboration of the Department of Nephrology, Mymensingh Medical College Hospital, Mymensingh, from January 2021 to December 2021. The subjects were selected on the basis of inclusion and exclusion criteria by purposive and convenient sampling method. A total of 110 subjects were included in this study. Among them, 55 were diagnosed CKD patients denoted as Group I and 55 were normal healthy individuals denoted as Group II. In this study, serum albumin and CRP levels were measured. All values were expressed as Mean±SD. All statistical analysis was done by using SPSS (statistical package for social science) windows package version 21.0. Statistical significance of difference between Group I and Group II were evaluated by using student's unpaired 't'-test and the significance was defined as p<0.05. Correlation was done by using Pearson's correlation coefficient test. Mean age of Group I was 52.65±4.93 and Group II was 51.15±6.32 (p=0.165). The mean±SD of BMI was 24.46±1.84 for Group I and 24.50±1.05 for Group II (p=0.886). The mean±SD values of serum albumin were 3.62±0.26g/dl and 4.16±0.69g/dl in Group I and Group II respectively. We found highly significant (p<0.001) decrease in serum albumin. The mean±SD values of CRP were 24.00±16.73mg/L and <6.0±0.00mg/L in Group I and Group II respectively. So, we found significant (p<0.05) increase in CRP levels. There was negative correlation between serum albumin and CRP. Analyzing the findings of this study, significant decrease in serum albumin and significant increase in CRP levels were observed in CKD patients.

Antibacterial Effects of Henna (Lawsonia inermis) Leaf Extracts (Chloroform) Against Two Food Borne and Nosocomial Infection Causing Pathogens: Staphylococcus aureus and Escherichia coli.

Evaluation of the in vitro antibacterial activity of Chloroform extracts isolated from Henna (Lawsonia inermis) leaf against two food born & nosocomial infection causing pathogens, gram-positive Staphylococcus aureus and gram-negative Escherichia coli. This interventional study was carried out during the period of January 2021 to December 2021 in the Department of Pharmacology and Therapeutics in collaboration with the Department of Microbiology, Mymensingh Medical College, Mymensingh, Bangladesh. The antibacterial activity was tested at different concentrations of Chloroform Henna leaf extracts by using disc diffusion and broth dilution method. The extract was prepared by using solvents chloroform and 0.1% DMSO. The test microorganisms were also tested for their activity against a standard antibiotic Ciprofloxacin by broth dilution method and the result was compared with that of Chloroform extracts. Chloroform Henna Extracts (CHE) were used initially in nine different concentrations (2.5, 5, 10, 20, 50, 100, 200, 500 and 1000mg/ml) and later in selected concentrations as needed to confirm the more precise margin of antimicrobial sensitivity of the extracts. Among different concentrations of the CHE, 100mg/ml and above concentrations showed inhibitory effect against Staphylococcus aureus and 300mg/ml and above concentrations showed inhibitory effect against Escherichia coli. The MIC for Staphylococcus aureus and Escherichia coli were 100 and 350mg/ml in CHE respectively. The MIC of Ciprofloxacin was 1µg/ml against both Staphylococcus aureus and Escherichia coli. The MIC of Ciprofloxacin was the lowest in comparison to MICs of CHE for the test organisms. The present study showed that Chloroform Henna extracts demonstrated antibacterial effects against food borne pathogens. From this study, it is clearly observed that there is definite antibacterial effect of the chloroform extract of Henna leaves against Staphylococcus aureus and Escherichia coli.

A Single Amino Acid Substitution in Elongation Factor G Can Confer Low-Level Gentamicin Resistance in Neisseria gonorrhoeae.

The continued emergence of Neisseria gonorrhoeae isolates which are resistant to first-line antibiotics has reinvigorated interest in alternative therapies such as expanded use of gentamicin (Gen). We hypothesized that expanded use of Gen promotes emergence of gonococci with clinical resistance to this aminoglycoside. To understand how decreased susceptibility of gonococci to Gen might develop, we selected spontaneous low-level Gen-resistant (GenR) mutants (Gen MIC = 32 µg/mL) of the Gen-susceptible strain FA19. Consequently, we identified a novel missense mutation in fusA, which encodes elongation factor G (EF-G), causing an alanine (A) to valine (V) substitution at amino acid position 563 in domain IV of EF-G; the mutant allele was termed fusA2. Transformation analysis showed that fusA2 could increase the Gen MIC by 4-fold. While possession of fusA2 did not impair either in vitro gonococcal growth or protein synthesis, it did result in a fitness defect during experimental infection of the lower genital tract in female mice. Through bioinformatic analysis of whole-genome sequences of 10,634 international gonococcal clinical isolates, other fusA alleles were frequently detected, but genetic studies revealed that they could not decrease Gen susceptibility in a similar manner to fusA2. In contrast to these diverse international fusA alleles, the fusA2-encoded A563V substitution was detected in only a single gonococcal clinical isolate. We hypothesize that the rare occurrence of fusA2 in N. gonorrhoeae clinical isolates is likely due to a fitness cost during infection, but compensatory mutations which alleviate this fitness cost could emerge and promote GenR in global strains.

Meningococcal Detoxified Outer Membrane Vesicle Vaccines Enhance Gonococcal Clearance in a Murine Infection Model.

BACKGROUND: Despite decades of research efforts, development of a gonorrhea vaccine has remained elusive. Epidemiological studies suggest that detoxified outer membrane vesicle (dOMV) vaccines from Neisseria meningitidis (Nm) may protect against infection with Neisseria gonorrhoeae (Ng). We recently reported that Nm dOMVs lacking the major outer membrane proteins (OMPs) PorA, PorB, and RmpM induced greater antibody cross-reactivity against heterologous Nm strains than wild-type (WT) dOMVs and may represent an improved vaccine against gonorrhea. METHODS: We prepared dOMV vaccines from meningococcal strains that were sufficient or deleted for PorA, PorB, and RmpM. Vaccines were tested in a murine genital tract infection model and antisera were used to identify vaccine targets. RESULTS: Immunization with Nm dOMVs significantly and reproducibly enhanced gonococcal clearance for mice immunized with OMP-deficient dOMVs; significant clearance for WT dOMV-immunized mice was observed in one of two experiments. Clearance was associated with serum and vaginal anti-Nm dOMV immunoglobulin G (IgG) antibodies that cross-reacted with Ng. Serum IgG was used to identify putative Ng vaccine targets, including PilQ, MtrE, NlpD, and GuaB. CONCLUSIONS: Meningococcal dOMVs elicited a protective effect against experimental gonococcal infection. Recognition and identification of Ng vaccine targets by Nm dOMV-induced antibodies supports the development of a cross-protective Neisseria vaccine.

cis- and trans-Acting Factors Influence Expression of the norM-Encoded Efflux Pump of Neisseria gonorrhoeae and Levels of Gonococcal Susceptibility to Substrate Antimicrobials.

The gonococcal NorM efflux pump exports substrates with a cationic moiety, including quaternary ammonium compounds such as berberine (BE) and ethidium bromide (EB) as well as antibiotics such as ciprofloxacin and solithromycin. The norM gene is part of a four-gene operon that is transcribed from a promoter containing a polynucleotide tract of 6 or 7 thymidines (T's) between the -10 and -35 hexamers; the majority of gonococcal strains analyzed in this study contained a T-6 sequence. Primer extension analysis showed that regardless of the length of the poly(T) tract, the same transcriptional start site (TSS) was used for expression of norM Interestingly, the T-6 tract correlated with a higher level of both norM expression and gonococcal resistance to NorM substrates BE and EB. Analysis of expression of genes downstream of norM showed that the product of the tetR-like gene has the capacity to activate expression of norM as well as murB, which encodes an acetylenolpyroylglucosamine reductase predicted to be involved in the early steps of peptidoglycan synthesis. Moreover, loss of the TetR-like transcriptional regulator modestly increased gonococcal susceptibility to NorM substrates EB and BE. We conclude that both cis- and trans-acting regulatory systems can regulate expression of the norM operon and influence levels of gonococcal susceptibility to antimicrobials exported by NorM.

Control of gdhR Expression in Neisseria gonorrhoeae via Autoregulation and a Master Repressor (MtrR) of a Drug Efflux Pump Operon.

The MtrCDE efflux pump of Neisseria gonorrhoeae contributes to gonococcal resistance to a number of antibiotics used previously or currently in treatment of gonorrhea, as well as to host-derived antimicrobials that participate in innate defense. Overexpression of the MtrCDE efflux pump increases gonococcal survival and fitness during experimental lower genital tract infection of female mice. Transcription of mtrCDE can be repressed by the DNA-binding protein MtrR, which also acts as a global regulator of genes involved in important metabolic, physiologic, or regulatory processes. Here, we investigated whether a gene downstream of mtrCDE, previously annotated gdhR in Neisseria meningitidis, is a target for regulation by MtrR. In meningococci, GdhR serves as a regulator of genes involved in glucose catabolism, amino acid transport, and biosynthesis, including gdhA, which encodes an l-glutamate dehydrogenase and is located next to gdhR but is transcriptionally divergent. We report here that in N. gonorrhoeae, expression of gdhR is subject to autoregulation by GdhR and direct repression by MtrR. Importantly, loss of GdhR significantly increased gonococcal fitness compared to a complemented mutant strain during experimental murine infection. Interestingly, loss of GdhR did not influence expression of gdhA, as reported for meningococci. This variance is most likely due to differences in promoter localization and utilization between gonococci and meningococci. We propose that transcriptional control of gonococcal genes through the action of MtrR and GdhR contributes to fitness of N. gonorrhoeae during infection.IMPORTANCE The pathogenic Neisseria species are strict human pathogens that can cause a sexually transmitted infection (N. gonorrhoeae) or meningitis or fulminant septicemia (N. meningitidis). Although they share considerable genetic information, little attention has been directed to comparing transcriptional regulatory systems that modulate expression of their conserved genes. We hypothesized that transcriptional regulatory differences exist between these two pathogens, and we used the gdh locus as a model to test this idea. For this purpose, we studied two conserved genes (gdhR and gdhA) within the locus. Despite general conservation of the gdh locus in gonococci and meningococci, differences exist in noncoding sequences that correspond to promoter elements or potential sites for interacting with DNA-binding proteins, such as GdhR and MtrR. Our results indicate that implications drawn from studying regulation of conserved genes in one pathogen are not necessarily translatable to a genetically related pathogen.

Phosphoethanolamine decoration of Neisseria gonorrhoeae lipid A plays a dual immunostimulatory and protective role during experimental genital tract infection.

The induction of an intense inflammatory response by Neisseria gonorrhoeae and the persistence of this pathogen in the presence of innate effectors is a fascinating aspect of gonorrhea. Phosphoethanolamine (PEA) decoration of lipid A increases gonococcal resistance to complement-mediated bacteriolysis and cationic antimicrobial peptides (CAMPs), and recently we reported that wild-type N. gonorrhoeae strain FA1090 has a survival advantage relative to a PEA transferase A (lptA) mutant in the human urethral-challenge and murine lower genital tract infection models. Here we tested the immunostimulatory role of this lipid A modification. Purified lipooligosaccharide (LOS) containing lipid A devoid of the PEA modification and an lptA mutant of strain FA19 induced significantly lower levels of NF-κB in human embryonic kidney Toll-like receptor 4 (TLR4) cells and murine embryonic fibroblasts than wild-type LOS of the parent strain. Moreover, vaginal proinflammatory cytokines and chemokines were not elevated in female mice infected with the isogenic lptA mutant, in contrast to mice infected with the wild-type and complemented lptA mutant bacteria. We also demonstrated that lptA mutant bacteria were more susceptible to human and murine cathelicidins due to increased binding by these peptides and that the differential induction of NF-κB by wild-type and unmodified lipid A was more pronounced in the presence of CAMPs. This work demonstrates that PEA decoration of lipid A plays both protective and immunostimulatory roles and that host-derived CAMPs may further reduce the capacity of PEA-deficient lipid A to interact with TLR4 during infection.

Lipid A's structure mediates Neisseria gonorrhoeae fitness during experimental infection of mice and men.

UNLABELLED: Phosphoethanolamine (PEA) on Neisseria gonorrhoeae lipid A influences gonococcal inflammatory signaling and susceptibility to innate host defenses in in vitro models. Here, we evaluated the role of PEA-decorated gonococcal lipid A in competitive infections in female mice and in male volunteers. We inoculated mice and men with mixtures of wild-type N. gonorrhoeae and an isogenic mutant that lacks the PEA transferase, LptA. LptA production conferred a marked survival advantage for wild-type gonococci in the murine female genital tract and in the human male urethra. Our studies translate results from test tube to animal model and into the human host and demonstrate the utility of the mouse model for studies of virulence factors of the human-specific pathogen N. gonorrhoeae that interact with non-host-restricted elements of innate immunity. These results validate the use of gonococcal LptA as a potential target for development of novel immunoprophylactic strategies or antimicrobial treatments. IMPORTANCE: Gonorrhea is one of the most common bacterial sexually transmitted infections, and increasing antibiotic resistance threatens the use of currently available antimicrobial therapies. In this work, encompassing in vitro studies and in vivo studies of animal and human models of experimental genital tract infection, we document the importance of lipid A's structure, mediated by a single bacterial enzyme, LptA, in enhancing the fitness of Neisseria gonorrhoeae. The results of these studies suggest that novel agents targeting LptA may offer urgently needed prevention or treatment strategies for gonorrhea.

Impact of fluoroquinolone resistance mutations on gonococcal fitness and in vivo selection for compensatory mutations.

BACKGROUND: Quinolone-resistant Neisseria gonorrhoeae (QRNG) arise from mutations in gyrA (intermediate resistance) or gyrA and parC (resistance). Here we tested the consequence of commonly isolated gyrA(91/95) and parC86 mutations on gonococcal fitness. METHODS: Mutant gyrA(91/95) and parC86 alleles were introduced into wild-type gonococci or an isogenic mutant that is resistant to macrolides due to an mtrR(-79) mutation. Wild-type and mutant bacteria were compared for growth in vitro and in competitive murine infection. RESULTS: In vitro growth was reduced with increasing numbers of mutations. Interestingly, the gyrA(91/95) mutation conferred an in vivo fitness benefit to wild-type and mtrR(-79) mutant gonococci. The gyrA(91/95), parC86 mutant, in contrast, showed a slight fitness defect in vivo, and the gyrA(91/95), parC86, mtrR(-79) mutant was markedly less fit relative to the parent strains. A ciprofloxacin-resistant (Cip(R)) mutant was selected during infection with the gyrA(91/95), parC86, mtrR(-79) mutant in which the mtrR(-79) mutation was repaired and the gyrA(91) mutation was altered. This in vivo-selected mutant grew as well as the wild-type strain in vitro. CONCLUSIONS: gyrA(91/95) mutations may contribute to the spread of QRNG. Further acquisition of a parC86 mutation abrogates this fitness advantage; however, compensatory mutations can occur that restore in vivo fitness and maintain Cip(R).

Estradiol-Treated Female Mice as Surrogate Hosts for Neisseria gonorrhoeae Genital Tract Infections.

Historically, animal modeling of gonorrhea has been hampered by the exclusive adaptation of Neisseria gonorrhoeae to humans. Genital tract infection can be established in female mice that are treated with 17ß-estradiol, however, and many features of experimental murine infection mimic human infection. Here we review the colonization kinetics and host response to experimental murine gonococcal infection, including mouse strain differences and evidence that IL-17 responses, toll-like receptor 4, and T regulatory cells play a role in infection. We also discuss the strengths and limitations of the mouse system and the potential of transgenic mice to circumvent host restrictions. Additionally, we review studies with genetically defined mutants that demonstrated a role for sialyltransferase and the MtrC-MtrD-MtrE active efflux pump in evading innate defenses in vivo, but not for factors hypothesized to protect against the phagocytic respiratory burst and H(2)O(2)-producing lactobacilli. Studies using estradiol-treated mice have also revealed the existence of non-host-restricted iron sources in the female genital tract and the influence of hormonal factors on colonization kinetics and selection for opacity (Opa) protein expression. Recent work by others with estradiol-treated mice that are transgenic for human carcinoembryonic adhesion molecules (CEACAMs) supports a role for Opa proteins in enhancing cellular attachment and thus reduced shedding of N. gonorrhoeae. Finally we discuss the use of the mouse model in product testing and a recently developed gonorrhea chlamydia coinfection model.