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The salicylidene acylhydrazide INP0341 inhibits growth of Chlamydia in HeLa cells, has negligible cell toxicity, and does not inhibit the growth of lactobacilli. The antichlamydial activity of INP0341 was retained when tested in vaginal and semen simulants. Vaginal tissue from INP0341-treated mice appeared similar to control sham-treated mice. To determine whether INP0341 can protect mice from a vaginal challenge, C3H/HeJ mice were either sham or INP0341 treated intravaginally pre- and postinoculation with 5 × 102 inclusion-forming units (IFUs) of Chlamydia trachomatis serovar D. Vaginal cultures taken over a month-long period showed a significant difference in the number of control mice that were culture positive versus the number in the INP0341-treated group, 100% (25/25) and 31% (8/26), respectively (P < .05). The quantity of IFUs shed and antibody titers to Chlamydia were significantly higher for the control group (P < .05). In summary, INP0341 is a promising compound to be considered for formulation as a vaginal microbicide.

Introduction

Salicylidene acylhydrazide compounds have been shown to inhibit bacterial pathogens, including Chlamydia and Neisseria gonorrhoeae. If such compounds could also target HIV-1, their potential use as topical microbicides to prevent sexually transmitted infections would be considerable. We determined the in vitro anti-HIV-1 activity, cytotoxicity and mechanism of action of several salicylidene acylhydrazides.

Methods

Inhibitory activity was assessed using TZMbl cells and primary peripheral blood mononuclear cells (PBMCs) as targets for HIV-1 infection. Anti-viral activity was measured against cell-free and cell-associated virus and in vaginal fluid and semen simulants. Since the anti-bacterial activity of salicylidene acylhydrazides is reversible by Fe2+, we determined whether Fe2+ and other cations could reverse the anti-HIV-1 activity of the compounds. We also employed real-time PCR to determine the stage affected in the HIV-1 replication cycle.

Results

We identified four compounds with 50% HIV-1 inhibitory concentrations of 1 to 7 μM. In vitro toxicity varied but was generally limited. Activity was similar against three R5 clade B primary isolates and whether targets for virus replication were TZMbl cells or PBMCs. Compounds inhibited cell-free and cell-associated virus and were active in vaginal fluid and semen simulants. Fe2+, but not other cations, reversed the anti-HIV-1 effect. Finally, inhibitory effect of the compounds occurred at a post-integration step.

Conclusions

We identified salicylidene acylhydrazides with in vitro anti-HIV-1 activity in the μM range. The activity of these compounds against other sexually transmitted pathogens makes them potential candidates to formulate for use as a broad-spectrum topical genital microbicide.

INPs, which are chemically synthesized compounds belonging to a class of acylated hydrazones of salicylaldehydes, can inhibit the growth of Chlamydiaceae. Evidence has been presented that in Yersinia and Chlamydia INPs may affect the type III secretion (T3S) system. In the present study 25 INPs were screened for antichlamydial activity at a concentration of 50 μM, and 14 were able to completely inhibit the growth of Chlamydia trachomatis serovar D in McCoy and HeLa 229 cells. The antichlamydial activities of two of these INPs, INPs 0341 and 0400, were further characterized due to their low cytotoxicity. These compounds were found to inhibit C. trachomatis in a dose-dependent manner; were not toxic to elementary bodies; were cidal at a concentration of ≥20 μM; inhibited all Chlamydiaceae tested; and could inhibit the development of C. trachomatis as determined by the yield of progeny when they were added up to 24 h postinfection. INP 0341 was able to affect the expression of several T3S genes. Compared to the expression in control cultures, lcrH-1, copB, and incA, all middle- to late-expressed T3S genes, were not expressed in the INP 0341-treated cultures 24 to 36 h postinfection. Iron, supplied as ferrous sulfate, as ferric chloride, or as holo-transferrin, was able to negate the antichlamydial properties of the INPs. In contrast, apo-transferrin and other divalent metal ions tested were not able to reverse the inhibitory effect of the INPs. In conclusion, the potent antichlamydial activity of INPs is directly or indirectly linked with iron, and this inhibition of Chlamydia has an effect on the T3S system of this intracellular pathogen.

A topical microbicide that women can use to prevent sexually transmitted diseases (STDs) is essential, and many microbicide candidates are being tested for activity against human immunodeficiency virus and other STDs, including Chlamydia trachomatis. Screening assays for assessing the activity of microbicides against C. trachomatis are typically done with laboratory-adapted strains, but it is possible that recent clinical isolates may have different susceptibilities to microbicides, as has been seen with Neisseria gonorrhoeae and Lactobacillus spp. (B. J. Moncla and S. L. Hillier, Sex. Transm. Dis. 32:491-494, 2005). We utilized three types of microbicides to help define this aspect of our assay to test microbicides against C. trachomatis in vitro. To simulate conditions of transmission, we used an assay that we previously developed in which we exposed chlamydial elementary bodies to microbicides prior to contact with epithelial cells. We first determined the toxicity of microbicides to the cells used to culture Chlamydia trachomatis in the assay and, if necessary, modified the assay to eliminate toxicity at the concentrations tested. We compared the sensitivities of recent clinical isolates of Chlamydia trachomatis versus laboratory strains of the same serovar and found major differences in sensitivity to nonoxynol-9 (non-9), but only minor differences were seen with the other microbicides. We thus conclude that when assessing activity of potential topical microbicides versus the obligate intracellular bacteria C. trachomatis, the use of recent clinical isolates may not be necessary to draw a conclusion about a microbicide's effectiveness. However, it is important to keep in mind that differences (like those seen with non-9) are possible and that clinical isolates could be included in later stages of testing.

Salicylidene acylhydrazides identified as inhibitors of virulence-mediating type III secretion systems (T3SSs) potentially target their inner membrane export apparatus. They also lead to inhibition of flagellar T3SS-mediated swimming motility in Salmonella enterica serovar. Typhimurium. We show that INP0404 and INP0405 act by reducing the number of flagella/cell. These molecules still inhibit motility of a Salmonella ΔfliH-fliI-fliJ/flhB(P28T) strain, which lacks three soluble components of the flagellar T3S apparatus, suggesting that they are not the target of this drug family. We implemented a genetic screen to search for the inhibitors' molecular target(s) using motility assays in the ΔfliH-fliI/flhB(P28T) background. Both mutants identified were more motile than the background strain in the absence of the drugs, although HM18 was considerably more so. HM18 was more motile than its parent strain in the presence of both drugs while DI15 was only insensitive to INP0405. HM18 was hypermotile due to hyperflagellation, whereas DI15 was not hyperflagellated. HM18 was also resistant to a growth defect induced by high concentrations of the drugs. Whole-genome resequencing of HM18 indicated two alterations within protein coding regions, including one within atpB, which encodes the inner membrane a-subunit of the FOF1-ATP synthase. Reverse genetics indicated that the alteration in atpB was responsible for all of HM18's phenotypes. Genome sequencing of DI15 uncovered a single A562P mutation within a gene encoding the flagellar inner membrane protein FlhA, the direct role of which in mediating drug insensitivity could not be confirmed. We discuss the implications of these findings in terms of T3SS export apparatus function and drug target identification.

Salicylidene acylhydrazides belong to a class of compounds shown to inhibit bacterial type III secretion (T3S) in pathogenic Gram-negative bacteria. This class of compounds also inhibits growth and replication of Chlamydiae, strict intracellular bacteria that possess a T3S system. In this study a library of 58 salicylidene acylhydrazides was screened to identify inhibitors of Chlamydia growth. Compounds inhibiting growth of both Chlamydia trachomatis and Chlamydophila pneumoniae were tested for cell toxicity and seven compounds were selected for preliminary pharmacokinetic analysis in mice using cassette dosing. Two compounds, ME0177 and ME0192, were further investigated by individual pharmacokinetic analysis. Compound ME0177 had a relatively high peak plasma concentration (Cmax) and area under curve and therefore may be considered for systemic treatment of Chlamydia infections. The other compound, ME0192, had poor pharmacokinetic properties but the highest anti-chlamydial activity in vitro and therefore was tested for topical treatment in a mouse vaginal infection model. ME0192 administered vaginally significantly reduced the infectious burden of C. trachomatis and the number of infected mice.

OBJECTIVE: To investigate the in vitro microbicidal and cytocidal potency of monocaprin dissolved in pharmaceutical hydrogel formulations and to evaluate their potential use as vaginal microbicides against sexually transmitted pathogens such as herpes simplex virus type 2 (HSV-2), human immunodeficiency virus type 1 (HIV-1), Chlamydia trachomatis, and Neisseria gonorrhoeae. METHODS: Gel formulations were mixed with equal volumes of virus/bacteria suspensions in culture medium and incubated for 1 and 5 minutes. The reduction in virus/bacteria titre was used as a measure of microbicidal activity. Similarly, gels were mixed with human semen to study their effect on leucocytes. The toxicity of the gels was tested in rabbits by the standard vaginal irritation test. RESULTS: Gels containing 20 mM of monocaprin caused a greater than 100,000-fold inactivation of HSV-2 and Neisseria in 1 minute and of Chlamydia in 5 minutes. Similarly, the gels caused a greater than 10,000-fold inactivation of HIV-1 in semen in 1 minute. They caused more than a 10,000-fold reduction in the number of viable leucocytes in semen in 1 minute. No toxic effect on the vaginal mucosa of rabbits was observed after daily exposure for 10 days. CONCLUSIONS: Hydrogels containing monocaprin are potent inactivators of sexually transmitted viruses and bacteria in vitro. This simple lipid seems to be a feasible choice as a mucosal microbicide for prevention of sexually transmitted infections. It is a natural compound found in certain foodstuffs such as milk and is therefore unlikely to cause harmful side effects in the concentrations used. 




Background

Infection due to Chlamydia trachomatis is the most common sexually transmitted bacterial disease of global health significance, and especially the L-serovars causing lymphogranuloma venereum are increasingly being found in Europe in men who have sex with men.

Results

The design and evaluation of a rapid, multiplex, real-time PCR targeting the major outer membrane protein (omp-1) -gene and a L-serovar-specific region of the polymorphic protein H (pmp-H) -gene for the detection of Chlamydia trachomatis is reported here. The PCR takes place as a single reaction with an internal control. For L1-, L2- and L3-serovar differentiation a second set of real-time PCRs was evaluated based on the amplification of serovar-specific omp-1-regions. The detection limit of each real-time PCR, multiplexed or not, was 50 genome copies per reaction with an efficiency ranging from 90,5–95,2%.

In a retrospective analysis of 50 ocular, rectal and urogenital specimens formerly tested to be positive for C. trachomatis we identified six L2-serovars in rectal specimens of HIV-positive men, one in a double-infection with L3, and one L2 in a urethral specimen of an HIV-negative male.

Conclusion

This unique real-time PCR is specific and convenient for the rapid routine-diagnostic detection of lymphogranuloma venereum-associated L-serovars and enables the subsequent differentiation of L1, L2 and L3 for epidemiologic studies.

Women who attended the gynaecology clinic or were admitted to the postpartum ward of Korle Bu Hospital, Accra, Ghana were tested for infection with Chlamydia trachomatis and Neisseria gonorrhoeae. Eight (4.9%) of 162 gynaecological patients were infected with C trachomatis and five (3.1%) with N gonorrhoeae, and respective prevalences among 148 postpartum women were 7.7% (3/39) and 3.4% (5/148). Among 40 gynaecological patients who were not pregnant and whose principal complaint was of lower abdominal pain, 4 (10%) were infected with C trachomatis and none with N gonorrhoeae. Antibodies against serovars D, E, F, and G were common, and three typable isolates were serovar G. C trachomatis would appear to be more common than N gonorrhoeae in obstetric and gynaecological patients in Ghana.

The in vitro activity of tosufloxacin (A-61827; T-3262) against 15 clinical isolates of Chlamydia trachomatis and 31 recent clinical isolates of Neisseria gonorrhoeae, including 15 spectinomycin-resistant N. gonorrhoeae and 16 penicillinase-producing N. gonorrhoeae, was determined. Tosufloxacin was compared with doxycycline, ciprofloxacin, and temafloxacin against C. trachomatis. Susceptibility testing was performed by using McCoy cell monolayers. The in vitro activity of tosufloxacin against N. gonorrhoeae was compared with that of ciprofloxacin, temafloxacin, doxycycline, ceftriaxone, and spectinomycin by using an agar dilution method. Tosufloxacin was as active as temafloxacin against C. trachomatis (MIC for 90% of strains tested [MIC90], 0.25 microgram/ml; MBC for 90% of strains tested [MBC90], 0.25 microgram/ml) and was almost as active as doxycycline (MIC90, 0.06 microgram/ml; MBC90, 0.125 microgram/ml). Ciprofloxacin, temafloxacin, and tosufloxacin were extremely active against N. gonorrhoeae, including spectinomycin-resistant N. gonorrhoeae and penicillinase-producing N. gonorrhoeae, with MIC90s of 0.004, 0.015, and 0.008 microgram/ml, respectively. Ceftriaxone was slightly less active (MIC90, 0.03 microgram/ml), and doxycycline was the least active drug tested (MIC90, 4.0 micrograms/ml). Tosufloxacin and temafloxacin had excellent activity against the C. trachomatis and N. gonorrhoeae strains tested.

Objectives

Compared to urogenital infections, little is known of serovar distribution in rectal chlamydial infection. The aim of this study was to explore possible relations between demographics, sexual behaviour, clinical manifestations, rectal symptoms, and chlamydial serovars including L2 (lymphogranuloma venereum).

Methods

Genotyping was done prospectively in all rectal chlamydial infections since the outbreak of proctitis caused by lymphogranuloma venereum in February 2003. 33 (15.1%) rectal Chlamydia trachomatis infections from the years 2001 and 2002 were genotyped retrospectively.

Results

Of all 219 rectal chlamydial infections, detected in the period July 2001 to August 2005, a total of 149 (68.0%) were successfully genotyped including 21 (14.1%) infections with serovar L2. In univariable and multivariable analyses, L2 serovar positive patients were significantly more often HIV positive (p = 0.002; OR: 6.5; 95% CI: 2.0 to 21.1), and had had sex in the past 6 months with more partners compared to other serovars. Furthermore, patients with L2 proctitis presented far more often with self reported rectal symptoms (p<0.005; OR: 19.4; 95% CI: 4.9 to 77.0) and clinical manifestations (p<0.005; OR: 15.4; 95% CI: 4.5 to 52.5).

Conclusions

Chlamydial infections with serovar L2 show a different clinical and epidemiological pattern compared to serovar D‐K. LGV proctitis is significantly associated with HIV positivity and a high number of sexual partners and causes more rectal symptoms and clinical manifestations. Neither young age nor ethnicity were identified as risk factors for any of the serovars investigated in this study.

We evaluated the analytical, work flow, and clinical performance of the Versant CT/GC DNA 1.0 assay (Versant CT/GC assay, where “CT” represents Chlamydia trachomatis and “GC” represents Neisseria gonorrhoeae). The assay simultaneously detects Chlamydia trachomatis and Neisseria gonorrhoeae in swab and first-catch urine (FCU) specimens. The limit of detection (LoD) was determined to be 342 copies/ml for C. trachomatis and 137 copies/ml for GC. The Versant CT/GC assay detected 15 C. trachomatis serovars and 46 GC strains. The Versant CT/GC assay demonstrated no cross-reactivity with 136 potentially cross-reacting organisms. Clinical concordance of the Versant CT/GC assay to the Aptima Combo 2 (AC2) assay from Gen-Probe was demonstrated using 1,129 patient specimens, including 589 urine and 540 swab specimens. Discrepant specimens were subjected to DNA sequencing to identify the presence of amplified targets and to identify false-positive and false-negative results. Overall percent agreement was greater than 98%. Positive and negative percent agreements for detection of C. trachomatis were 94.4% and 99.1%, respectively, in urine specimens and 95.8% and 99.8%, respectively, in swab specimens. Positive percent agreement for the detection of N. gonorrhoeae was 100% in both urine and swab specimens, and negative percent agreements were 99.6% and 99% in urine and swab specimens, respectively. In conclusion, the performance of the Versant CT/GC assay was comparable to that of the AC2 assay. The Versant CT/GC assay can be recommended for the detection of C. trachomatis and N. gonorrhoeae in swab and urine specimens of symptomatic and asymptomatic individuals.

We developed a murine model of systemic infection with Chlamydia trachomatis biovar lymphogranuloma venereum (LGV). The pathological features of this infection resemble those of human LGV infection since both are characterized by granuloma formation. Mice developed resistance to reinfection with LGV, and this resistance was based on cellular immune mechanisms since it was transferable with immune spleen cells but not with immune serum. Resistance required viable organisms for induction. We compared LGV biovar infection with trachoma biovar infection. Trachoma biovar produced similar but less marked microbiological and pathological features. Cross-immunity was less apparent between serovars from trachoma and LGV biovars than it was between serovars within the same biovar. This model of systemic C. trachomatis infection will be useful in exploring virulence features of LGV.

We present the case of a 26 year old HIV positive homosexual man who was managed for suspected Crohn's disease for over 1 year before lymphogranuloma venereum (LGV) was clinically diagnosed. He had presented with constipation, secondary to acute haemorrhagic proctitis, and subsequently had two chlamydia negative rectal smears, using direct fluorescent antibody (DFA) Chlamydia trachomatis staining. Positive chlamydial serology guided retrospective testing of an early rectal biopsy, which was found to have C trachomatis by polymerase chain reaction (Roche Cobas) and identified as LGV serovar L2 by the Sexually Transmitted Bacteria Reference Laboratory (STBRL), Health Protection Agency (HPA), Colindale, London. Chlamydial serology may have a role in identifying late stage LGV infection. Although no standardised test currently exists, consideration should be given to evaluating the role of chlamydial serology in establishing a diagnosis of LGV.

Objective:

To determine the prevalence of lymphogranuloma venereum (LGV) and non-LGV associated serovars of urethral and rectal Chlamydia trachomatis (CT) infection in men who have sex with men (MSM).

Design:

Multicentre cross-sectional survey.

Setting:

Four genitourinary medicine clinics in the United Kingdom from 2006–7.

Subjects:

4825 urethral and 6778 rectal samples from consecutive MSM attending for sexual health screening.

Methods:

Urethral swabs or urine and rectal swabs were tested for CT using standard nucleic acid amplification tests. Chlamydia-positive specimens were sent to the reference laboratory for serovar determination.

Main outcome:

Positivity for both LGV and non-LGV associated CT serovars; proportion of cases that were symptomatic.

Results:

The positivity (with 95% confidence intervals) in rectal samples was 6.06% (5.51% to 6.66%) for non-LGV CT and 0.90% (0.69% to 1.16%) for LGV; for urethral samples 3.21% (2.74% to 3.76%) for non-LGV CT and 0.04% (0.01% to 0.16%) for LGV. The majority of LGV was symptomatic (95% of rectal, one of two urethral cases); non-LGV chlamydia was mostly symptomatic in the urethra (68%) but not in the rectum (16%).

Conclusions:

Chlamydial infections are common in MSM attending for sexual health screening, and the majority are non-LGV associated serovars. We did not identify a large reservoir of asymptomatic LGV in the rectum or urethra. Testing for chlamydia from the rectum and urethra should be included for MSM requesting a sexual health screen, but serovar-typing is not indicated in the absence of symptoms. We have yet to identify the source of most cases of LGV in the UK.

A DNA probe assay (PACE; Gen-Probe, San Diego, Calif.) was compared with a culture reference method for the detection of Chlamydia trachomatis. Using stock isolates of each of the 15 serovars (A to K, Ba, L1, L2, and L3) of C. trachomatis, the lower limit of sensitivity for the DNA probe ranged between 1,086 inclusion-forming units (IFU) for serovar E (Bour) to 2,930 IFU for serovar L1 (440), with the only exception being serovar C (TW-3), with which 99 IFU was detected. There was no cross-reactivity with Chlamydia psittaci (Texas turkey) and Chlamydia pneumoniae (TWAR-183). Bacterial and fungal isolates representing 14 species of normal vaginal flora as well as Neisseria gonorrhoeae gave negative results with the DNA probe when tested at a level of 1.5 X 10(7) CFU/ml. In addition, the DNA probe, a direct fluorescent-antibody stain (DFA) (MicroTrak; Syva Corp., Palo Alto, Calif.), and an enzyme-linked immunosorbent assay (Chlamydiazyme; Abbott Laboratories, North Chicago, Ill.) were compared with culture for the detection of C. trachomatis, using 196 clinical cervical samples. Of the 196 samples, 20 (10%) were culture positive. Of the 176 culture-negative samples, 1 was not evaluated by DNA probe and 4, because of a lack of cellular material, were not evaluated by DFA. The sensitivities of the DNA probe, DFA, and enzyme-linked immunosorbent assay were 60, 75, and 85%, respectively, and specificities were 95, 99, and 97%, respectively. Of the false-positive direct results, there was only one specimen with which more than one direct method was positive, and with this specimen all three direct methods were positive. The majority of false-negative results by the direct methods were from specimens which by the culture method gave <100 IFU per culture.

Images

There has been an increasing prevalence of lymphogranuloma venereum (LGV) or Chlamydia trachomatis (C. trachomatis) cases among the men who have sex with men (MSM) population, particularly in Europe and North America. These cases may present with an incomplete or undisclosed history and proctosigmoiditis without characteristic adenopathy syndrome. During the initial evaluation and colonoscopy, there is a strong clinical and endoscopic suspicion of inflammatory bowel disease (IBD) by virtue of presentation and endoscopic and histological findings. The diagnosis of IBD is subsequently modified to LGV proctosigmoiditis when one or more of the following transpire: (1) there is failure of response to IBD therapy; (2) additional components of history (MSM/travel) may be identified; (3) return of initially performed Chlamydia antibody test is positive; and (4) response to antibiotics effective against Chlamydia. We describe three such cases initially suspected to be an inflammatory bowel disease and subsequently identified as C. trachomatis proctosigmoiditis.

Lymphogranuloma venereum (LGV) is a sexually transmitted infection that is causing an ongoing epidemic in men who have sex with men (MSM) in Europe, the United Kingdom, and North America. Twenty-nine rectal swabs positive for Chlamydia trachomatis were analyzed by real-time PCR for the presence of LGV serovars. Genotyping revealed an identical L2b serovar from four specimens. All patients were MSM and human immunodeficiency virus infected. Three of the four presented with severe ulcerative proctitis. We report a cluster of rectal LGV serovar L2b infections in Sydney, Australia.

Human immunodeficiency virus type 1 (HIV-1) infection continues to spread in developing countries, mostly through heterosexual transmission. The development of a safe and cost-effective topical microbicide, effective against a range of STDs including HIV-1, would greatly impact the ongoing epidemic. When formulated in a vehicle, a micronized form of cellulose acetate phthalate (CAP), which is an inactive pharmaceutical excipient, has been shown to inactivate HIV-1, herpes simplex virus types 1 and 2, cytomegalovirus, Neisseria gonorrhoeae, Trichomonas vaginalis, Haemophilus ducreyi, and Chlamydia trachomatis in vitro. Formulated CAP was also shown to be effective against herpes simplex virus type 2 in vivo. Here we show that a formulation of CAP protected four of six rhesus monkeys from vaginal infection with simian immunodeficiency virus. Thus, CAP may be a candidate for use as a topical microbicide for preventing HIV-1 infection in humans.

Difloxacin is a new quinolone antimicrobial agent with in vitro activity against both Neisseria gonorrhoeae and Chlamydia trachomatis and a long (26-h) half-life. A single oral dose of 200 mg of difloxacin was used to treat 30 men with uncomplicated urethral gonorrhea in an open trial. Of the isolates of N. gonorrhoeae, three produced penicillinase and two were resistant to tetracycline. N. gonorrhoeae was eradicated from all 29 evaluable patients. The geometric mean MIC of difloxacin for 30 pretreatment N. gonorrhoeae isolates was 0.014 (range, less than or equal to 0.0039 to 0.03) microgram/ml. Four (13.3%) of the 30 subjects with gonococcal urethritis also had C. trachomatis recovered from their pretreatment cultures. Treatment with difloxacin was associated with the eradication of C. trachomatis from all four men. In addition, C. trachomatis was isolated from the posttreatment culture of only one man who had a negative culture before treatment. Nineteen patients (65.5%) reported adverse experiences, and 17 of them (58.6%) developed symptoms suggestive of central nervous system dysfunction. An oral dose of 200 mg of difloxacin is effective treatment for uncomplicated urethral gonorrhea and may also eliminate a coexisting infection with C. trachomatis. Side effects may limit the utility of this agent.

Background

Vaginal lactobacilli protect the female genital tract by producing lactic acid, bacteriocins, hydrogen peroxide or a local immune response. In bacterial vaginosis, normal lactobacilli are replaced by an anaerobic flora and this may increase susceptibility to Neisseria gonorrhoeae, a facultative anaerobe. Bacterial interference between vaginal lactobacilli and N. gonorrhoeae has not been studied in liquid medium under anaerobic conditions. By co-cultivating N. gonorrhoeae in the presence of lactobacilli we sought to identify the relative contributions of acidification and hydrogen peroxide production to any growth inhibition of N. gonorrhoeae.

Methods

Three strains of N. gonorrhoeae distinguishable by auxotyping were grown in the presence of high concentrations (107-108 cfu/mL) of three vaginal lactobacilli (L. crispatus, L. gasseri and L. jensenii) in an anerobic liquid medium with and without 2-(N-morpholino)-ethanesulfonic (MES) buffer. Fusobacterium nucleatum was used as an indicator of anaerobiosis. Bacterial counts were performed at 15, 20 and 25 h; at 25 h pH and hydrogen peroxide concentrations were measured.

Results

Growth of F. nucleatum to >108 cfu/mL at 25 h confirmed anaerobiosis. All bacteria grew in the anaerobic liquid medium and the addition of MES buffer had negligible effect on growth. L. crispatus and L. gasseri produced significant acidification and a corresponding reduction in growth of N. gonorrhoeae. This inhibition was abrogated by the addition of MES. L. jensenii produced less acidification and did not inhibit N. gonorrhoeae. Hydrogen peroxide was not detected in any experiment.

Conclusions

During anaerobic growth, inhibition of N. gonorrhoeae by the vaginal lactobacilli tested was primarily due to acidification and abrogated by the presence of a buffer. There was no evidence of a specific mechanism of inhibition other than acid production under these conditions and, in particular, hydrogen peroxide was not produced. The acidification potential of vaginal lactobacilli under anaerobic conditions may be their most important characteristic conferring protection against N. gonorrhoeae infection.

Autophagy, a eukaryotic cellular activity leading to the degradation of cellular components, serves as a defense mechanism against facultative intracellular bacteria as well as a growth niche for the obligate intracellular bacterium Coxiella burnetii. We here demonstrate that the obligate intracellular bacterial pathogen Chlamydia trachomatis lymphogranuloma venereum strongly induced autophagy in the middle of the chlamydial developmental cycle (24 h after infection), a time point with maximal level of chlamydial replication, but not during the early stages with low overall chlamydial metabolism (before 8 h). No autophagy induction was evident in cells exposed to heat- and ultraviolet-inactivated elementary bodies (EBs, the infectious form of Chlamydia) nor to inocula from which EBs had been removed prior to inoculation. Blocking chlamydial development with chloramphenicol also prevented autophagy induction in cells infected with infectious EBs. It appears that autophagy is activated primarily in response to the metabolic stress consequent to chlamydial replication. However, autophagy-defective ATG5−/− cells supported chlamydial development as efficiently as autophagy-proficient ATG5+/+ cells.

Background

Knowledge regarding characteristics and transmission of Neisseria gonorrhoeae, Chlamydia trachomatis and Mycoplasma genitalium and antibiotic resistance in N gonorrhoeae in Guinea-Bissau, West Africa, is entirely lacking.

Objectives

To characterise N gonorrhoeae, C trachomatis and M genitalium samples from Guinea-Bissau and to define bacterial populations, possible transmission chains and for N gonorrhoeae spread of antibiotic-resistant isolates.

Design

Prospective cohort study.

Setting

Two sexual health and family planning clinics, Bissau, Guinea-Bissau.

Participants

Positive samples from 711 women and 27 men.

Material and methods

Positive samples for N gonorrhoeae (n=31), C trachomatis (n=60) and M genitalium (n=30) were examined. The gonococcal isolates were characterised with antibiograms, serovar determination and N gonorrhoeae multiantigen sequence typing (NG-MAST). The C trachomatis ompA gene and the M genitalium mgpB gene were sequenced, and phylogenetic analyses were performed.

Results

For N gonorrhoeae, the levels of resistance (intermediate susceptibility) to ciprofloxacin, erythromycin, rifampicin, ampicillin, tetracycline, penicillin G and cefuroxime were 10% (0%), 6% (10%), 13% (10%), 68% (0%), 74% (0%), 68% (16%) and 0% (84%), respectively. All isolates were susceptible to cefixime, ceftriaxone, spectinomycin and azithromycin, and the minimum inhibitory concentrations of kanamycin (range: 8–32 mg/l) and gentamicin (range: 0.75–6 mg/l) were low (no resistance breakpoints exist for these antimicrobials). 19 NG-MAST sequence types (STs) (84% novel STs) were identified. Phylogenetic analysis of the C trachomatis ompA gene revealed genovar G as most prevalent (37%), followed by genovar D (19%). 23 mgpB STs were found among the M genitalium isolates, and 67% of isolates had unique STs.

Conclusions

The diversity among the sexually transmitted infection (STI) pathogens may be associated with suboptimal diagnostics, contact tracing, case reporting and epidemiological surveillance. In Guinea-Bissau, additional STI studies are vital to estimate the STI burden and form the basis for a national sexual health strategy for prevention, diagnosis and surveillance of STIs.

Article summary

Article focus

Knowledge regarding characteristics and transmission of Neisseria gonorrhoeae, Chlamydia trachomatis and Mycoplasma genitalium and antibiotic resistance in N gonorrhoeae in Guinea-Bissau, West Africa, is entirely lacking.

We aimed to phenotypically and genetically characterise N gonorrhoeae, and genetically characterise C trachomatis and M genitalium samples from women attending two sexual health clinics in Bissau, Guinea-Bissau and to define the bacterial populations, possible transmission chains and for N gonorrhoeae also to define the presence and spread of antibiotic-resistant isolates from women and additionally a group of symptomatic men.

Key messages

In Guinea-Bissau, N gonorrhoeae isolates displayed high level of resistance to traditional gonorrhoea antimicrobials but have remained susceptible to extended-spectrum cephalosporins, spectinomycin and azithromycin.

Genovar G, D and F were the most prevalent C trachomatis genovars, the usually most common genovar among heterosexuals, that is, genovar E, was rare.

The diversity among the bacterial STI pathogens may be associated with suboptimal diagnostics, contact tracing, case reporting and epidemiological surveillance. Additional studies are vital to estimate the STI burden and form the basis for a national sexual health strategy for prevention, diagnosis and surveillance.

Strengths and limitations of this study

This is the first time N gonorrhoeae, C trachomatis and M genitalium samples from Bissau, Guinea-Bissau, have been phenotypically and genetically characterised to define the bacterial populations, possible transmission chains and antibiotic resistance in N gonorrhoeae.

The study sample was relatively small, and in this setting, it is difficult to perform appropriate sample transportation and storage, as well as optimised diagnostics, which may have resulted in that true-positive samples were lost.

The inhibitory effects of hinokitiol (β-thujaplicin) on Chlamydia trachomatis D/UW-3/Cx were shown by MIC, minimum lethal concentration (MLC), and preinoculation minimal microbicidal concentration assays using HeLa 229 cells. The MIC and the MLC were both 32 μg/ml. Further evaluation of hinokitiol as a topical agent against C. trachomatis is warranted.

The in vitro activity of the quinolone carboxylic acid, ciprofloxacin, against a variety of genital tract pathogens was examined. Each of 35 isolates of Neisseria gonorrhoeae, including some beta-lactamase producing strains and strains resistant to tetracycline, was inhibited at a concentration of 0.01 mg/l. Most (13 of 20) strains of Gardnerella vaginalis were inhibited at 1 mg/l but three isolates had minimum inhibitory concentrations (MICs) of 8 mg/l or more. Each of seven strains of Chlamydia trachomatis was completely inhibited at a concentration of 2 mg/l. Prolonged (72 hours) exposure of the chlamydiae to ciprofloxacin was required for inhibition at this concentration.