Jo Kitawaki

Is Neisseria gonorrhoeae Initiating a Future Era of Untreatable Gonorrhea?: Detailed Characterization of the First Strain with High-Level Resistance to Ceftriaxone

ABSTRACT Recently, the first Neisseria gonorrhoeae strain (H041) that is highly resistant to the extended-spectrum cephalosporin (ESC) ceftriaxone, the last remaining option for empirical first-line treatment, was isolated. We performed a detailed characterization of H041, phenotypically and genetically, to confirm the finding, examine its antimicrobial resistance (AMR), and elucidate the resistance mechanisms. H041 was examined using seven species-confirmatory tests, antibiograms (30 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of ESC resistance determinants (penA, mtrR, penB, ponA, and pilQ). Transformation, using appropriate recipient strains, was performed to confirm the ESC resistance determinants. H041 was assigned to serovar Bpyust, MLST sequence type (ST) ST7363, and the new NG-MAST ST4220. H041 proved highly resistant to ceftriaxone (2 to 4 μg/ml, which is 4- to 8-fold higher than any previously described isolate) and all other cephalosporins, as well as most other antimicrobials tested. A new penA mosaic allele caused the ceftriaxone resistance. In conclusion, N. gonorrhoeae has now shown its ability to also develop ceftriaxone resistance. Although the biological fitness of ceftriaxone resistance in N. gonorrhoeae remains unknown, N. gonorrhoeae may soon become a true superbug, causing untreatable gonorrhea. A reduction in the global gonorrhea burden by enhanced disease control activities, combined with wider strategies for general AMR control and enhanced understanding of the mechanisms of emergence and spread of AMR, which need to be monitored globally, and public health response plans for global (and national) perspectives are important. Ultimately, the development of new drugs for efficacious gonorrhea treatment is necessary.

Endometriosis: the pathophysiology as an estrogen-dependent disease.

Endometriosis, defined as the presence of endometrial glands and stroma outside of the uterine cavity, develops mostly in women of reproductive age and regresses after menopause or ovariectomy, suggesting that the growth is estrogen-dependent. Indeed, the lesions contain estrogen receptors (ER) as well as aromatase, an enzyme that catalyses the conversion of androgens to estrogens, suggesting that local estrogen production may stimulate the growth of lesions. The expression patterns of ER and progesterone receptors in endometriotic lesions are different from those in the eutopic endometrium. Moreover, estrogen metabolism, including the expression pattern of aromatase and the regulation of 17 beta-hydroxysteroid dehydrogenase type 2 (an enzyme responsible for the inactivation of estradiol to estrone), is altered in the eutopic endometrium of women with endometriosis, adenomyosis, and/or leiomyomas compared to that in the eutopic endometrium of women without disease. Immunostaining for P450arom in endometrial biopsy specimens diagnosed these diseases with sensitivity and specificity of 91 and 100%, respectively. This is applicable to the clinical diagnosis of endometriosis. The polymorphisms in the ER-alpha gene, the CYP19 gene encoding aromatase, and several other genes are associated with the risk of endometriosis. Studies of these will lead to better understandings of the etiology and pathophysiology of endometriosis.

Supplementation of hydrogen-rich water improves lipid and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance.

Oxidative stress is recognized widely as being associated with various disorders including diabetes, hypertension, and atherosclerosis. It is well established that hydrogen has a reducing action. We therefore investigated the effects of hydrogen-rich water intake on lipid and glucose metabolism in patients with either type 2 diabetes mellitus (T2DM) or impaired glucose tolerance (IGT). We performed a randomized, double-blind, placebo-controlled, crossover study in 30 patients with T2DM controlled by diet and exercise therapy and 6 patients with IGT. The patients consumed either 900 mL/d of hydrogen-rich pure water or 900 mL of placebo pure water for 8 weeks, with a 12-week washout period. Several biomarkers of oxidative stress, insulin resistance, and glucose metabolism, assessed by an oral glucose tolerance test, were evaluated at baseline and at 8 weeks. Intake of hydrogen-rich water was associated with significant decreases in the levels of modified low-density lipoprotein (LDL) cholesterol (ie, modifications that increase the net negative charge of LDL), small dense LDL, and urinary 8-isoprostanes by 15.5% (P < .01), 5.7% (P < .05), and 6.6% (P < .05), respectively. Hydrogen-rich water intake was also associated with a trend of decreased serum concentrations of oxidized LDL and free fatty acids, and increased plasma levels of adiponectin and extracellular-superoxide dismutase. In 4 of 6 patients with IGT, intake of hydrogen-rich water normalized the oral glucose tolerance test. In conclusion, these results suggest that supplementation with hydrogen-rich water may have a beneficial role in prevention of T2DM and insulin resistance.

Ceftriaxone-Resistant Neisseria gonorrhoeae, Japan

To the Editor: Spread of multidrug-resistant Neisseria gonorrhoeae is a major public health concern. Effective antimicrobial therapy is a key element in gonorrhea control. However, N. gonorrhoeae has developed resistance to multiple classes of antimicrobial drugs, including β-lactams, tetracyclines, and fluoroquinolones (1–3). Even an extended-spectrum oral cephalosporin-resistant, cefixime-resistant N. gonorrhoeae has emerged, and cefixime has now been withdrawn from use in Japan. Best practice treatment is limited to injectable extended-spectrum cephalosporins, such as ceftriaxone and spectinomycin. The emergence of ceftriaxone-resistant N. gonorrhoeae threatens effective disease control. We identified a novel ceftriaxone-resistant N. gonorrhoeae isolated from a 31-year-old female commercial sex worker; MIC of ceftriaxone for this isolate was high (2 µg/mL). The woman visited a clinic in Kyoto for a routine examination for sexually transmitted infections in January 2009. Although she had no obvious symptoms or signs, a throat sample collected on her first visit yielded a positive result for N. gonorrhoeae by the strand displacement amplification test (ProbeTec ET, Becton Dickinson, Franklin Lakes, NJ, USA), but a vaginal sample taken at the same time was negative. After 2 weeks, another throat sample was positive for N. gonorrhoeae when cultured on Thayer-Martin medium, and the patient subsequently received 1 g ceftriaxone intravenously. Her pharyngeal sample was also N. gonorrhoeae positive by strand displacement amplification test on the third visit 2 weeks later, and further ceftriaxone treatment was prescribed. However, a culture for test of cure was not conducted because reinfection was considered. A negative result was finally obtained in April 2009. The culture showed positive reactions in oxidase and catalase tests. Gram staining showed gram-negative diplococci. The ID-test HN-20 Rapid system (Nissui, Tokyo, Japan) classified the bacterium as N. gonorrhoeae. Susceptibility was determined by the agar dilution method (4). For this strain, named H041, MIC of ceftriaxone was high (2 µg/mL), and the strain was highly resistant to penicillin G (4 µg/mL), cefixime (8 µg/mL), and levofloxacin (32 µg/mL). However, it demonstrated susceptibility to spectinomycin (16 µg/mL) and reduced susceptibility to azithromycin (0.5 µg/mL). To characterize the ceftriaxone-resistant N. gonorrhoeae H041, multilocus sequence typing characterized the strain as ST7363 (5), which is the predominant sequence type (ST) among cefixime-resistant clones (6). N. gonorrhoea multiantigen sequence typing (NG-MAST) was also performed (7). The NG-MAST strategy uses 2 genes, por and tbpB, for porin and a transferrin-binding protein, respectively. NG-MAST indicated that the strain H041 was ST4220 and contained the por2594 allele and the tbpB10 allele. NG-MAST 4220 is a novel ST. However, the tbpB10 allele is the most frequently observed allele (76.5%) among multilocus sequence typing-ST7363 N. gonorrhoeae strains (n = 81) (M. Ohnishi, unpub. data). Molecular typing suggested that the novel ceftriaxone-resistant N. gonorrhoeae, H041, is closely related to the ST7363 cefixime-resistant N. gonorrhoeae. Therefore, we compared SpeI-digested genomic DNA banding patterns of strain H041 with those of other N. gonorrhoeae strains by using pulsed-field gel electrophoresis as described (8). Four ST7363 strains, including N. gonorrhoeae H041, and 4 ST1901 strains (another major ST among cefixime-resistant N. gonorrhoeae strains) (6) were analyzed. The banding pattern of SpeI digested H041 genomic DNA was similar to that of other ST7363 strains and indistinguishable from that of cefixime-resistant but ceftriaxone-susceptible NG0207 (Figure). Figure Pulsed-field gel electrophoresis patterns of ceftriaxone-resistant Neisseria gonorrhoeae strain H041 and other multilocus sequence typing (MLST) ST7363 and ST1901 strains. SpeI-digested genomic DNA from ceftriaxone-resistant N. gonorrhoeae H041, 3 of ... We describe the emergence of ceftriaxone-resistant N. gonorrhoeae, isolated from a pharyngeal specimen from a female commercial sex worker. At 2 µg/mL, the MIC was 4-fold higher than that of the previously reported ceftriaxone nonsusceptible strain (9). Our susceptibility testing suggests that only azithromycin and spectinomycin are effective drugs for treating this strain. In this case, eradication was successful, although N. gonorrhoeae colonization of the pharynx may just be tempory because the pharynx is not an ideal site for N. gonorrhoeae growth. From the routine examinations of commercial sex workers during January–March 2009, 40 N. gonorrhoeae were isolated in the clinic, but no other ceftriaxone-resistant strains were isolated. There is no evidence of dissemination of this strain in Kyoto. Three independent molecular subtyping methods indicated that the ceftriaxone-resistant H041 strain was N. gonorrhoeae, and it might originate from an ST7363 cefixime-resistant N. gonorrhoeae clone. There are several possible mechanisms for the acquisition of resistance, including formation of a new mosaic type penA allele as penA-X cefixime resistance and acquisition of an extended-spectrum β-lactamase gene. The H041 strain did not produce β-lactamase in a nitrocephin test. Further molecular analysis is needed to elucidate the precise mechanism of the ceftriaxone resistance of the H041 strain. The emergence of ceftriaxone-resistant N. gonorrhoeae raises concerns for controlling gonorrhea because ceftriaxone is widely recommended and the first-line treatment for gonorrhea around the world. N. gonorrhoeae has a potential to gain an extraordinarily high MIC to ceftriaxone. Surveillance for ceftriaxone-resistant N. gonorrhoeae should be strengthened.

Detection of aromatase cytochrome P-450 in endometrial biopsy specimens as a diagnostic test for endometriosis

OBJECTIVE To evaluate the clinical usefulness of examining endometrial biopsy specimens for aromatase cytochrome P-450 as a diagnostic test for endometriosis. DESIGN Retrospective, case-controlled study. SETTING Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, Kyoto, Japan. PATIENT(S) One hundred five women of reproductive age with normal menstrual cycles underwent endometrial biopsy laparotomy or laparoscopy, and examination of their tissue revealed endometriosis, adenomyosis, and/or leiomyomas. Patients who had cervical carcinoma in situ but no other gynecologic disease were considered to be disease-free. INTERVENTION(S) Endometrial biopsy specimens were collected. MAIN OUTCOME MEASURE(S) The expression of aromatase cytochrome P-450 was examined by reverse transcription-polymerase chain reaction and immunohistochemical analysis. The distribution and intensity of the immunostaining was assessed using a semiquantitative index designed H-score. RESULT(S) Immunostaining for aromatase cytochrome P-450 was detected in biopsy specimens obtained from patients with endometriosis, adenomyosis, and/or leiomyomas but not in specimens obtained from disease-free patients (H-score <20), with a sensitivity and specificity of 91% and 100%, respectively. CONCLUSION(S) The expression of aromatase cytochrome P-450 in biopsy specimens of eutopic endometrium distinguishes between disease-free women and women with endometriosis, adenomyosis, and/or leiomyomas. This technique can be used at outpatient infertility clinics as an initial screening procedure to rule out the presence of estrogen-dependent disease.

Immunoaffinity purification of aromatase cytochrome P-450 from human placental microsomes, metabolic switching from aromatization to 1β and 2β-monohydroxylation, and recognition of aromatase isozymes

Microsomal estrogen synthetase (aromatase) cytochrome P-450 was purified from fresh human placental microsomes by monoclonal anti-aromatase P-450 antibody-Sepharose 4B chromatography. The purified P-450 showed a single band of 55 kDa on SDS-polyacrylamide gel electrophoresis and the aromatase specific activity on reconstitution was 70 nmol/min/mg protein. The purified P-450 was stable with a t 1/2 of approximately 2 years on storage at -90 degrees C and showed Km = 43 nM for androstenedione aromatization. However, it was unstable under spectral measurement conditions in the presence of sodium dithionite and carbon monoxide and the carbon monoxide difference spectra showed a maximum at 450 nm and a specific content of 9.1 nmol of P-450/mg protein, giving a turnover number of approximately 7.7 per min for the purified aromatase. The one-step immunochemical purification method gave a 490-fold increase of specific activity with 55% yield of aromatase activity of the original microsomes. Analysis of androgen metabolism by the purified aromatase and an apparent large kinetic isotope effect found at the secondary positions when using [19(-3)H3, 4(-14)C] androgens revealed metabolic switching from the first 19-hydroxylation to 1 beta- and 2 beta- monohydroxylation by aromatase. Substrate specificity for [19(-3)H3]androstenedione and testosterone was indicated by differences in the extent of metabolic switching (18% and 30%) and in the 2 beta/1 beta ratio (60/40 and 10/90, respectively). The mouse monoclonal antibody used for immunoaffinity purification suppresses aromatase activity of human placenta, but was totally ineffective for aromatase in goldfish brain and rat ovary. Rabbit polyclonal antibodies to human placental aromatase P-450 suppressed both human placental and rat ovarian aromatase but were ineffective for goldfish brain aromatase. The study indicates that they are isozymes of aromatase based on different structures of P-450.

Dienogest inhibits aromatase and cyclooxygenase-2 expression and prostaglandin E2 production in human endometriotic stromal cells in spheroid culture

OBJECTIVE To determine the effect of dienogest (DNG) on the expression of aromatase and cyclooxygenase-2 (COX-2) and the production of prostaglandin E(2) (PGE(2)) in human endometriotic stromal cells (ESCs). DESIGN Experimental study in vitro. SETTING University hospital. PATIENT(S) Seventeen patients with ovarian endometrioma. INTERVENTION(S) ESCs from chocolate cyst linings of ovaries were treated with DNG. MAIN OUTCOME MEASURE(S) Expression of aromatase and COX-2 evaluated in spheroid cultures of human ESCs by real-time quantitative polymerase chain-reaction and immunocytochemistry, production of PGE(2) quantified by enzyme-linked immunosorbent assay (ELISA), and nuclear factor kappa B (NF-κB) DNA-binding examined by ELISA and immunocytochemistry. RESULT(S) The pharmaceutical actions of DNG on the expression of aromatase and COX-2 and the production of PGE(2) were examined using spheroid cultures of human ESCs. More aromatase, COX-2, and PGE(2) were expressed in spheroid cultures than in conventional ESCs monolayers. In the spheroid cultures, DNG (10(-7) M) and progesterone (10(-7) M) inhibited the expression of aromatase, COX-2, and PGE(2). DNG also inhibited NF-κB DNA-binding activity and reduced the immunocytochemical protein expression of aromatase, COX-2, and NF-κB p50 nuclear localization. CONCLUSION(S) Because DNG inhibits aromatase and COX-2 expression as well as PGE(2) production in ESCs, these pharmacologic features might contribute to a therapeutic effect of DNG on endometriosis.

Endometrial receptivity defects during IVF cycles with and without letrozole

BACKGROUND Our aim was to study ways to improve IVF success rates in women with suspected endometrial receptivity defects. METHODS We conducted a retrospective cohort study examining the effect of letrozole (aromatase inhibitor) on integrin expression as a marker of endometrial receptivity. We compared IVF outcomes in 97 infertile women who had undergone ανβ3 integrin assessment by immunohistochemistry in mid-luteal endometrial biopsies. Of 79 women undergoing standard IVF, 29 (36.7%) lacked normal integrin expression. Eighteen other women with low integrin were studied after receiving letrozole during early IVF stimulation. An independent set of ανβ3 integrin-negative patients (n = 15) who had undergone repeat endometrial biopsy for integrin testing while taking letrozole were re-evaluated. RESULTS Clinical pregnancy and delivery rates were higher in women with normal ανβ3 integrin expression compared with those who were integrin negative [20/50 (40%) versus 4/29 (13.8%); P = 0.02 and 19/50 (38%) versus 2/29 (7%); P < 0.01, respectively]. In 18 women who received letrozole early in IVF, 11 conceived (61.1%; P < 0.001) compared with integrin-negative patients who did not receive letrozole. In integrin-negative women who were rebiopsied on letrozole, 66.7% reverted to normal integrin expression. Positive endometrial aromatase immunostaining using a polyclonal antibody was a common finding in infertile patients compared with controls. CONCLUSIONS Lack of endometrial ανβ3 integrin expression is associated with a poor prognosis for IVF that might be improved with letrozole co-treatment. Prospective studies are needed to confirm and extend these findings but the data suggest that aromatase expression may contribute to implantation failure in some women.

Contribution of aromatase to the deoxyribonucleic acid synthesis of MCF-7 human breast cancer cells and its suppression by aromatase inhibitors ☆

We have studied the effects of various steroids on DNA synthesis in MCF-7 human breast carcinoma cells, which have aromatase activity and which exert an oestrogen receptor-mediated growth, to assess the significance of intracellular aromatase on growth stimulation as well as inhibition by aromatase inhibitors. The cells were cultured for 96 h in phenol red-free medium containing 10% charcoal-treated fetal bovine serum and test reagents and pulse-labelled with [3H]thymidine. Physiological concentrations of oestradiol, oestrone, testosterone (T) and androstenedione (AD) stimulated thymidine incorporation. However, oestrone-sulphate and dihydrotestosterone (DHT) only stimulated at concentrations greater than the physiological levels. T and DHT stimulation was blocked by tamoxifen, but not by cyproterone acetate, suggesting that the stimulation was mediated via the oestrogen receptor but not by the androgen receptor. Stimulation by T and AD was reduced by aminoglutethimide and 14 alpha-hydroxy-4-androstene-3,6,17-trione, both of which inhibit aromatase activity, however, stimulation by nonaromatizable DHT was not reduced by the inhibitors, suggesting that androgens were converted by the intracellular aromatase to oestrogens which stimulated the thymidine incorporation. It is suggested that intracellular aromatase significantly contributes to the stimulation of DNA synthesis and that aromatase inhibitors suppress the stimulation.

Telmisartan, an angiotensin II type 1 receptor blocker, prevents the development of diabetes in male Spontaneously Diabetic Torii rats

To assess the beneficial effects of the angiotensin II type 1 receptor blocker telmisartan on a non-obese animal model of reduced function and mass of islet beta-cells prior to the development of diabetes, Spontaneously Diabetic Torii (SDT) rats were treated with telmisartan at 8 weeks of age. At 24 weeks of age, the treatment with telmisartan dose-dependently ameliorated hyperglycemia and hypoinsulinemia, and high-dose (5 mg/kg/day) treated SDT rats did not developed diabetes. Real-time RT-PCR analysis revealed that treatment with high-dose telmisartan reduced mRNA expression of local renin-angiotensin system (RAS) components, components of NAD(P)H oxidase, transforming growth factor-beta1 and vascular endothelial growth factor in the pancreas of male SDT rats. Immunohistochemical and Western blot analyses revealed that treatment with telmisartan also reduced expression of p47(phox). These results suggest that treatment with telmisartan reduces oxidative stress by local RAS activation and protects against islet beta-cell damage and dysfunction. These findings provide at least a partial explanation for the reduced incidence of new-onset diabetes that has been observed in several clinical trials involving angiotensin II type 1 receptor blockers and ACE inhibitors.