Shigeaki Yokoi

Emergence and Spread of Neisseria gonorrhoeae Clinical Isolates Harboring Mosaic-Like Structure of Penicillin-Binding Protein 2 in Central Japan

ABSTRACT Of 150 clinical isolates of Neisseria gonorrhoeae recovered in 2001, we examined 55 clinical isolates of N. gonorrhoeae for which cefixime MICs were ≥0.125 μg/ml and randomly selected 15 isolates for which cefixime MICs were ≤0.06 μg/ml for analysis of alterations in the penicillin-binding protein 2 (PBP 2) gene. We found insertion of an extra codon (Asp-345a) in the transpeptidase domain of PBP 2, and this insertion occurred alone or in conjunction with other amino acid substitutions. We also found a mosaic PBP 2 that was composed of fragments of the PBP 2 proteins from Neisseria cinera and Neisseria perflava. This mosaic PBP 2 was significantly associated with decreased susceptibilities to penicillin and cephalosporins, especially oral cephalosporins. For most of the isolates with a mosaic PBP 2, the cefixime MICs were ≥0.5 μg/ml and the cefdinir MICs were ≥1 μg/ml. Analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis revealed that most isolates with the mosaic PBP 2 were genetically similar. The recombination events that generated the mosaic PBP 2 would likely have contributed to the decreased sensitivities to cephalosporins. Isolates with the mosaic PBP 2 appear to threaten the efficacy of the currently recommended regimen with cefixime. The emergence of such strains may be the result of the in vivo generation of clones in which interspecies recombination occurred between the penA genes of N. gonorrhoeae and commensal Neisseria species.

Threat to Cefixime Treatment for Gonorrhea

To the Editor: From November 2002 through May 2003, 4 Japanese men, ranging in age from 23 to 45 years, visited the Department of Urology at Toyota Memorial Hospital, Toyota, Japan. Physical examinations showed urethral discharge and dysuria. Each had had sexual contact with sex workers in central Japan. Four strains of Neisseria gonorrhoeae were isolated from urethral specimens. Treatment comprised 200 mg cefixime, twice a day for 3 days. However, all 4 patients returned to the clinic with continuing symptoms, despite having completed the prescribed course of cefixime and abstaining from sexual activity. N. gonorrhoeae was again isolated from urethral swabs. Each patient was then treated with 1 g intravenous ceftriaxone. In the 3 patients who returned to the clinic for followup, the ceftriaxone treatment resulted in clinical and microbiologic cure. Pulsed-field gel electrophoresis (PFGE) analysis of the SpeI-digested DNAs of N. gonorrhoeae was performed to assess the relatedness of pre- and posttreatment isolates (1). For these 8 isolates, MICs of penicillin G, tetracycline, cefixime, cefdinir, cefodizime, ceftriaxone, levofloxacin, azithromycin, and spectinomycin were determined on chocolate agar (GC) medium base supplemented with 1% IsoVital X (Becton Dickinson, Franklin Lakes, NJ, USA) and containing serial 2-fold dilutions of each agent (2). Media were inoculated with 104 CFU and incubated at 35°C in 5% CO2 overnight. The MIC was defined as the lowest concentration inhibiting growth to <1 CFU. β-lactamase activity of the isolates was tested with a nitrocefin disk. The nucleotide sequences of the full-length penA gene encoding the penicillin-binding protein 2 (PBP 2) were identified in the isolates (1). Briefly, genomic DNAs from each isolate were subjected to PCR to amplify 3 fragments of the penA gene of N. gonorrrhoeae. PCR products were sequenced by the dye terminator method and with an automatic sequencer. In each of the 4 cases, the PFGE patterns of the pre- and posttreatment isolates had the same numbers of bands (12–16 fragments), and the corresponding bands were the same apparent size; the pre- and posttreatment isolates were indistinguishable (3). MICs of antimicrobial agents for the 8 isolates are shown in the Table. All isolates were enzymatically negative for β-lactamase and possessed identical mosaic alterations in PBP 2. The mosaic PBP 2 was composed of fragments of PBP 2 from N. cinerea and N. perflava and was identical to that identified in our previous study (1). Table Antimicrobial drug susceptibilities of clinical isolates of Neisseria gonorrhoeae from patients with gonococcal urethritis treated unsuccessfully with a 3-day cefixime regimen* Until recently, Japanese guidelines recommended oral administration of cefixime, 200 mg twice a day for 3 days to prolong the period of time for which the serum drug concentration remains above the MIC (4). However, treatment failure with this cefixime regimen was observed in our 4 cases of gonorrhea. The isolates showed cefixime MICs of 0.5–1 μg/mL and harbored mosaic alterations in PBP 2. Most recently, the emergence and spread of such strains in Japan (1,5,6) have led to the recommendation that ceftriaxone and spectinomycin should be used as the primary therapy for gonorrhea instead of oral cephalosporins (7). In 2001, treatment failure was reported in a Caucasian man residing in Hawaii who had been given a single 400-mg dose of cefixime for gonorrhea (8). Pre- and posttreatment strains of N. gonorrhoeae were recovered from this patient, and 1 strain was isolated from his Japanese female sex partner who had visited Hawaii from Japan. Another pretreatment strain was isolated from a Micronesian man with gonorrhea residing in Hawaii, who had had sex with a woman from Malaysia or the Marshall Islands. This man was successfully treated with a single 400-mg dose of cefixime. For these strains, MICs of penicillin, tetracycline, spectinomycin, cefixime, ceftriaxone, ciprofloxacin, and azithromycin were 8.0, 4.0–8.0, <32, 0.25–0.5, 0.125, 8–16, and 0.125–0.25 μg/mL, respectively. The antibiograms of these strains in Hawaii were similar to those of strains with mosaic PBP 2 found in our 4 patients. The introduction to Hawaii of such multidrug resistant strains might be related to sex partners from Asia. Although the strains were not analyzed for alterations in PBP 2, they could have been derived from strains with cefixime resistance-associated mosaic PBP 2. The strains with mosaic PBP 2 showed such decreased susceptibility to cefixime that they were not effectively eradicated by the 3-day treatment. Although it is not clear whether these strains are also resistant to the single 400-mg dose of cefixime (8, 9), their emergence and spread could threaten treatment for gonorrhea with cefixime (10). Global emergence and spread of such multidrug-resistant strains of N. gonorrhoeae would be a matter of serious concern. The antimicrobial susceptibilities of current gonococcal isolates must be monitored periodically. In particular, posttreatment isolates from patients treated unsuccessfully with cefixime should be surveyed.

Remarkable Increase in Central Japan in 2001-2002 of Neisseria gonorrhoeae Isolates with Decreased Susceptibility to Penicillin, Tetracycline, Oral Cephalosporins, and Fluoroquinolones

ABSTRACT Four hundred sixty-two clinical isolates of Neisseria gonorrhoeae recovered from 1999 through 2002 in central Japan were examined for MICs of antimicrobial agents. The majority was sensitive to ceftriaxone and spectinomycin, but a remarkable increase in isolates with decreased susceptibility to penicillin, tetracycline, oral cephalosporins, and fluoroquinolones was observed from 2001 through 2002.

Laparoscopic Radical Nephroureterectomy: A Multicenter Analysis in Japan

BACKGROUND Laparoscopic nephroureterectomy (LNUx) is prevalent in Japan and throughout the world, but long-term outcome data remain limited. OBJECTIVE To understand the present state of LNUx in Japan, we conducted a multicenter analysis of clinical outcome and long-term cancer control for patients who underwent the procedure. DESIGN, SETTING, AND PARTICIPANTS Between January 1995 and December 2005, 1003 patients with urothelial cancer in the upper urinary tract were treated with LNUx at 51 institutions in Japan, and patient data were collected retrospectively. MEASUREMENTS Patient profiles were gathered and analyzed for survival, intravesical recurrence, and risk factors influencing them. RESULTS AND LIMITATIONS Median operative time was 320 min. Median bleeding volume was 232 ml. Complications occurred in 93 cases (9.3%) intraoperatively and in 107 cases (10.7%) postoperatively. Overall survival rate was 70% at 5 yr. Grade 3, pT3 or pT4, multifocal tumor, lymph-node metastasis, and previous or coexistent bladder tumor were independent risk factors for overall survival. Intravesical recurrence rate was 43% at 5 yr. Intravesical recurrence occurred more frequently in males, in patients with multifocal tumors, in patients with previous or coexistent bladder tumors, and in patients who underwent the hand-assisted approach. CONCLUSIONS Our report represents the largest multicenter analysis of LNUx reported to date. Male sex and the use of the hand-assisted approach were shown for the first time to be risk factors for recurrence-free survival and intravesical recurrence. To further analyze the effectiveness of LNUx, a long-term outcome comparison with risk stratification must be made between LNUx and open nephroureterectomy.

Emergence of clinical strains of Mycoplasma genitalium harbouring alterations in ParC associated with fluoroquinolone resistance

Surveillance for antimicrobial resistance in Mycoplasma genitalium clinical strains is extremely limited as culturing of strains from clinical specimens is still difficult. We therefore conducted a non-cultural assessment of fluoroquinolone resistance of M. genitalium clinical strains by analysing the quinolone-resistance determining regions (QRDRs) of the gyrA and parC genes. The QRDRs amplified from M. genitalium DNA taken from urine specimens of 28 men with non-gonococcal urethritis positive for M. genitalium by polymerase chain reaction were sequenced. An amino acid change (Phe-108-->Iso) in GyrA was found in one specimen, and the same change was accompanied by an amino acid change (Lys-97-->Arg) in ParC in another specimen. A single amino acid change (Ser-83-->Asn, Asp-87-->Tyr or Asp-87-->Val) in ParC was also found in three other respective specimens without alterations in GyrA. No alterations in GyrA and ParC were found in the remaining 23 specimens. The alterations of Ser-83-->Asn, Asp-87-->Tyr and Asp-87-->Val in ParC found in 3 (10.7%) of 28 specimens were analogous to those commonly observed in fluoroquinolone-resistant mutants of other Mycoplasma and Ureaplasma spp. M. genitalium harbouring mutations associated with fluoroquinolone resistance in the parC gene may have emerged clinically and the prevalence may be ca. 10% in Japan.

Selection of Mycoplasma genitalium strains harbouring macrolide resistance-associated 23S rRNA mutations by treatment with a single 1 g dose of azithromycin

Objective A single 1 g dose regimen of azithromycin has been recommended for the treatment of Mycoplasma genitalium infections. The authors evaluated whether this regimen could select M genitalium strains with macrolide resistance after treatment for M genitalium-positive non-gonococcal urethritis. Methods In seven men with non-gonococcal urethritis, who were infected with M genitalium without macrolide resistance-associated mutations but experienced microbiological azithromycin treatment failure, M genitalium DNAs in their post-treatment urine specimens were examined for mutations in the 23S rRNA gene and the ribosomal protein genes of L4 and L22. To assess the relatedness of M genitalium strains before and after treatment, their DNAs in pretreatment and post-treatment urine were genotyped by analysing short tandem repeats of an AGT/AAT unit in the MG309 gene and single nucleotide polymorphisms in the MG191 gene. Results In four of seven patients, M genitalium in post-treatment urine had an A-to-G transition at nucleotide position 2071 or 2072, corresponding to 2058 or 2059 in the 23S rRNA gene of Escherichia coli. In one of the four strains, Pro81Ser in the ribosomal protein L4 accompanied the mutation in the 23S rRNA gene. The genotyping of M genitalium DNAs suggested that these four post-treatment strains were selected from the respective closely related or identical pretreatment strains without macrolide resistance-associated mutations by the treatment. Conclusions The single 1 g dose treatment of azithromycin could select M genitalium strains harbouring macrolide resistance-associated mutations. For M genitalium, this regimen might increase the risk of macrolide resistance selection after treatment.

Macrolide Resistance–associated 23S rRNA Mutation in Mycoplasma genitalium, Japan

To the Editor: Mycoplasma genitalium is now recognized as a serious pathogen in sexually transmitted infections (1,2). Azithromycin regimens have been commonly used for treatment of M. genitalium infections (3). However, failure of azithromycin treatment has been reported in cases of M. genitalium–positive nongonococcal urethritis (NGU) (4,5), and macrolide-resistant strains of M. genitalium have been isolated from case-patients in Australia, Sweden, and Norway for whom azithromycin treatment has failed (4,5). In these strains, mutations in the 23S rRNA gene were associated with macrolide resistance, and mutations in ribosomal protein genes L4 and L22 were also found (5). Surveillance for antimicrobial resistance of M. genitalium is essential to identify antimicrobial resistant strains and to then determine appropriate treatment. Coculture of patient specimens with Vero cells has improved the primary isolation rate of M. genitalium from clinical specimens and offered some current clinical strains for antimicrobial drug susceptibility testing (6). To determine their antimicrobial susceptibilities, a molecular real-time PCR method has been developed (7,8). However, isolating M. genitalium from clinical specimens and antimicrobial drug susceptibility testing of clinical isolates remain labor-intensive, time-consuming tasks. In addition, no methods are available to directly determine antimicrobial drug susceptibilities of M. genitalium in clinical specimens. To monitor macrolide susceptibilities in clinical strains of M. genitalium in Japan, therefore, we examined M. genitalium DNA found in the urine of men with NGU for the presence of macrolide resistance–associated mutations in the 23S rRNA gene and the ribosomal protein genes L4 and L22. This retrospective study was approved by the Institutional Review Board of the Graduate School of Medicine, Gifu University, Gifu, Japan. We collected pretreatment urine specimens from 308 men with NGU who had visited a urologic clinic (iClinic) in Sendai, Japan, during 2006 through 2008 and stored the specimens at –70°C. Each man gave informed consent. Twenty-five of 58 urine specimens confirmed to be positive for M. genitalium by PCR-based assay were randomly chosen for this study and subjected to DNA purification. The 23S rRNA gene and the ribosomal proteins genes L4 and L22 of M. genitalium were amplified from the purified DNA by PCR as reported previously and then sequenced (5). In 1 specimen, we found an A-to-G transition at nucleotide position 2072 in the 23S rRNA gene of M. genitalium, corresponding to position 2059 in Escherichia coli (Table). An A2059 (E. coli numbering) residue in region V of the 23S rRNA gene is critical for the binding of macrolides (9). Mutations of A2058, A2059, and other 23S rRNA residues within the macrolide-binding site can confer a high-level resistance to macrolides in several bacterial species, including M. genitalium (5,9). Therefore, M. genitalium strains that harbor the A2059G (E. coli numbering) mutation in the 23S rRNA gene could be highly macrolide resistant. We also found a T-to-G transition at nucleotide position 2199 in the 23S rRNA gene of M. genitalium, corresponding to position 2185 in E. coli, in 3 specimens, but this mutation has not been associated with macrolide resistance in other bacterial species (9). Table Mutations in the 23S rRNA gene and amino acid changes in L4 and L22 ribosomal proteins of 25 Mycoplasma genitalium strains in the pretreatment urine specimens of men with nongonococcal urethritis, Japan We found amino acid changes in L4 and L22 ribosomal proteins in M. genitalium in 9 specimens. L4 and L22 ribosomal proteins each have extended loops, which converge to form a narrowing in the exit tunnel adjacent to the macrolide-binding site (10). Therefore, macrolide resistance–associated missense mutations in L4 and L22 tend to be localized to Gln62–Gly66 in L4 and Arg88-Ala93 in L22 of E. coli, which are closest to the macrolide-binding site (10). All of the amino acid changes in L4 of M. genitalium found in this study corresponded to those at the downstream regions from Gln62-Gly66 in L4 of E. coli. Of the amino acid changes in L22 of M. genitalium, the only Gly93Glu change found in M. genitalium harboring the A2059G (E. coli numbering) mutation in the 23S rRNA gene was located within the region corresponding to Arg88-Ala93 in L22 of E. coli. In this strain, therefore, the Gly93Glu change in L22 might contribute to the increase of macrolide resistance. The patient with NGU, whose specimen exhibited this strain of M. genitalium that harbored both the A2059G (E. coli numbering) mutation in the 23S rRNA gene and in which the Gly93Glu change in L22 was detected, was given a single dose of 1 g azithromycin and was clinically cured of NGU. However, the present study suggests that M. genitalium strains with high-level macrolide resistance might have already emerged in clinical settings in Japan. The emergence and spread of such a clinical mutant could threaten the ability of macrolides to treat M. genitalium infections. We should continue monitoring macrolide resistance of M. genitalium clinical strains. The nonculture approach used in our study will be useful until culturing of mycoplasmas from clinical specimens and antimicrobial drug susceptibility testing can be performed easily in laboratories.

Quantitative detection of Ureaplasma parvum (biovar 1) and Ureaplasma urealyticum (biovar 2) in urine specimens from men with and without urethritis by real-time polymerase chain reaction.

Background: We previously reported a significant association between Ureaplasma urealyticum (biovar 2) and nongonococcal urethritis (NGU). We also found that the presence of Ureaplasma parvum (biovar 1) in the male urethra might be the result of colonization. Objective: The objective of this study was to clarify the pathogenic role of human Ureaplasma in NGU by assessing the association of bacterial loads with clinical findings and inflammatory responses in the urethra. Study Design: The 16S rRNA gene of Ureaplasma was quantified by a TaqMan-based real-time polymerase chain reaction assay in first-pass urine from 37 men with Ureaplasma-positive nonmycoplasmal nonchlamydial NGU (NMNCNGU) and 30 Ureaplasma-positive men without urethritis. Results: U. urealyticum (biovar 2) loads in 23 men with NMNCNGU were significantly higher than those in 14 men without urethritis. However, U. parvum (biovar 1) loads did not differ significantly between 14 men with NMNCNGU and 20 men without urethritis. Conclusion: The association of increased U. urealyticum (biovar 2) loads with symptomatic urethritis suggests that U. urealyticum (biovar 2) may be a pathogen of NGU. Our results also suggest that the presence of U. parvum (biovar 1) may not be significant in the development of NGU.

Longitudinal Quantitative Detection by Real-Time PCR of Mycoplasma genitalium in First-Pass Urine of Men with Recurrent Nongonococcal Urethritis

ABSTRACT By using a TaqMan assay we monitored longitudinal changes in Mycoplasma genitalium loads in five men with recurrent M. genitalium-positive nongonococcal urethritis. We observed regrowth of M. genitalium persisting in hosts after treatment and a possible association of the increase in the M. genitalium load with emergence of symptoms and signs of nongonococcal urethritis in four of these patients.

Renal cell carcinoma associated with Xp11.2 translocation/TFE3 gene fusion: radiological findings mimicking papillary subtype.

The authors describe the computed tomography (CT) and magnetic resonance imaging (MRI) findings of an 18‐year‐old man with renal cell carcinoma (RCC) associated with the Xp11.2 translocation/transcription factor E3 (TFE3) gene fusion (Xp11 translocation carcinoma). The lesion was hyperdense on unenhanced CT, hypovascular on contrast‐enhanced studies, hypointense on T2‐weighted MR images, and hemosiderin deposition was suspected on phase‐shift gradient‐echo MR images. Histopathological specimens revealed pathological findings resembling papillary RCC predominantly and exhibited immunoreactivity for TFE3. Because there is often considerable morphological overlap between this carcinoma and papillary RCC, the imaging findings of Xp11 translocation carcinoma may be similar to those of the papillary subtype. Therefore, Xp11 translocation carcinoma should be considered, particularly in young patients when radiologic images demonstrate a renal tumor mimicking the papillary subtype. J. Magn. Reson. Imaging 2011;33:217–220.