Association between proline-requiring auxotype and fluoroquinolone resistance in Neisseria gonorrhoeae isolated in Japan.

We examined the association between auxotype and fluoroquinolone resistance in Neisseria gonorrhoeae isolated in Fukuoka, Japan, and investigated whether the prevalence of fluoroquinolone-resistant N. gonorrhoeae isolates was caused by the dissemination of the same clone in the community. We examined the antimicrobial susceptibility of 294 N. gonorrhoeae, isolates obtained during three different periods in Fukuoka, Japan, and 89 isolates of N. gonorrhoeae, classified by the presence of amino-acid substitutions in the quinolone resistance-determining regions (QRDRs) of GyrA and ParC proteins, to various agents, and we examined the auxotypes of the isolates. In 22 isolates with amino-acid substitutions within QRDRs in GyrA and ParC, pulsed-field gel electrophoresis analysis was performed. The proportion of fluoroquinolone-resistant isolates (ciprofloxacin, minimum inhibitory concentration [MIC] > or = 1 microg/ml) in 1998 (23.9%) was significantly higher than that in 1992-1993 (0%). The proportion of proline-requiring isolates increased significantly, from 4.4% in 1992-1993 to 54.5% in 1998. The ciprofloxacin MIC90 for the proline-requiring isolates were 32- and 128-fold, respectively, higher than those for the prototrophic isolates and the arginine-requiring isolates. The proportion of isolates with amino-acid substitutions within the QRDRs in GyrA and ParC in the proline-requiring group (55.5%) was significantly higher than that in the prototrophic group (0%). Of the 22 isolates with amino-acid substitutions within the QRDRs in GyrA and ParC, 16 showed the same pulsed-field gel electrophoresis pattern. These results suggest that a close association exists between the increase in the proline-requiring isolates and the increase in the fluoroquinolone-resistant isolates in the gonococci isolated in Fukuoka, and that the prevalence of fluoroquinolone-resistant N. gonorrhoeae isolates with GyrA and ParC substitutions may be mainly caused by the dissemination of a single clone in the community.

Antimicrobial activity of gemifloxacin (SB-265805), a newer fluoroquinolone, against clinical isolates of Neisseria gonorrhoeae, including fluoroquinolone-resistant isolates.

Antimicrobial activity of gemifloxacin (SB-265805), a newly developed fluoroquinolone, to Japanese isolates of Neisseria gonorrhoeae was compared with those of various fluoroquinolones, including norfloxacin, ciprofloxacin, tosufloxacin, levofloxacin, sparfloxacin, and trovafloxacin. Among the fluoroquinolones tested, gemifloxacin was most active against N. gonorrhoeae isolates. The MIC90 values of gemifloxacin for 94 N. gonorrhoeae isolated from 1992 through 1993 and 100 isolated from 1996 through 1997 were 0.03 and 0.125 microg/ml, respectively. On the other hand, MIC90 values of the other fluoroquinolone for the 1992-1993 isolates and the 1996-1997 isolates ranged from 0.125 to 2 microg/ml and from 0.5 to 8 microg/ml, respectively. Gemifloxacin was also the most potent fluoroquinolone against 31 ciprofloxacin-resistant isolates with the ciprofloxacin MIC of 1 to 16 microg/ml, for which the gemifloxacin MIC50 and MIC90 values were 0.25 and 2 microg/ml, respectively. Moreover, the activity of gemifloxacin against fluoroquinolone-resistant gonococcal isolates containing multiple amino acid substitutions in both GyrA and ParC proteins was superior to those of the other compounds.

High prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium in female commercial sex workers in Japan.

Our objectives were to explore the prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium in Japanese female commercial sex workers (CSWs), in comparison with pregnant women as controls. A high-risk group of 174 female CSWs and 90 asymptomatic pregnant women were enrolled in this study. Detection of C. trachomatis, N. gonorrhoeae, and M. genitalium on the endocervix of the women was performed mainly by using polymerase chain reaction (PCR)-based assays. The prevalence rates of C. trachomatis, N. gonorrhoeae, and M. genitalium were 19.0%, 32.8%, and 12.6%, respectively, in the CSWs, compared with 5.6%, 0%, and 1.1% respectively, in the pregnant women. These results suggest a high prevalence of C. trachomatis, N. gonorrhoeae, and M. genitalium in Japanese CSWs. We conclude that continued close monitoring of the prevalence of C. trachomatis, N. gonorrhoeae, and M. genitalium infection in CSWs is important for preventing the dissemination of these microorganisms, and that further investigation of M. genitalium as a sexually transmitted pathogen in women is needed.

Effect of urine component on leukocyte chemiluminescence response.

In the urine, the function of polymorphonuclear leukocytes (PMNs) is thought to be impaired because of the high osmolality and low pH along with a high concentration of inorganic salts. We investigated the effect of the properties of urine and its components on the chemiluminescence (CL) response of PMNs. This was helped by using an artificial urine. The CL response was measured by automatic luminometer following stimulation of phorbol myristate acetate. We found the CL response of PMNs to be significantly suppressed at a pH of 6 or 5, but not suppressed at a pH of 7 or 8. The CL response was significantly reduced when the osmolality was increased to 580 or 800 mOsm/kg H2O by the addition of urea as compared to the response in the standard artificial urine at an osmolality of 425 mOsm/kg H2O. A change in the osmolality by the addition of mannitol only minimally influenced the CL responses. In addition, the CL response was significantly impaired by both low and high concentrations of sodium at 12 and 300 mEq/L as compared to 77.5 mEq/L in the standard artificial urine. Potassium significantly reduced the CL response in a concentration-dependent manner in the range of from 4 to 31 mEq/L as compared to 52.3 mEq/L in the standard artificial urine. A high concentration of calcium at 8.1 or 9.9 mg/dL reduced CL response as compared to 3.9 mg/dL, whereas CL response was not reduced by the change in the concentration of magnesium. A high concentration of creatinine significantly reduced the CL response as compared to the standard artificial urine. We conclude that the function of PMNs in urine is reduced mainly by urine pH, concentration of urea, sodium, potassium, and creatinine. We suggest that reversal of these change in urine may restore functions of PMNs to clear bacteria in patients with urinary tract infections.

Preventive effect of dapsone on renal scarring following mannose-sensitive piliated bacterial infection.

Renal scarring has been thought to occur in the later stages of chronic pyelonephritis. We previously reported that mannose-sensitive (MS) piliated bacteria promoted renal scarring, which was prevented by antioxidants. The preventive effect of diaphenylsulfone (dapsone), which has a scavenging activity on active oxygen species, on renal scarring was examined. Female Sprague-Dawley rats were inoculated with clinical isolates of Serratia marcescens which had both MS and mannose-resistant pili or with recombinant strains which had MS pili on their surface; they were then administered 20 mg/kg of dapsone or not. Dapsone significantly suppressed scarring following infection of the kidney. The bacterial counts in the kidneys were not different in dapsone-treated and nontreated rats. We conclude that dapsone is effective in preventing renal scarring, and it is suggested that the clinical use of this drug may prevent renal scar formation following pyelonephritis.

Chemiluminescence response of whole blood in patients undergoing urological operations.

Polymorphonuclear leukocytes (PMNs) are one of the most important components of the defence mechanisms against bacterial infection. The functions of PMNs are believed to be impaired in patients during the perioperative period. Bactericidal function of PMNs was investigated together with the luminol-dependent chemiluminescence (CL) reaction of whole blood in 23 patients, 12 undergoing open surgery and 11 undergoing endoscopic surgery. Blood samples were collected one day before surgery (day -1) and 2 hours (day 0), 24 hours (day 1) and 7 days (day 7) after surgery. Counts of whole white blood cells (WBCs), PMNs and lymphocytes were not different between the two surgery groups. CL responses in the open surgery group were increased on days 0, 1 and 7. In the endoscopic surgery group, CL response was increased on day 1, but not on day 0 or day 7. These results suggest that the PMN function during the perioperative period was not impaired, but increased just after surgery, mainly due to an increasing number of WBC caused by the surgical intervention.

Renal scarring by mannose-sensitive adhesin of Escherichia coli type 1 pili.

Most Escherichia coli isolates from patients with pyelonephritis possess both pap (mannose-resistant) pili and type 1 (mannose-sensitive) pili. In the experimental pyelonephritis model of rats, the mannose-sensitive-piliated strain caused severe renal scarring, whereas the mannose-resistant or nonpiliated strain did not. Type 1 pili consist of several subunits; one major subunit and other minor subunits. One of the minor subunits, adhesin, is responsible for mannose-sensitive adhesion to eukaryotic cells. The role of adhesin was examined in scar formation after infection with a newly constructed adhesin-deficient mutant which has pilus structure but cannot agglutinate guinea pig erythrocytes. A mutant plasmid, pYMZ84, containing a deletion in the adhesin gene of type 1 pili, failed to agglutinate guinea pig erythrocytes even though the bacteria expressed pili morphologically indistinguishable from those produced by plasmid pSH2, carrying the intact genes for the type 1 pili. E. coli harboring pYMZ84 caused negligible or minimal renal scarring, whereas E. coli harboring pSH2 caused severe renal scarring in rats. These data suggest that the mannose-sensitive adhesin of type 1 pili stimulates renal scarring.

Distribution of a methicillin-resistance gene in urinary isolates of methicillin-resistant staphylococci examined by enzymatic detection of the polymerase chain reaction.

We tried to examine the susceptibility to various antimicrobial agents and to detect the mec A gene using enzymatic detection of the polymerase chain reaction in methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA) and Staphylococcus epidermidis isolated from patients with complicated urinary tract infections (UTIs). All the strains of MRSA and MSSA showed a low sensitivity to imipenem (IPM), ceftazidime (CAZ), flomoxef (FMOX), amikacin (AMK), ciprofloxacin (CPFX) and ofloxacin (OFLX). Although all the strains of MRSA had the mec A gene, none of the MSSA strains had it. 74% of S. epidermidis had the mec A gene and strains resistant to methicillin were seen in 72% of them. The mec A-positive S. epidermidis showed a lower susceptibility to IPM, CAZ, FMOX, AMK, CPFX and OFLX than the mec A-negative strains. These results suggest that methicillin resistance was due to the mec A gene in MRSA and methicillin-resistant S. epidermidis (MRSE), and that MRSEs were very common among the bacteria causing complicated UTI. When we try to control nosocomial infections due to MRSA, it should also be noted that MRSE can be a reservoir of the mec A gene.

Fleroxacin enhancement of superoxide production by polymorphonuclear leukocytes: the role of protein kinases.

New quinolone (NQ) antimicrobials may influence the functions of polymorphonuclear leukocyes (PMNs). Fleroxacin (FLRX), one of the newer NQs which has a long half-life in blood and a strong bactericidal effect, was examined for its influence on superoxide production by PMNs. Augmentation of superoxide production by PMNs when stimulated with phorbol myristate acetate (PMA) and formyl-methionyl-leucyl-phenylalanine (fMLP) was observed following the addition of 25, 50, 100 and 200 micrograms/ml of FLRX. In addition, the effects of staurosporine and H-7, inhibitors of protein kinase C (PKC), and of genistein, a tyrosine kinase (TK) inhibitor, on FLRX-enhanced superoxide production were examined. Superoxide production augmented by FLRX was diminished by the addition of staurosporine and H-7, when PMNs were stimulated with PMA, and by the addition of genistein, when PMNs were stimulated with fMLP. These results suggest that FLRX augments superoxide production by PMNs through enhancing the activities of phosphorylation by PKC or TK within the signal transduction pathway in PMNs.

Inhibition of chemiluminescence response of human polymorphonuclear leukocytes by three different stimulations in hyperosmotic condition comparable to the renal medulla.

Hyperosmolality in renal medulla inhibits functions of polymorphonuclear leukocyte (PMN) such as phagocytosis, intracellular killing, and superoxide generation. The main factors of hyperosmolality in the renal medulla are thought to be urea and NaCl. We studied the luminol-dependent chemiluminescence (CL) response of PMNs to three different stimulators: phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP), and opsonized zymosan (OZ). When PMNs were incubated or preincubated with hyperosmotic urea or NaCl solutions, CL responses were significantly reduced following stimulation by each of PMA, FMLP, or OZ. Reduction of CL response was concentration and osmolality dependent in hyperosmotic urea and NaCl solution. These results suggest that respiratory burst and production of active oxygen species of PMNs through three different signal transduction pathways are inhibited in hyperosomotic conditions comparable to the renal medulla.