Multi-drug resistance pattern and genome-wide SNP detection in levofloxacin-resistant uropathogenic Escherichia coli strains.

OBJECTIVES: Antibiotic treatment is extremely stressful for bacteria and has profound effects on their viability. Such administration induces physiological changes in bacterial cells, with considerable impact on their genome structure that induces mutations throughout the entire genome. This study investigated drug resistance profiles and structural changes in the entire genome of uropathogenic Escherichia coli (UPEC) strains isolated from six adapted clones that had evolved under laboratory conditions. METHODS: Eight UPEC strains, including two parental strains and six adapted clones, with different fluoroquinolone resistance levels originally isolated from two patients were used. The minimum inhibitory concentration (MIC) of 28 different antibiotics including levofloxacin was determined for each of the eight strains. In addition, the effects of mutations acquired with increased drug resistance in the levofloxacin-resistant strains on expression of genes implicated to be involved in drug resistance were examined. RESULTS: Of the eight UPEC strains used to test the MIC of 28 different antibiotics, two highly fluoroquinolone-resistant strains showed increased MIC in association with many of the antibiotics. As drug resistance increased, some genes acquired mutations, including the transcriptional regulator acrR and DNA-binding transcriptional repressor marR. Two strain groups with genetically different backgrounds (GUC9 and GFCS1) commonly acquired mutations in acrR and marR. Notably, acquired mutations related to efflux pump upregulation also contributed to increases in MIC for various antibiotics other than fluoroquinolone. CONCLUSIONS: The present results obtained using strains with artificially acquired drug resistance clarify the underlying mechanism of resistance to fluoroquinolones and other types of antibiotics.

Study of Normal Flora in the Pharynx of Healthy Children.

To improve our current understanding of normal flora in children, we investigated bacterial isolates from the pharynx and nasopharynx of 173 and 233 healthy children, respectively. The bacterial isolation rates were compared among three age groups: infants (<1 year), toddlers (1-5 years), and school-aged children (6-15 years). Gram-positive cocci were the predominant bacteria in the pharynx (Streptococcus mitis/oralis, 87.3%; Streptococcus salivarius, 54.3%; Rothia mucilaginosa, 41.6%; Staphylococcus aureus, 39.3%). Among infants, S. salivarius and Neisseria subflava, which are related to the development of teeth, were significantly lower than in the other age groups (P <0.0001, S. salivarius; P <0.01, N. subflava). With the exception of Corynebacterium pseudodiphtheriticum (44.2%, gram-positive rods), gram-negative rods largely predominated the nasopharynx (Moraxella catarrhalis, 32.1%; Moraxella nonliquefaciens, 28.3%). Among toddlers, M. catarrhalis and Streptococcus pneumoniae, which are the most common pathogens in acute otitis media, were significantly higher than in the infant group (P <0.05). Among the bacterial species implicated in pediatric respiratory infections, Streptococcus pyogenes was isolated in 3.5% of the pharyngeal samples. S. pneumoniae and Haemophilus influenzae were isolated in 22.3% and 17.2% of the nasopharyngeal samples, respectively. In conclusion, the normal flora of the respiratory tract differs not only by the sampling site but also by the age group.

Induction of plasmid-mediated AmpC ß-lactamase DHA-1 by piperacillin/tazobactam and other ß-lactams in Enterobacteriaceae.

Chromosomal AmpC ß-lactamase induction by several types of ß-lactams has been reported, but not enough data are available on DHA-1 ß-lactamase, a plasmid-mediated AmpC ß-lactamase. Therefore, we evaluated the DHA-1 ß-lactamase induction by various antibiotics including piperacillin/tazobactam (PIP/TZB) in this study. Six strains (Enterobacter cloacae 2 strains, Citrobacter freundii 1 strain, Serratia marcescens 2 strain, and Morganella morganii 1 strain) possessing chromosomal inducible AmpC ß-lactamase were used as controls. Four strains (Escherichia coli 2 strains, Klebsiella pneumoniae 1 strain, and C. koseri 1 strain) possessing DHA-1 ß-lactamase were used. The ß-lactamase activities were determined by a spectrophotometer using nitrocefin. ß-lactamase induction by PIP, PIP/TZB was not observed in any strains and ß-lactamase induction by third- and fourth-generation cephems was not observed in most strains. The induction ratios of the chromosomal AmpC ß-lactamase in the reference group by PIP/TZB were <1.51, and those of the DHA-1 ß-lactamase were <1.36, except for K. pneumoniae Rkp2004 (2.22). The ß-lactamase induction by first- and second-generation cephems, flomoxef, and carbapenem differed in each strain. Cefmetazole (CMZ) strongly induced ß-lactamase. This study demonstrated that the induction of DHA-1 ß-lactamase was similar to that of chromosomal AmpC using various Enterobacteriaceae, although the induction of ß-lactamase in both groups by PIP/TZB was low. We also reported that the induction of PIP/TZB, a ß-lactamase inhibitor combination antibiotic, against various AmpC-producing Enterobacteriaceae, including DHA-1 producers, was low.

Molecular epidemiology and clinical features of extended-spectrum beta-lactamase- or carbapenemase-producing Escherichia coli bacteremia in Japan.

OBJECTIVES: To identify risk factors and clinical outcomes in patients with bacteremia due to extended-spectrum beta-lactamase (ESBL) or carbapenemase-producing Escherichia coli, as well as to determine the prevalence and genetic background of such isolates. METHODS: Case control study was performed with patients with E. coli bacteremia between January 2008 and May 2013 (n = 115) at a tertiary university hospital in Japan. Cases had ESBL-producing E. coli (ESBL-EC) whereas controls had non-ESBL-producing E. coli (non-ESBL-EC) isolates. A retrospective chart review was performed to identify risk factors and clinical outcomes. Isolates were characterized by antimicrobial susceptibility testing, polymerase chain reaction analysis for beta-lactamase genes, and multi-locus sequence typing. RESULTS: Of 115 unique cases of E. coli bacteremia, 30 (26.1%) were due to ESBL-EC and three (2.6%) were due to carbapenemase-producing E. coli. All three carbapenemase-producing E. coli isolates were IMP-6 and concurrently produced ESBL (ESBL/IMP-6-EC). ESBL-EC isolates showed multidrug resistance. Of the ESBL-EC isolates, CTX-M-27 was the most prevalent (33.3%), followed by CTX-M-14 (30%). Multi-locus sequence typing revealed that 19 (63.3%) isolates were ST131. The multivariate analysis identified nursing home-associated infections and antibiotic administration in the preceding 30 days as risk factors for ESBL-EC bacteremia. The 14-day mortality non-ESBL-EC, ESBL-EC, and ESBL/IMP-6-EC was 4.7% (4/85), 20% (6/30), and 66.7% (2/3), respectively. CONCLUSIONS: CTX-M-27, CTX-M-14, and ST131 were the most prevalent ESBL-EC isolates from bacteremic patients in a Japanese hospital. Further studies with larger sample sizes are warranted to investigate the clinical significance of ESBL-EC and ESBL/IMP-6-EC.

Antimicrobial susceptibility of Haemophilus influenzae strains isolated from the urethra of men with acute urethritis and/or epididymitis.

We determined minimum inhibitory concentrations (MICs) of 41 antimicrobial agents for 73 clinical strains of Haemophilus influenzae isolated from the urethra of men with acute urethritis and/or epididymitis and examined the strains for the production of ß-lactamase. We also compared their antimicrobial susceptibilities with those of H. influenzae strains from respiratory tract or otorhinolaryngological infections that were reported in Japan. The proportion of ß-lactamase-nonproducing ampicillin-resistant strains from acute urethritis and/or epididymitis appeared to be lower, but that of ß-lactamase-producing ampicillin-resistant strains appeared to be higher, compared with those from respiratory tract or otorhinolaryngological infections. However, their antimicrobial susceptibilities to a variety of other antimicrobial agents would be similar to those from respiratory tract or otorhinolaryngological infections. Almost all of the strains of H. influenzae from acute urethritis and/or epididymitis were susceptible to the agents, including ceftriaxone, quinolones, macrolides, and tetracyclines, commonly prescribed for treatment of acute urethritis based on the MIC breakpoints recommended by the Clinical and Laboratory Standards Institute. Ceftriaxone and quinolones could be effective on H. influenzae-induced urethritis. However, azithromycin treatment failures were reported in acute urethritis caused by H. influenzae strains considered susceptible to azithromycin. Further studies will be needed to determine MIC breakpoints of antimicrobial agents, which are recommended for treatment of urogenital infections, for H. influenzae strains causing these infections. Nevertheless, this study provides useful data regarding antimicrobial susceptibilities of H. influenzae strains isolated from the urogenital tract, which have rarely been studied.

The first case report of emphysematous pyelonephritis and bacteremia due to Oligella urethralis.

Oligella urethralis (O. urethralis) is an organism that rarely causes infections in humans. We report the case of a 90-year-old bedridden woman with progressive dementia who was placed in a long-term-care facility. She was admitted to our hospital due to fever and unconsciousness with pyuria. The abdominal computed tomography showed left pneumatosis and urinary stone. Fluoroquinolones-resistant O. urethralis, which was identified by the Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and the 16S rRNA gene sequencing, was isolated form the blood and urine cultures at admission. To the best of our knowledge, this is the first case of emphysematous pyelonephritis caused by O. urethralis.

Serotype 35B Streptococcus pneumoniae, Japan, 2002-2012.

Among 641 pneumococcal isolates recovered from 2002 to 2012 in Japan, 19 (3.0%) were serotype 35B. Twelve of the 19 were ST558 (single-locus variant of Utah35B-24-ST377) and seven were ST2755. Continuous monitoring of serotypes and their clonal association is important, especially in Japan where PCV7 was licensed only in 2010.

The mechanism of high-level carbapenem resistance in Klebsiella pneumoniae: underlying Ompk36-deficient strains represent a threat of emerging high-level carbapenem-resistant K. pneumoniae with IMP-1 ß-lactamase production in Japan.

The mechanisms of high-level carbapenem resistance in Klebsiella pneumoniae isolated in Japan were investigated. High-level carbapenem-resistant K. pneumoniae Mkp4437 and a less carbapenem-sensitive K. pneumoniae strain, Mkp4365, were recovered from the same patient. These two strains were found to be homologous by pulsed-field gel electrophoresis, and both strains contained blaIMP-1, blaDHA-1, blaCTXM-14, blaTEM-1, and blaSHV-1. Based on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the lack of Ompk36 was observed in Mkp4437. Direct sequencing of the ompK36 gene demonstrated that a new insertional sequence in the open reading frame of the ompK36 gene was found in Mkp4437. Three clinical isolates (minimum inhibitory concentration [MIC] 2-4 mg/L to imipenem) were identified upon screening the strains of K. pneumoniae isolated in the University hospital with MICs of ≥ 1 mg/L to imipenem. Interestingly, these three isolates all lacked OmpK36. Conjugation of the plasmid harboring IMP-1 to these three OmpK36-deficient strains led to the isolation of high-level carbapenem-resistant transconjugants. In conclusion, the mechanisms of high-level carbapenem resistance in K. pneumoniae entail not only the production of IMP-1 ß-lactamase but also the lack of OmpK36. It is vital to monitor for the presence of less carbapenem-sensitive K. pneumoniae strains, which lack OmpK36, because blaIMP-1 transmission to these strains may result in isolates with a high-level carbapenem-resistant phenotype.

[Usefulness of real-time PCR in detecting Chlamydia trachomatis and Neisseria gonorrhoeae in endocervical swabs and first-voided urine specimens].

We evaluated performance of Abbott RealTime CT/NG assay (real-time PCR, Abbott Japan) for detect Chlamydia trachomatis and Neisseria gonorrhoeae by real-time PCR in 88 female patients with cervicitis symptoms seen at gynecological clinics and 100 male patients with urethritis symptoms seen at urological or dermatology clinics in Kitakyushu, Japan. Endocervical swab and first-voided urine (FVU) specimens were then collected from women and FVU specimens from men. Detection rates of C. trachomatis and N. gonorrhoeae by real-time PCR in the 3 types of specimens were compared to those by ProbeTec ET assay (ProbeTec, BD Diagnostic System). The overall positive concordance between real-time PCR and ProbTec were 97.1% (66/68) for C. trachomatis and 100% (33/33) for N. gonorrhoeae, C. trachomatis detection yielded 3 discordant results in endocervical specimens and 1 discordant result in male FVU by real-time PCR and ProbTec. Three of 4 reexamined using Aptime Combo 2 Assay (Fuji Rebio Inc.) were positive for C. trachomatis. Endocervical swab and FVU specimen results for C. trachomatis were discordant in 3 cases in real-time PCR and 4 in ProbeTec. Subjects with 2 or more positive endocervical awab results in female or male FVU specimens were assumed to be "true positive" for C. trachomatis. The sensitivities of real-time PCR for detecting C. trachomatis was 94.4% in endocervical swabs, 77.8% in female FVU and 97.4% in the male FVU. The sensitivities for real-time PCR for detectig N. gonorrhoeae was 100% in all 3 specimen types. Abbott RealTime CT/NG assay was useful for detecting C. trachomatis using endocervical swabs or male FVU specimens and for detecting N. gonorrhoeae using endocervical swabs and all FVU specimens.

The efficacy of sequential therapy using pazufloxacin followed by oral fluoroquinolones for treatment of pyelonephritis.

The efficacy of sequential therapy of pazufloxacin (PZFX), which is a parenteral fluoroquinolone, followed by oral fluoroquinolones [tosufloxacin tosilate (TFLX) or levofloxacin (LVFX)] for treatment of pyelonephritis, was evaluated. Patients with pyelonephritis who had fever (≥37.5 °C), pyuria (≥10 white blood cells/high-power field), and bacteriuria (≥10(4) colony-forming units/ml) were eligible for this study. PZFX (500 mg) was given intravenously twice a day for at least 3 days. If the patients were clinically improved, TFLX (150 mg) or LVFX (100 mg) was then administered orally three times a day for at least 5 days. Patients underwent clinical and microbiological evaluation at 5-9 days after final drug administration. Clinical and microbiological efficacy could be assessed in 21 of 25 cases enrolled. Both clinical and microbiological efficacy rates were 81.0 % (17/21 cases). In the effective cases, the mean administration time was 4.2 days for PZFX and 6.0 days for oral fluoroquinolones. The mean time to defervescence was 3.4 days for the effective cases. In the four treatment failure cases, three quinolone-resistant Escherichia coli and a quinolone-resistant Enterococcus faecalis were isolated. This sequential therapy seemed to be clinically effective in the treatment of pyelonephritis; however, the prevalence of quinolone-resistant E. coli should be taken into account.

Nationwide surveillance of bacterial respiratory pathogens conducted by the Surveillance Committee of Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Clinical Microbiology in 2009: general view of the pathogens' antibacterial susceptibility.

For the purpose of nationwide surveillance of antimicrobial susceptibility of bacterial respiratory pathogens from patients in Japan, the Japanese Society of Chemotherapy (JSC) started a survey in 2006. From 2009, JSC continued the survey in collaboration with the Japanese Association for Infectious Diseases and the Japanese Society for Clinical Microbiology. The fourth-year survey was conducted during the period from January and April 2009 by the three societies. A total of 684 strains were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections. Susceptibility testing was evaluable with 635 strains (130 Staphylococcus aureus, 127 Streptococcus pneumoniae, 4 Streptococcus pyogenes, 123 Haemophilus influenzae, 70 Moraxella catarrhalis, 78 Klebsiella pneumoniae, and 103 Pseudomonas aeruginosa). A maximum of 45 antibacterial agents including 26 ß-lactams (four penicillins, three penicillins in combination with ß-lactamase inhibitors, four oral cephems, eight parenteral cephems, one monobactam, five carbapenems, and one penem), four aminoglycosides, four macrolides (including ketolide), one lincosamide, one tetracycline, two glycopeptides, six fluoroquinolones, and one oxazolidinone were used for the study. Analysis was conducted at the central reference laboratory according to the method recommended by the Clinical and Laboratory Standard Institute (CLSI). Incidence of methicillin-resistant S. aureus (MRSA) was as high as 58.5 %, and that of penicillin-intermediate and penicillin-resistant S. pneumoniae (PISP and PRSP) was 6.3 % and 0.0 %, respectively. Among H. influenzae, 21.1 % of them were found to be ß-lactamase-non-producing ampicillin (ABPC)-intermediately resistant (BLNAI), 18.7 % to be ß-lactamase-non-producing ABPC-resistant (BLNAR), and 5.7 % to be ß-lactamase-producing ABPC-resistant (BLPAR) strains. A high frequency (76.5 %) of ß-lactamase-producing strains has been suspected in Moraxella catarrhalis isolates. Four (3.2 %) extended-spectrum ß-lactamase-producing K. pneumoniae were found among 126 strains. Four isolates (2.5 %) of P. aeruginosa were found to be metallo-ß-lactamase-producing strains, including three (1.9 %) suspected multi-drug resistant strains showing resistance against imipenem, amikacin, and ciprofloxacin. Continuous national surveillance of the antimicrobial susceptibility of respiratory pathogens is crucial to monitor changing patterns of susceptibility and to be able to update treatment recommendations on a regular basis.

Sensitivities of major causative organisms isolated from patients with acute uncomplicated cystitis against various antibacterial agents: results of subanalysis based on the presence of menopause.

We investigated whether the presence of menopause influenced the species and distribution of causative bacteria isolated from patients with acute uncomplicated cystitis (the most common urinary tract infection), and we also investigated the sensitivity of the isolated species to antibacterial agents. Using multivariate analysis, we also investigated risk factors for infection with quinolone-resistant Escherichia coli, because its frequency has increased and it is now a clinical problem in Japan. Six hundred and thirty-four strains were isolated from 489 premenopausal patients (mean age 32.3 ± 10.1 years). Major causative bacteria detected were Escherichia coli (65.0 %), Enterococcus faecalis (12.0 %), Streptococcus agalactiae (5.5 %), and Klebsiella pneumoniae (1.6 %). From 501 postmenopausal patients (mean age 68.7 ± 10.29 years), 657 strains were isolated, and the major causative bacteria detected were E. coli (61.5 %), E. faecalis (13.7 %), K. pneumoniae (5.2 %), and S. agalactiae (4.0 %). The sensitivities to fluoroquinolones (FQs) and cephems of E. coli isolated from premenopausal patients were both ≥90 %, while the sensitivities to FQs of E. coli isolated from postmenopausal patients were about 5 % lower. In regard to infection with quinolone-resistant E. coli (minimal inhibitory concentration of levofloxacin [LVFX] ≥4 µg/mL), significant risk factors were observed in patients with more than two episodes of cystitis within a year (p = 0.0002), patients to whom antibacterial agents were used previously for this episode of cystitis (p = 0.0175), and patients who had a history of FQ administration within 1 month. Although the species and distribution of causative bacteria of acute uncomplicated cystitis were the same regardless of the presence of menopause, the sensitivities to FQs of E. coli detected in postmenopausal patients were significantly lower than those in the premenopausal women. The major risk factors for infection with quinolone-resistant E. coli were a history of FQ administration and the morbidity of cystitis rather than the menopausal status. It was considered that taking an appropriate history including the morbidity of cystitis and history of FQ administration, and the appropriate selection of an antibacterial agent, would be important when empirical therapy is required.

[Usefulness of real-time PCR in detecting Chlamydia trachomatis and Neisseria gonorrhoeae in endocervical swabs and first-voided urine specimens].

We evaluated performance of Abbott RealTime CT/NG assay (real-time PCR, Abbott Japan) for detect Chlamydia trachomatis and Neisseria gonorrhoeae by real-time PCR in 88 female patients with cervicitis symptoms seen at gynecological clinics and 100 male patients with urethritis symptoms seen at urological or dermatology clinics in Kitakyushu, Japan. Endocervical swab and first-voided urine (FVU) specimens were then collected from women and FVU specimens from men. Detection rates of C. trachomatis and N. gonorrhoeae by real-time PCR in the 3 types of specimens were compared to those by ProbeTec ET assay (ProbeTec, BD Diagnostic System). The overall positive concordance between real-time PCR and ProbTec were 97.1% (66/68) for C. trachomatis and 100% (33/33) for N. gonorrhoeae, C. trachomatis detection yielded 3 discordant results in endocervical specimens and 1 discordant result in male FVU by real-time PCR and ProbTec. Three of 4 reexamined using Aptime Combo 2 Assay (Fuji Rebio Inc.) were positive for C. trachomatis. Endocervical swab and FVU specimen results for C. trachomatis were discordant in 3 cases in real-time PCR and 4 in ProbeTec. Subjects with 2 or more positive endocervical awab results in female or male FVU specimens were assumed to be "true positive" for C. trachomatis. The sensitivities of real-time PCR for detecting C. trachomatis was 94.4% in endocervical swabs, 77.8% in female FVU and 97.4% in the male FVU. The sensitivities for real-time PCR for detecting N. gonorrhoeae was 100% in all 3 specimentypes. Abbott RealTime CT/NG assay was useful for detecting C. trachomatis using endocervical swabs or male FVU specimens and for detecting N. gonorrhoeae using endocervical swabs and all FVU specimens.

Nationwide survey of antibacterial activity against clinical isolates from urinary tract infections in Japan (2008).

In this study, the causative bacteria and their sensitivity to various antimicrobial agents as well as risk factors for quinolone-resistant Escherichia coli were investigated in patients with acute uncomplicated cystitis or complicated cystitis by isolation and culture of bacteria from urine samples. In total, 1312 strains were isolated from 1009 patients with acute uncomplicated cystitis, including E. coli (63.3%), Enterococcus faecalis (12.8%) and Streptococcus agalactiae (4.6%). In addition, 994 strains were isolated from 725 patients with complicated cystitis, including E. coli (36.4%), E. faecalis (19.2%), Klebsiella pneumoniae (5.0%), S. agalactiae (4.7%) and Pseudomonas aeruginosa (4.5%). At least 90% of E. coli isolates from acute uncomplicated cystitis showed sensitivity to fluoroquinolones and cephems, whilst 70.4-88.4% of E. coli isolates from complicated cystitis were sensitive to fluoroquinolones and 85.4-89.5% were sensitive to cephems. Factors associated with quinolone-resistant E. coli included two or more episodes of cystitis within 1 year, failure of quinolone therapy, underlying urinary tract disease, prior quinolone treatment within 1 month and age ≥ 75 years. It is important to confirm the sensitivity of causative bacteria for optimal antimicrobial therapy, and empirical antimicrobial agents should be selected by considering patient characteristics and other factors.

The nationwide study of bacterial pathogens associated with urinary tract infections conducted by the Japanese Society of Chemotherapy.

This study was conducted by the Japanese Society of Chemotherapy and is the first nationwide study on bacterial pathogens isolated from patients with urinary tract infections at 28 hospitals throughout Japan between January 2008 and June 2008. A total of 688 bacterial strains were isolated from adult patients with urinary tract infections. The strains investigated in this study are as follows: Enterococcus faecalis (n = 140), Escherichia coli (n = 255), Klebsiella pneumoniae (n = 93), Proteus mirabilis (n = 42), Serratia marcescens (n = 44), and Pseudomonas aeruginosa (n = 114). The minimum inhibitory concentrations of 39 antibacterial agents used for these strains were determined according to the Clinical and Laboratory Standards Institute (CLSI) manual. All Enterococcus faecalis strains were susceptible to ampicillin and vancomycin. Although a majority of the E. faecalis strains were susceptible to linezolid, 11 strains (7.8%) were found to be intermediately resistant. The proportions of fluoroquinolone-resistant Enterococcus faecalis, Escherichia coli, Proteus mirabilis, and S. marcescens strains were 35.7%, 29.3%, 18.3%, and 15.2%, respectively. The proportions of E. coli, P. mirabilis, K. pneumoniae, and S. marcescens strains producing extended-spectrum ß-lactamase were 5.1%, 11.9%, 0%, and 0%, respectively. The proportions of Pseudomonas aeruginosa strains resistant to carbapenems, aminoglycosides, and fluoroquinolones were 9.2%, 4.4%, and 34.8%, respectively, and among them, 2 strains (1.8%) were found to be multidrug resistant. These data present important information for the proper treatment of urinary tract infections and will serve as a useful reference for periodic surveillance studies in the future.

Clinical effects of 2 days of treatment by fosfomycin calcium for acute uncomplicated cystitis in women.

Fosfomycin calcium is a fosfomycin antimicrobial agent with a characteristic structure. After oral administration, the drug is absorbed and excreted via the kidneys in the unchanged form, without being metabolized in the body. It is, therefore, indicated for the treatment of urinary tract diseases, including cystitis and pyelonephritis. In the present study, the clinical usefulness of fosfomycin calcium (FOSMICIN® TABLETS 500) administered orally at the dosage of 1 g (two tablets) three times daily for 2 days was examined in female patients, who were at least 20 years of age, with acute uncomplicated cystitis of bacterial origin. Of the 48 patients enrolled between February 2008 and August 2008, 39 were evaluable for efficacy and safety. Overall evaluation of the cure revealed that microbiological eradication rate (microbiological outcome) and clinical efficacy rate (clinical outcome) at 5-9 days after drug administration (visit 2) were 94.9%. Determination of the microbiological and clinical outcomes for the evaluation of recurrence at 4-6 weeks after drug administration (visit 3) were 75.8 and 85.7%, respectively. Of the 48 patients, 40 (83.3%) returned to the clinic at visit 3. The causative bacterial species for cystitis was Escherichia coli in 31 (79.5%) of the 39 patients evaluable for efficacy and safety. Adverse drug reactions observed during the administration and follow-up periods included mild diarrhea and loose stools in 1 patient each, neither requiring any specific treatment. Evaluation of cure at visit 2 in patients in whom the causative bacterial species for the infection was E. coli revealed a microbiological outcome of 93.5%, and clinical outcome was 96.8%. Furthermore, evaluation for recurrence at visit 3 revealed a microbiological outcome of 74.1% and clinical outcome of 82.1%. When the patients were divided by age into an under 60 years of age group and an over 60 years of age group, the microbiological and clinical outcomes determined for evaluation of cure at visit 2 were 96.4 and 92.9%, respectively, and the corresponding rates determined for the evaluation of recurrence at visit 3 were 87.0 and 96.0%, respectively, in the under 60 years of age group. In the over 60 years of age group, the corresponding microbiological outcome and clinical outcome rates evaluated for cure were 90.9 and 100%, respectively, and those evaluated for recurrence were 50.0 and 60.0%, respectively. These results indicate the usefulness of fosfomycin calcium administered at 1 g three times daily for 2 days for acute uncomplicated cystitis.

Molecular mechanisms of fosfomycin resistance in clinical isolates of Escherichia coli.

To clarify the molecular mechanisms of fosfomycin resistance in clinical isolates of Escherichia coli, the murA, glpT, uhpT, uhpA, ptsI and cyaA genes were sequenced from six fosfomycin-resistant isolates. Two strains were found to harbour a mutation in the murA gene that leads to an amino acid substitution (Asp369Asn or Leu370Ile) in the target protein. The remaining four strains carried specific mutations in the glpT gene; one strain possessed a mutation and the other three strains possessed truncated versions of the GlpT transporter owing either to the presence of insertion sequences or a deletion in the coding region of the gene. Two of the strains with truncated GlpT had also lost the entire uhpT gene, which encodes another fosfomycin transporter. Uptake of specific substrates for the transporters was either totally blocked or reduced in strains possessing truncated forms of GlpT or those lacking the uhpT gene. Escherichia coli strains expressing an amino-acid-substituted MurA were at least eight-fold more resistant to fosfomycin than the strain overproducing wild-type MurA. In conclusion, novel amino acid substitutions in MurA or the loss of function of transporters were identified as mechanisms of fosfomycin resistance in clinical isolates of E. coli.