'Haemophilus quentini' in the urethra of men complaining of urethritis symptoms.

We isolated a cryptic genospecies of Haemophilus influenzae referred to as 'Haemophilus quentini' in the urethra of 3 men complaining of urethritis symptoms. H. influenzae strains, which had been isolated from the urethra in 77 of 1518 men complaining of urethritis symptoms, identified by the conventional test, and stored, were re-cultured for this study. Sixty-seven strains surviving storage were screened by a PCR-based assay specific for the cryptic genital Haemophilus genospecies. Three strains (HI09003, HI11006, and HI14016) were screened by PCR and identified as 'H. quentini' by 16S rRNA sequencing. The men positive for HI09003 and HI11006 were diagnosed as having non-chlamydial non-gonococcal urethritis (NGU), and their demographic and clinical features were similar to those of NGU caused by other pathogens. The man positive for HI14016 was ultimately diagnosed as having condyloma acuminatum on the glans. The 3 strains of 'H. quentini' produced no ß-lactamase and were susceptible to ampicillin and other antimicrobial agents, including cephalosporins, fluoroquinolones, tetracyclines, and macrolides, recommended for treatment for urethritis. 'H. quentini' would be an uncommon pathogen in men with urogenital infections. Based on the clinical features of the two patients with 'H. quentini'-positive NGU, it would be difficult to predict the presence of 'H. quentini' in the urethra. The 3 strains of 'H. quentini' were susceptible to a variety of antimicrobial agents. Further accumulation of data regarding 'H. quentini' infections is needed to characterize the pathogenic roles of this genospecies in urogenital infections and to establish appropriate management of 'H. quentini' infections.

Phylogenetics of family Enterobacteriaceae and proposal to reclassify Escherichia hermannii and Salmonella subterranea as Atlantibacter hermannii and Atlantibacter subterranea gen. nov., comb. nov.

Multilocus sequence analysis based on hypervariable housekeeping proteins was utilized to differentiate closely related species in the family Enterobacteriaceae. Of 150 housekeeping proteins, the top 10 hypervariable proteins were selected and concatenated to obtain distance data. Distances between concatenated proteins within the family were 0.9-41.2%, whereas the 16S rRNA and atpD-gyrB-infB-rpoB concatenated sequence (4MLSA) distances were 0.8-6.0% and 0.9-22.1%, respectively. These data indicate that phylogenetic analysis by concatenation of hypervariable proteins is a powerful tool for discriminating species in the family Enterobacteriaceae. To confirm the discriminatory power of the 10 chosen concatenated hypervariable proteins (C10HKP), phylogenetic trees based on C10HKP, 4MLSA, and the 16S rRNA gene were constructed. Comparison of average bootstrap values among C10HKP, 4MLSA and 16S rRNA genes indicated that the C10HKP tree was the most reliable. Location via the C10HKP tree was consistent with existing assignments for almost all species in the family Enterobacteriaceae. However, the C10HKP tree suggested that several species (including Enterobacter massiliensis, Escherichia vulneris, Escherichia hermannii, and Salmonella subterranea) should be reassigned to different clusters than those defined in previous analyses. Furthermore, E. hermannii and S. subterranea appeared to fall onto a branch independent from those occupied by the other Enterobacteriaceae. Therefore, we propose Atlantibacter gen. nov., such that E. hermannii and S. subterranea would be transferred to genus Atlantibacter as Atlantibacter hermannii, comb. nov. and Atlantibacter subterranea. comb. nov., respectively.

Development of a rapid diagnostic method for identification of Staphylococcus aureus and antimicrobial resistance in positive blood culture bottles using a PCR-DNA-chromatography method.

Blood culturing and the rapid reporting of results are essential for infectious disease clinics to obtain bacterial information that can affect patient prognosis. When gram-positive coccoid cells are observed in blood culture bottles, it is important to determine whether the strain is Staphylococcus aureus and whether the strain has resistance genes, such as mecA and blaZ, for proper antibiotic selection. Previous work led to the development of a PCR method that is useful for rapid identification of bacterial species and antimicrobial susceptibility. However, that method has not yet been adopted in community hospitals due to the high cost and methodological complexity. We report here the development of a quick PCR and DNA-chromatography test, based on single-tag hybridization chromatography, that permits detection of S. aureus and the mecA and blaZ genes; results can be obtained within 1 h for positive blood culture bottles. We evaluated this method using 42 clinical isolates. Detection of S. aureus and the resistance genes by the PCR-DNA-chromatography method was compared with that obtained via the conventional identification method and actual antimicrobial susceptibility testing. Our method had a sensitivity of 97.0% and a specificity of 100% for the identification of the bacterial species. For the detection of the mecA gene of S. aureus, the sensitivity was 100% and the specificity was 95.2%. For the detection of the blaZ gene of S. aureus, the sensitivity was 100% and the specificity was 88.9%. The speed and simplicity of this PCR-DNA-chromatography method suggest that our method will facilitate rapid diagnoses.

Remarkable increase in fluoroquinolone-resistant Mycoplasma genitalium in Japan.

OBJECTIVES: We determined the prevalence of macrolide and fluoroquinolone resistance-associated mutations in Mycoplasma genitalium DNA specimens from men with non-gonococcal urethritis (NGU) and analysed their effects on antibiotic treatments of M. genitalium infections. METHODS: In this retrospective study, we examined antibiotic resistance-associated mutations in the 23S rRNA, gyrA and parC genes of M. genitalium and the association of the mutations with microbiological outcomes of antibiotic treatments in men with M. genitalium-positive NGU. RESULTS: No macrolide resistance-associated mutations in the 23S rRNA gene were observed in 27 M. genitalium DNA specimens in 2011 and in 24 in 2012. However, 5 of 17 in 2013 had 23S rRNA mutations. Three of 15 in 2011, 6 of 19 in 2012 and 8 of 17 in 2013 had fluoroquinolone resistance-associated alterations in ParC. Three in 2013 had both the antibiotic resistance-associated alterations coincidentally. In two men with M. genitalium harbouring 23S rRNA mutations, the mycoplasma persisted after treatment with a regimen of 2 g of extended-release azithromycin (AZM-SR) once daily for 1 day. All nine men with mycoplasma harbouring ParC alterations were microbiologically cured with a regimen of 100 mg of sitafloxacin twice daily for 7 days. CONCLUSIONS: Macrolide- or fluoroquinolone-resistant M. genitalium appears to be increasing, and the increase in fluoroquinolone-resistant mycoplasmas is especially remarkable in Japan. Mycoplasmas harbouring 23S rRNA mutations would be resistant to the AZM-SR regimen, but those harbouring ParC alterations would still be susceptible to the sitafloxacin regimen.

Comparative analysis of MRSA strains isolated from cases of mupirocin ointment treatment in which eradication was successful and in which eradication failed.

Nasal decolonization in methicillin-resistant Staphylococcus aureus (MRSA) carriers using mupirocin (MUP) is a strategy that complements barrier precautions and contact isolation. However, eradication failure cases have been observed despite isolates being susceptible to MUP. This would suggest that the minimum inhibitory concentration (MIC) alone is not the only determinant of successful eradication. In this study, we undertook a comparative analysis of MRSA isolates from cases of successful and unsuccessful MUP-eradication treatment. The analyses we carried out were: determination of mupirocin MICs, sequencing of the isoleucyl-tRNA synthetase (ileS) gene, staphylococcal cassette chromosome mec typing, and the assessment of slime production. MICs for all 14 of the successful nasal decolonization cases showed susceptibility to MUP, whereas 21 (87.5 %) of the 24 unsuccessful cases were MUP-susceptible, with low-level resistance seen in 3 (12.5 %) strains. In the analysis of mutations in the ileS gene, one strain with an MIC of 4 µg/ml exhibited a G1778A point mutation that has not been previously reported. In the 14 successful nasal decolonization cases, only 1 strain (7.1 %) was an MRSA slime-producer, compared with 19 (79.7 %) of the 24 MRSA strains that could not be eradicated after MUP treatment (p < 0.05). For the eradication of MRSA by MUP, it is possible that slime may affect drug penetration. In conclusion, slime production was the only significant difference between isolates recovered from successful and unsuccessful eradication cases.

Two cases of bacteremia caused by Leptotrichia trevisanii in patients with febrile neutropenia.

We present two cases of bacteremia caused by Leptotrichia trevisanii: a 12-year-old girl with recurrent myeloid leukemia of the mandible and a 66-year-old man with esophageal carcinoma. As this filamentous bacillus showed indefinite Gram staining and the identification based on biochemical enzymatic reactions was not definitive, identification required 16s rRNA analysis. For this organism, drug sensitivity testing showed susceptiblity to each ß-lactam antibiotics and clindamycin, but resistance to fluoroquinolone and erythromycin. This filamentous bacillus needs careful identification and appropriate antibiotic treatment.

Milk and dairy product intakes, intestinal bacteria, and respiratory infections in children of elementary school age and older in Japan.

OBJECTIVES: The aim of this study was to examine the associations between milk and dairy product intakes, intestinal bacteria, and respiratory infections in children of elementary school age and older in Japan. METHODS: We conducted cross-sectional surveys each year from 2013 to 2015 for grades 2, 5, and 8 students of an elementary and junior high school (n = 1020). Exclusion owing to ineligibility regarding data on dietary intake, respiratory infections, and intestinal bacteria led to 922 participants for the analyses. Dietary intake was assessed with a self-administered food frequency questionnaire. Respiratory infections occurring ≥ 4 episodes over the past year were determined based on the caregivers' reports. Intestinal bacteria (species and counts) were analyzed with real-time polymerase chain reaction. Logistic regression models were used to estimate the odds ratios (ORs) and 95% CIs. RESULTS: The odds of ≥ 4 respiratory infection episodes decreased with higher milk intake after adjusting for potential confounders, and the ORs (95% CIs) for the second and third tertile categories, compared with the first tertile category, were 0.91 (0.58-1.42) and 0.48 (0.29-0.77), respectively (P for trend = 0.001). A decreasing trend in the ORs for lactic acid drink intake was observed only in those with a low count of intestinal Faecalibacterium prausnitzii. CONCLUSIONS: We found that higher milk intake was inversely associated with respiratory infections in children older than preschool age. Higher lactic acid drink intake could be inversely associated only in children with a low F. prausnitzii count in the intestine.

Lactobacillus acidophilus strain L-92 induces CD4(+)CD25(+)Foxp3(+) regulatory T cells and suppresses allergic contact dermatitis.

The anti-allergic mechanism of heat-killed Lactobacillus acidophilus strain L-92 has not been fully investigated. Recent studies have reported that CD4(+)CD25(+)Foxp3(+) (forkhead box P3) T regulatory (Treg) cells play important roles in controlling allergic diseases. Hence, we examined the effect of orally administered L-92 on CD4(+)CD25(+)Foxp3(+) cell populations. BALB/c mice were supplemented daily with L-92 by gavage for 5 weeks. 2,4-Dinitrofluorobenzene (DNFB) was used to induce allergic contact dermatitis (ACD) in mice. Fluorescent-activated cell sorter (FACS) analysis was used to determine CD4(+)CD25(+)Foxp3(+) T cell populations in spleen and cervical lymph nodes (CLN). Interleukin-10 (IL-10), transforming growth factor-ß (TGF-ß), and Foxp3 mRNA expressions in mouse ear skin were investigated by real-time reverse transcription-polymerase chain reaction (RT-PCR). The percentage of CD4(+)CD25(+)Foxp3(+) T cell populations were significantly increased in both spleen and CLN of L-92-fed group than vehicle and control. In addition, L-92 produced higher levels of Foxp3, IL-10 and TGF-ß compared to control mice. These results suggest that L-92 can up-regulate the number of Treg cells to suppress the progression of DNFB-induced contact dermatitis in mice.

Nocardia elegans infection involving purulent arthritis in humans.

Nocardia elegans infection in humans is rare and is predominantly associated with pulmonary infections. We describe the first case of N. elegans infection associated with purulent arthritis in humans. The patient was a 66-year-old woman without underlying disease. She had swelling in her left ankle that was increasing in size, but it did not cause the patient substantial pain. Punctual discharge was collected for Gram staining and Kinyoun's acid-fast staining. The results of microscopic findings were suggestive of the genus Nocardia. The 16S rRNA sequence of the isolate was completely identical (100%) with that of N. elegans, indicating that the isolate was N. elegans. All the previously reported 4 cases of N. elegans infection in humans were associated with respiratory infections; we present the first case of the infection involving purulent arthritis.

Comparative molecular and microbiological diagnosis of 19 infective endocarditis cases in which causative microbes were identified by PCR-based DNA sequencing from the excised heart valves.

Infective endocarditis (IE) is traditionally diagnosed by microbiological analysis of blood cultures, following which therapeutic antibiotics are chosen based on antimicrobial sensitivity tests. However, such conventional techniques do not always lead to an accurate etiological diagnosis. Recently, PCR analysis of the 16S rRNA gene has been employed to identify organisms isolated from excised heart valves. In this study, we analyzed 19 valve samples from patients with confirmed IE, as identified by Duke's criteria. Using broad-range PCR amplification, followed by direct gene sequencing, pathological agents were identified in all samples. Although blood cultures yielded negative results in 4 cases, PCR analysis of valve samples showed positive identification of causative organisms. In 3 cases, there was a difference between blood culture and PCR in identification of pathological agents, which are likely to be misidentified by the conventional method based on the phenotypic database. Postoperative antibiotics were chosen considering the severity of lesions and the results of PCR, Gram staining, and valve cultures. All patients were cured without relapse. The broad-range PCR method was therefore beneficial for the management of IE because it enabled us to identify pathogens directly from the site of infection, even organisms that were difficult to culture or likely to be misidentified by the conventional culture method. Identification of the agents provided precise knowledge of the microbiological spectrum involved in the cases of IE.

Mycobacterium shinjukuense sp. nov., a slowly growing, non-chromogenic species isolated from human clinical specimens.

Seven isolates of a slowly growing, non-chromogenic Mycobacterium species were obtained from sputum and bronchial lavage fluid samples from elderly patients in different regions of Japan. These isolates were distinguished from related non-tuberculous species by colony morphology, positive results for Tween hydrolysis, catalase at 68 °C, nitrate reductase and pyrazinamidase and negative results for semi-quantitative catalase, urease and arylsulfatase. The mycolic acid pattern obtained by HPLC revealed a single cluster of late-eluting mycolic acids similar to but different from those of Mycobacterium malmoense ATCC 29571(T). The 16S rRNA gene, 16S-23S internal transcribed spacer (ITS), rpoB and hsp65 sequences were unique in comparison with those of other mycobacteria. Comparison of 16S rRNA gene sequences showed that the isolates were most closely related to Mycobacterium tuberculosis H37Rv(T) (21 base differences in 1508 bp; 98.6 % 16S rRNA gene sequence similarity). A representative strain, GTC 2738(T), showed 91.9 % rpoB sequence similarity with Mycobacterium marinum strain M, 95 % hsp65 sequence similarity with Mycobacterium kansasii CIP 104589(T) and 81.1 % 16S-23S ITS sequence similarity with Mycobacterium gordonae ATCC 14470(T). Phylogenetic analysis of concatenated sequences of the 16S rRNA, rpoB and hsp65 genes showed that strain GTC 2738(T) was located on a distinct clade adjacent to M. tuberculosis, M. ulcerans and M. marinum, with bootstrap values of 81 %. DNA-DNA hybridization demonstrated less than 70 % reassociation with type strains of genetically related species and supported the novel species status of the isolates. On the basis of this evidence, a novel species with the name Mycobacterium shinjukuense sp. nov. is proposed. The type strain, isolated from a sputum sample, is strain GTC 2738(T)( = JCM 14233(T) = CCUG 53584(T)).

[A case of infective Aerococcus urinae endocarditis successfully treated by aortic valve replacement].

Aerococcus urinae is a endocarditis rare causative organism with low virulene. We report an A. urinae endocarditis case treated by aortic valve replacement. An 80-year-old woman hospitalized for urinary tract infection and hydronephrosis due to three-week renal calculi. Blood culture on admission isolated Streptococcus acidominimus. During the course, she was transferred to our care for surgical intervention after developing congestive heart failure due to severe aortic regurgitation. Echocardiographic findings indicated infective endocarditis. She underwent aortic valve replacement, and gram staining of the resected valve tissue showed gram-positive cocci, although valve culture was negative. PCR amplification and DNA sequencing using the valve material matched an A. urinae sequence. The woman recovered and was discharged six weeks after antibiotic treatment.

Molecular epidemiological analysis of methicillin-resistant staphylococci in a neonatal intensive care unit.

To investigate the contamination by and transmission of MRSA/MRCNS in the old NICU of Hospital A and the relocated NICU (new NICU), we isolated and evaluated staphylococci from nurses' palms, towels under the heads of neonates, infant incubators (including portholes and infant covers), and room air. Detection rates of MRSA/MRCNS isolated from different sample locations were 52.6% in the old NICU and 53.4% in the new NICU, which demonstrates that the nurses' palms in both the old and new NICUs and indoor environment were contaminated with MRSA/MRCNS. In the old NICU, numerous MRSA and MRCNS strains (Log 3.17 ± 0.19 cfu/10 cm²) were identified from towels, and the implementation of improvement plans resulted in a decrease in the number of MRSA/MRCNS isolates (Log 1.95 ± 0.57 cfu/10cm²) from the towels used in the new NICU. A homology study of MRSA/MRCNS strains by PFGE DNA restriction patterns identified genotypes that showed similar patterns in the nurses' palms, towels, infant incubators, and room air.

First case of bloodstream infection caused by Rhodococcus erythropolis.

We describe the first case of bloodstream infection caused by Rhodococcus erythropolis. The identification was performed using 16S rRNA sequencing. This case illustrates that non-equi Rhodococcus infections may be underdiagnosed due to difficulties in identification in the routine clinical microbiology laboratory.

First report of acute cholecystitis with sepsis caused by Cellulomonas denverensis.

Cellulomonas denverensis is a small and thin gram-positive rod-shaped bacterium that was proposed as a new species in 2005. Here we report a female case of acute cholecystitis and sepsis in which C. denverensis was determined to be causative.

Failure to detect Mycoplasma genitalium in the pharynges of female sex workers in Japan.

To determine the prevalence of genital mycoplasmas and ureaplasmas in the pharynges of Japanese female sex workers practicing fellatio on their clients, vaginal swabs and throat washings were collected from 403 female sex workers attending a clinic in Kyoto, Japan, for regular screening of gonococcal and chlamydial infections. Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum in vaginal and throat specimens were tested by nucleic acid amplification tests. The prevalence of N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, U. parvum, and U. urealyticum in the genitals was 1.7%, 7.2%, 1.7%, 19.6%, 40.4%, and 10.2%, respectively, whereas their prevalence in the pharynges was 4.0%, 2.0%, 0%, 1.2%, 0.2%, and 0.7%, respectively. Gonococcal infection in the pharynx was significantly associated with gonococcal infection in the genitals. Chlamydial infection in the pharynx was also significantly associated with chlamydial infection in the genitals. M. hominis, U. parvum, and U. urealyticum were all detected in vaginal swabs and in throat washings; however, M. genitalium was detected in vaginal swabs but not in throat washings. For each of these genital mycoplasmas and ureaplasmas, a positive test result in the pharynx was not significantly associated with a positive result in the genitals. M. hominis, U. parvum, and U. urealyticum were detected in throat washings, but M. genitalium was not. These findings do not necessarily rule out the transmission of M. genitalium from the pharynx to the urethra by orogenital sex.

Expression and purification of 30 kilodalton protein antigen of Ara- Burkholderia pseudomallei.

The 30 kDa protein of B. pseudomallei is found in virulent Ara- but not avirulent Ara+ strain. The gene was cloned in Escherichia coli JM105 employing pInIII-C2 vector. The open reading frame was 870 nucleotides with a guanine plus cytosine content of 69.9%. Arginine was the most abundant amino acid in the protein, having a PI of 12.65. Nucleotide sequence of the gene was 96% identical to B. pseudomallei 1710b chromosome II sequence CP000125.1, encoding an oxidoreductase of the short chain dehydrogenase/reductase family. The 30 kDa antigen was expressed as a maltose-fusion protein with a yield of 5.25 mg/l of bacterial culture.

First case report of sepsis caused by Mycobacterium wolinskyi in chronic myelogenous leukemia.

Infections caused by Mycobacterium wolinskyi have rarely been reported, and essentially all were cellulitis and/or osteomyelitis related with traumatic event or surgical wound. Here, we present the 1st case of septic complication due to this organism in a patient with chronic myelogenous leukemia of the 1st but late chronic phase.

Corynebacterium ulcerans infection of the lung mimicking the histology of Churg-Strauss syndrome.

We report the first case of pulmonary Corynebacterium ulcerans infection mimicking Churg-Strauss syndrome (CSS). Productive cough, fever, general fatigue, and weight loss developed in a 50-year-old man. Laboratory data revealed prominent eosinophilia and elevated serum IgE. On chest images, multiple nodules and cavities were predominantly detected in the right lung. Histopathologic examination showed necrotizing granulomas and vasculitis with massive eosinophilic infiltration identical to the findings seen in CSS; however, clusters of Gram-positive, coryneform rods were observed in the alveolar spaces. A toxigenic strain of C ulcerans was isolated from lung tissue. The patient was treated with antibiotics, and a favorable clinical course ensued.