Trend of Invasive Streptococcus agalactiae at Tertiary Care Hospital in Japan

Streptococcus agalactiae causes various severe infectious diseases such as sepsis, meningitis, and streptococcal toxic shock-like syndrome. Though Streptococcus agalactiae infection has been increasing recently, the comprehensive characteristic investigation of invasive Streptococcus agalactiae isolated in tertiary care hospitals has not been nearly performed in Japan. In this study, we investigated the clinical characteristics and antimicrobial susceptible patterns of 88 Streptococcus agalactiae isolated at two tertiary care hospitals during 2009-2015 in Japan. There was no significant differences between genders in our study. Two-third Streptococcus agalactiae were isolated from over age 60. Total mortality rate was 19% and invasive Streptococcus agalactiae-associated death cases have occurred every year after 2011. All Streptococcus agalactiae were completely susceptible toampicillin. Total non-susceptible rates of erythromycin, minocycline, levofloxacin and trimethoprim-sulfamethoxazole in this study were approximately 30%, 44%, 37%, and 7%, respectively. Our results suggest the need for continuous antimicrobial susceptibility survey of Streptococcus agalactiae.

Trend of Respiratory Tract-Associated Streptococcus dysgalactiae Subsp. Equisimilius at General Japanese Hospital from 2013 to 2015

Streptococcus dysgalactiae subsp. equisimilius are pathogenic bacteria which cause various infectious diseases from acute pharyngitis to streptococcal toxic shock syndrome. Although Streptococcus dysgalactiae subsp. equisimilius infection has been increasing recently, the recent characteristic investigation of respiratory tract associated Streptococcus dysgalactiae subsp. equisimilius has not been performed in Japan. In this study, we analyzed the relationship between respiratory tract and norespiratory tract associated Streptococcus dysgalactiae subsp. equisimilius by investigating the recent clinical characteristics and antimicrobial susceptible patterns of 98Streptococcus dysgalactiae subsp. equisimilius isolated during 2013-2015 in Japan. There was significant difference of gender between respiratory tract and no respiratory tract associated Streptococcus dysgalactiae subsp. equisimilius. No Streptococcus dysgalactiae subsp. equisimilius from no respiratory tract were found under 18 years-old patients. The prevalence of clindamycin non susceptible Streptococcus dysgalactiae subsp. equisimilius isolates from respiratory tract was significant greater than those from non-respiratory tract. We also analyzed the relationship between respiratory tract associated Streptococcus dysgalactiae subsp. equisimilius and Streptococcus pyogenes in 2014.There was no significant difference of gender between Streptococcus dysgalactiae subsp. equisimilius and Streptococcus pyogenes. The numbers of Streptococcus pyogenes-infected patients were significantly greater than those of Streptococcus dysgalactiae subsp. equisimilius-infected patients in children from 1 to 18 years old. From antimicrobial resistance, we found that the numbers of clarithromycin resistant Streptococcus pyogenes were greater than those of clarithromycin resistant Streptococcus dysgalactiae subsp. equisimilius among respiratory tract isolation. Our results suggest that the clinical and antimicrobial susceptible patterns are useful for screening the respiratory tract associated Streptococcus dysgalactiae subsp. equisimilius infection.

Comparative Study of Urosepsis-Associated Escherichia coli in Tertiary Care University Hospital in the Central Region of Japan from 2008 to 2011

Escherichia coli infection is important cause of morbidity and mortality. Urosepsis is most commonly caused by Escherichia coli. It is generally reported to have low mortality rates and favorable outcomes compared with sepsis induced in other organ/ system or tissues. This study was conducted to find out the clinical characteristics of urosepsis-associated Escherichia coli isolates at tertiary care university hospital in the central region of Japan from 2008 to 2011. Escherichia coli was identified by standard laboratory procedure. Antimicrobial susceptibility testing was performed by micro dilution assay according to CLSI recommendation. We analyzed the relationship between medical records appended to clinical species and bacterial data by using a statistical method. Of one hundred five Escherichia coli, fifty-three were from urosepsis and fifty-two were from other disease. The ratio of male to female from urosepsis tended to be lower than that from others. Forty-two isolates were from outpatient and sixty-three were from inpatient. The ratio of inpatient to outpatient from urosepsis was significant lower than that from others. With respect to age, the numbers of elder patients were higher than those of others. Most of the Escherichia coli isolates were from the emergency room followed by urology, and general medicine. The ratio of urosepsis to no urosepsis at urology department was significant higher than that at other departments. With respect to antimicrobial susceptible pattern, the ratio of urosepsis to no urosepsis about only aztreonam resistant was significant higher than that about other antibiotics. We need enough attention to be paid to urosepsis, especially female and elder patients.

Clinical Characteristics of Haemophilus influenzae at General Hospital in the Central Region of Japan

Haemophilus influenzae is an important pathogen that caused several infection diseases, such as sinusitis, otitis media, sepsis, and meningitis. This study was conducted to find out the prevalence and antimicrobial susceptibility pattern of Haemophilus influenzae isolates at general hospital in the central region of Japan from December 2015 to January 2016. Haemophilus influenzae was identified by standard laboratory procedure. Antimicrobial susceptibility testing was performed by micro dilution assay according to CLSI recommendation. One hundred ninety-one Haemophilus influenzae were isolated, among which 95 (49.7%) were from male and 96 (50.3%) were from female. The age incidence of (0) years, (≤2) years, (≤5) years, and (6≤) years groups were 22(11.5%), 92(48.2%), 61(31.9%), and 16(8.4%), respectively. Positive samples were received mostly from the nasal discharge (177/92.7%), sputum (6/3.1%), tonsillar (6/3.1%), and pharynx (2/1.0%). Ceftriaxone was the most active antibiotics with 100% susceptible rates, followed by ciprofloxacin (99.5%) and minocycline (99%) in our study. Furthermore, we categorized four patterns: beta lactamase-negative ampicillin-sensitive strain (BLNAS), beta lactamase-negative ampicillin-re- sistant strain (BLNAR), beta lactamase-positive ampicillin resistant strain (BLPAR), and beta lactamase-positive amoxicillin-clavulanic acid-resistant strain (BLPACR) from those ampicillin susceptible results. The numbers of female were significant greater than those of male in BLPAR (p = 0.0336). With respect to antimicrobial susceptible pattern, there was no minocycline and piperacillin resistant strain in both BLNAS and BLNAR (p

Interleukin-17 levels in Helicobacter pylori-infected gastric mucosa and pathologic sequelae of colonization

AIM： To determine the role of interleukin （IL）-17 in gastric ulcerogenesis. METHODS： Thirty-six gastric ulcer （GU） patients and 29 non-ulcer （NU） patients were enrolled in this study. Mucosal biopsy samples were obtained from the gastric antrum and GU site during endoscopy. Samples were used in in situ stimulation for 48 h in the presence of 10 ug/mL phytohemagglutinin-P （PHA）, histological examination, and Helicobacter pylori（Hpylon） culture. IL-17 and IL-8 protein levels in culture supematants were assayed by ELISA. IL- 17 mRNA expression was analyzed by reverse transcriptasepolymerase chain reaction （RT-PCR）. Hpylori cagA and vacA status was assessed by reverse hybridization using a line probe assay （UPA）. IL-8 levels in culture supematants were assayed after AGS cells were co-cultured with Hpylori strain 26 695 or recombinant human （rh） IL-17. RESULTS： All 36 GU patients and 15 of 29 NU patients were found to be Hpy/or/-positive, while 14 NU patients were Hpylori-nogative. All 51 H pylori strains from both GU and NU patients were cagA- and vacAsl/ml-positive. Antral mucosal tissues from H pylori-positive patients contained significantly （H pylori-positive NU patients： median 467 pg/mg/protein, range 53-2 499; Hpylori negative NU patients： median 104 pg/mg/protein, range 16-312, P〈0.0005） higher levels of IL-17 than those from uninfected patients. IL-17 levels at the ulcer site were significantly （ulcer site： median 1 356 pcj/mg/protein, range 121-1 3730; antrum： median 761 pg/mg/protein, range 24-7 620, P〈0.005） higher than those at distant sites in the antrum. Biopsies from H pylori-positive GU and NU patients showed IL-17 mRNA expression in all samples whereas those from the antrum of the Hpylori-negativecontrols showed no detectable expression. A significant correlation was seen between IL-17 and IL-8 levels at each biopsy site （ulcer： r = 0.62,P〈0.0001; antrum： r = 0.61, P〈0.0001） in GU patients. RhIL-17 and Hpylori strain 26 695 each stimulated IL-8 production from AGS cells. CONCLUSION： IL-17 may play an important role in the inflammatory response to Hpyloricolonization, and may ultimately influence the outcome of H pylori-associated diseases that arise within the context of gastritis.

Cytomegalovirus infection in severe ulcerative colitis patients undergoing continuous intravenous cyclosporine treatment in Japan

AIM： TO investigate active cytomegalovirus （CMV） infection following the cydosporine A （CyA） treatment of steroid-refractory ulcerative colitis （UC）. METHODS： Twenty-three patients with severe UC not responding to steroid therapy （male 14, and female 9） enrolled at Nagoya University Hospital from 1999 to 2005. They received continuous intravenous infusion of CyA （average 4 mg/kg per day） for 1 mo. Serum and colonic biopsy samples were collected before CyA treatment and 4 d, 10 d, 20 d, and 30 d after treatment. Patients were evaluated for CMV by using serology （IgM antibody by ELISA）, quantitative real-time PCR for CMV DNA, and histopathological assessment of hematoxylin and eosin （HE）-stained colonic biopsies. CMV infection was indicated by positive results in any test. RESULTS： No patients had active CMV infection before CyA treatment. Eighteen of 23 UC patients treated with CyA were infected with active CMV （IgM antibody in 16/23 patients, 69.6%; CMV DNA in 18/23 patients, 78.2%; and inclusion bodies in 4/23 patients, 17.3%）. There was no difference in the active CMV-infection rate between males and females. Active CMV infection was observed after approximately 8 d of CyA treatment, leading to an exacerbation of colitis. Fifteen of these 18 patients with active CMV infection （83.3%） required surgical treatment because of severe deteriorating colitis. Treatment with ganciclovir rendered surgery avoidable in three patients. CONCLUSION： Our results suggest that active CMV infection in severe UC patients treated with CyA is associated with poor outcome. Further, ganciclovir is useful for treatment of CMV-associated UC after immunosuppressive therapy.

Analysis of Pyelonephritis-Associated Beta Hemolytic Streptococcus in Japan

Pyelonephritis is an inflammation of the kidney tissue, calyces, and renal pelvis. It is commonly caused by bacterial infection that has spread up the urinary tract. Common bacteria are Escherichia coli and Enterococcus faecalis. Beta hemolytic streptococci also cause pyelonephritis. Beta hemolytic streptococcus such as Streptococcus agalactiae, and S. dysgalactiae subsp. equisimilis, when grown on blood-agar, produce complete hemolysis around each colony, usually cause severe infections including many cases of cellulitis, pneumonia, meningitis and sepsis. However the analysis of pyelonephritis causing beta hemolytic streptococcus has seldom been investigated. In this study, we tried to clarify the relationship between beta hemolytic streptococcus and pyelonephritis patient in Japan. We investigated beta hemolytic streptococcus isolated from pyelonephritis patient at two tertiary care hospitals in the central region of Japan by analyzing the background and antimicrobial susceptibility of beta hemolytic streptococcus. We recovered total sixteen beta hemolytic streptococcus in nine years. In pyelonephritis patients, beta hemolytic streptococcus was isolated from 9 S. agalactiae and 7 S. dysgalactiae subsp. equisimilis, respectively. The numbers of streptococci from female patients were more than those from male patients. Bacteria were not isolated from under 30 year patients. All S. dysgalactiae subsp. equisimilis were isolated from 60-year-age patients. About one fifth of beta hemolytic streptococcus had erythromycin and minocycline-resistant ability. Four beta hemolytic streptococci that were all S. agalactiae, had levofloxacin-resistant ability. Only one S. agalactiae had trimethoprim-sulfamethoxazole-resistant ability. Ampicillin was effective against all beta hemolytic streptococci. In our results, beta hemolytic streptococcus caused pyelonephritis in aged people and the quarter of these bacteria had some antibiotics resistance.

Comparative Investigation of Alternative Negative Staining Reagents in Bacterial Morphological Study

Negative staining is an effective method that can be used for electron microscopic study to observe fine structural morphology without destruction of bacterial structure. Although uranium acetate is used worldwide as a general dyeing solution, it is extremely difficult to use it by a new purchase at a research institution because it falls under the nuclear regulation substance in Japan. Therefore, we examined alternative reagents for negative staining that could replace uranium acetate through bacterial observation with an electron microscope. Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pyogenes were examined by four stain reagents (phosphotungstic acid (PTA), EMstainer, TI blue, and uranium acetate). Pre cultured bacteria were stained with each stain reagents on a copper grid, washed with PBS, and observed with a transmission electron microscope. In the comparison between bacterial structures, the cell wall structure and bacterial flagella could be observed well in the order of PTA, EMstainer, and uranium acetate. With TI blue staining, flagella could be observed very poorly. In comparison between bacteria, gram negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, could be observed well as compared with gram positive cocci such as Staphylococcus aureus and Streptococcus pyogenes. The uranium acetate looked very coarse in background particles. Since crystals tend to precipitate, TI blue also required filtering, and electron beams were absorbed by the agglomerated crystals, and the frequency of electronic burning occurred high frequency. In this study, there was clear difference in the observation conditions depending on the type of bacteria and the kind of the staining reagents. Especially, it was confirmed that good negative staining features of Pseudomonas aeruginosa by electron microscope were obtained by PTA and EMstainer staining. These alternative reagents are considered to be a candidate for a negative staining.

Clinical Characteristics of Vaginal Discharge Associated Streptococcus pyogenes at General Japanese Hospital

Streptococcus pyogenes (S. pyogenes) is popular pathogen caused various infectious disease. This study was conducted to find out the recent clinical characteristics of S. pyogenes isolates from vaginal discharge at Japanese hospital. S. pyogenes was identified by standard laboratory procedure. Antimicrobial susceptibility testing was performed by microdilution assay according to CLSI recommendation. Total 61 S. pyogenes were isolated from vaginal discharge. The major age incidence among 0 - 10 years age group was 14 (23%), among 21 - 30 years age group, 7 (11.5%), among 31 - 40 years age group, 13 (21.3%), among 51 - 60 years age group, 8 (13.1%), and among 61 - 70 years age group, 11 (18%). The numbers of ciprofloxacin and clarithromycin no-susceptible S. pyogenes from 0 - 10, 31 - 40, and 61-70 years age group were higher than those from other groups. The numbers of clindamycin and minocycline no susceptible S. pyogenes from 0 - 10, and 21 - 40 years age group were higher than those from other group. Furthermore, our study revealed that clarithromycin was completely ineffective in 21 - 30 years age group (p S. pyogenes because the trend of infectious disease epidemiology is always changing drastically.

Clinical Characteristics of Invasive Methicillin-Resistant Staphylococcus aureus at General Hospital in the Central Region of Japan from July 2014 to June 2015

Methicillin-resistant Staphylococcus aureus is crucial pathogen caused severe invasive infection disease. This study was conducted to find out the prevalence and antimicrobial susceptibility pattern of invasive Methicillin-resistant Staphylococcus aureus isolates at general hospital in the central region of Japan from July 2014 to June 2015. Methicillin-resistant Staphylococcus aureus was identified by standard laboratory procedure. Antimicrobial susceptibility testing was performed by micro dilution assay according to CLSI recommendation. Invasive Methicillin-resistant Staphylococcus aureus disease was defined as isolation of bacteria from a normally sterile body site. One hundred seventy-one methicillin-resistant Staphylococcus aureus were isolated among which 95 (55.6%) were from inpatient and 76 (44.4%) were from outpatient. The age incidence of (0 - 1) years, (1 - 10) years, (11 - 40) years, (41 - 60) years and >60 years age groups were 18 (10.5%), 41 (24.0%), 15 (8.8%), 5 (2.9%), and 92 (53.8%) respectively. There was significant difference of age distribution between invasive and noninvasive disease in 0 - 1 years group and 11 - 40 years age group. Positive samples were received mostly from the pediatrics (56/32.7%), respiratory medicine (25/14.6%) and general medicine (25/14.6%). We also found the significant differences of department between invasive and noninvasive disease in pediatrics, dermatology, and surgery. Arbekacin, teicoplanin, and vancomycin were the most active antibiotics with 100% susceptible rates in our study. Our study revealed that erythromycin and gentamicin were more antimicrobial effective in invasive disease than in noninvasive disease significantly. Methicillin-resistant Staphylococcus aureus infection spreads worldwide easily and inadequate use of antibiotics contributes to uptake of their new antimicrobial resistance. Continuous antimicrobial surveys are need for guiding policy on the adequate use of antibiotics to reduce the morbidity and mortality.

Clinical Characteristics of Respiratory Tract-Associated Streptococcus pyogenes at General Japanese Hospital in 2014

Streptococcus pyogenesis most popularpathogen caused respiratory tract infection disease. This study was conducted to find out the recent clinical characteristics of Streptococcus pyogenes isolates from respiratory tract at Japanese hospital in 2014. Streptococcus pyogenes was identified by standard laboratory procedure. Antimicrobial susceptibility testing was performed by micro dilution assay according to CLSI recommendation. Respiratory tract isolates were defined as isolation from pharynx, nasal discharge, tonsillar, and sputum. Total one hundred sixty-one Streptococcus pyogenes were isolated among which 136 were from respiratory tract and 25 were from non- respiratory tract. Respiratory tract-associated Streptococcus pyogenes was isolated among which 102 were from male and 59 were from female. The age incidence among 0 - 1 years age group was 8, among 1 - 18 years age group, 131 (p < 0.01), among 19 - 64 years age group, 17 (p < 0.01), and in 65-years it was 5. Although we investigated the relationship between season and bacterial isolation, we did not find any significant differences between respiratory tract and no respiratory tract isolation. However, our study revealed that clarithromycin was less antimicrobial effective in respiratory tract disease than in no respiratory tract disease significantly (p < 0.01). Although several antibiotics such as penicillin are still effective against Streptococcus pyogenes, incidence of Streptococcus pyogenes infection is not decreasing worldwide. Our study suggests the need for continuous epidemiological surveillance of Streptococcus pyogenes.

Prevalence and Antimicrobial Susceptibility Pattern of Streptococcus pneumoniae at General Hospital in the Central Region of Japan from December 2013 to February 2014

Streptococcus pneumonia infection is important cause of morbidity and mortality. This study was conducted to find out the prevalence and antimicrobial susceptibility pattern of Streptococcus pneumoniae isolates at general hospitalin the central region of Japan from December 2013 to February 2014. Streptococcus pneumoniae was identified by standard laboratory procedure. Antimicrobial susceptibility testing was performed by micro dilution assay according to CLSI recommendation. One hundred fifty-three Streptococcus pneumoniae were isolated among which 80 (52.2%) were males and 73 (47.8%) were females. Nasal discharge (134%/87.6%) contributed more than other biological materials. The age incidence of (0 - 1) years, (1 - 10) years, (11 - 40) years, (41 - 60) years and >60 years age groups were 26 (17.0%), 110 (71.9%), 3 (2.0%), 10 (6.5%), and 4 (2.6%) respectively. Positive samples were received mostly from the pediatrics (137%/89.5%), respiratory medicine (12%/7.8%) and lowest from gastroenterology (1%/0.6%) and neurology (1/ 0.6%) department. Vancomycin and rifampicin were the most active antibiotics with 100% susceptibility rates. The next best were levofloxacin, penicillin G and ceftriaxone. Our study revealed that 82 Streptococcus pneumonia isolates had multidrug resistant ability (53.6%). Streptococcus pneumoniae infection spreads among community easily and inappropriate use of antibiotics contributes to their resistance. Continuous antimicrobial susceptible surveys are essential to guide policy on the adequate use of antibiotics to reduce the morbidity and mortality and reduce the emergency of antimicrobial resistance.

The Two-Component Sensor Protein CovS Affects Penicilling Susceptibility by Modulation of Cell-Wall Synthesis in Streptococcus pyogenes

In Streptococcus pyogenes, we have described the two-component signal transduction sensor and regulatory systems, CovR/S affect the antimicrobial susceptibility including penicillin G before. But the mechanism how two-component sensor protein CovS modulates penicillin G susceptibility has not been elucidated till date. This study aimed to determine how the CovS affected penicillin G susceptibility in Streptococcus pyogenes by northern blot analysis. At first, we investigated the covS mRNA expression under penicillin G induction. We found that the decrease of covS mRNA expression under Penicillin G stimulation. Next we investigated the expression of cell wall synthesis gene, pbp2a and glmM with use of covS knockout mutants from emm1 Streptococcus pyogenes strain 1529. We found that the cell-wall synthesis gene expression of the ?covS mutant strain were lower than that of the wild-type strain. Furthermore the expression of glmM mRNA gene was lower than the expression of pbp2a mRNA gene in the ?covS mutant strain. The covS-complemented strain almost restored the mRNA expression compared to covS mutant strain. The two-component sensor protein CovS affects the susceptibility to penicillin G in Streptococcus pyogenes by modulation of cell-wall synthesis.

LicT Modulates Biofilm Formation of Streptococcus pyogenes

Streptococcus pyogenes frequently causes purulent infections in humans. Biofilm formation is an important virulence property of S. pyogenes because of decreased susceptibility of bacteria to antibiotic treatment. Biofilm is composed of various types of matrix including glycocalyx which is an important exocellular matrix material related to bacterial sugar metabolism. A putative antiterminator protein, LicT (Spy0571), is one of the components of the glucose-independent β-gluco-side-specific phosphotransferase system (PTS). Although the PTS, a carbohydrate metabolic system, may play a role in biofilm formation, the relationship between LicT and biofilm formation has not yet been elucidated. Here, we evaluated whether LicT affected biofilm formation in modified chemically defined medium (CDMM) supplemented with glucose or β-glucoside:salicin. We created licT- and licT-complemented mutant strains from S. pyogenes 1529. Although the licT mutant strain tended to have higher growth rate than wild-typestrain in CDMM with glucose, it had a significant lower growth rate than the wild-type strain in CDMM with salicin. In addition, the licT mutant exhibited lower biofilm formation in CDMM containing salicin than the wild-type strain by 96 well plate analysis and confocal laser scanning microscopic analysis. Our results suggest that LicT plays an important role in biofilm formation of S. pyogenes.