There have been few reports on the epidemiological analysis of environmental Leptospira isolates. This is probably because the isolation of leptospires from the environment was usually unsuccessful due to the overgrowth of contaminants and the slow growth of Leptospira. In this study, we collected a total of 88 samples of soil and water from three sites: Metro Manila and Nueva Ecija, Philippines (an area where Leptospira is now endemic), and Fukuoka, Japan (an area where Leptospira was once endemic). We succeeded in isolating Leptospira from 37 samples by using the novel combination of five antimicrobial agents reported in 2011. The frequencies of positive isolation of Leptospira in the Philippines and Japan were 40 and 46%, respectively. For Leptospira-positive samples, five colonies from each sample were isolated and analyzed by pulsed-field gel electrophoresis (PFGE). The isolates from each area showed their respective characteristics in phylogenetic trees based on the PFGE patterns. Some isolates were closely related to each other across borders. Based on 16S rRNA gene-based phylogenetic analysis, four isolates in Fukuoka were identified as a pathogenic species, L. alstonii; however, its virulence had been lost. One isolate from Nueva Ecija was identified as the intermediate pathogenic species Leptospira licerasiae. Most of the isolates from the environment belonged to nonpathogenic Leptospira species. We also investigated the strain variation among the isolates in a puddle over 5 months. We demonstrated, using PFGE analysis, that Leptospira survived in the wet soil on dry days and appeared in the surface water on rainy days. These results showed that the soil could be a reservoir of leptospires in the environment.

Streptococcus pyogenes strains can be divided into two classes, one capable and the other incapable of producing H 2 O 2 (M. Saito, S. Ohga, M. Endoh, H. Nakayama, Y. Mizunoe, T. Hara, and S. Yoshida, Microbiology 147: [2469][2470][2471][2472][2473][2474][2475][2476][2477] 2001). In the present study, this dichotomy was shown to parallel the presence or absence of H 2 O 2 -producing lactate oxidase activity in permeabilized cells. Both lactate oxidase activity and H 2 O 2 production under aerobic conditions were detectable only after glucose in the medium was exhausted. Thus, the glucose-repressible lactate oxidase is likely responsible for H 2 O 2 production in S. pyogenes. Of the other two potential H 2 O 2 -producing enzymes of this bacterium, NADH and ␣-glycerophosphate oxidase, only the former exhibited low but significant activity in either class of strains. This activity was independent of the growth phase, suggesting that the protein may serve in vivo as a subunit of the H 2 O 2 -scavenging enzyme NAD(P)H-linked alkylhydroperoxide reductase. The activity of lactate oxidase was associated with the membrane while that of NADH oxidase was in the soluble fraction, findings consistent with their respective physiological roles, i.e., the production and scavenging of H 2 O 2 . Analyses of fermentation end products revealed that the concentration of lactate initially increased with time and decreased on glucose exhaustion, while that of acetate increased during the culture. These results suggest that the lactate oxidase activity of H 2 O 2 -producing cells oxidizes lactate to pyruvate, which is in turn converted to acetate. This latter process proceeds presumably via acetyl coenzyme A and acetyl phosphate with formation of extra ATP.The gram-positive microorganism Streptococcus pyogenes is the causative agent of a variety of important human diseases. These include not only the direct consequences of infections such as pharyngitis, impetigo, cellulitis, necrotizing fasciitis, and toxic-shock-like syndrome but also secondary pathologies called poststreptococcal sequelae. The physiological features of S. pyogenes place it as a member of lactic acid bacteria, which are generally believed to be almost totally dependent for growth on the lactic mode of fermentation under both aerobic and anaerobic conditions.In a number of species of lactic acid bacteria, it has been known that cells growing aerobically produce and excrete copious amounts of H 2 O 2. In a previous study, it was found that S. pyogenes strains could be divided into two classes with respect to the production of H 2 O 2 , i.e., producers and nonproducers (18). However, the metabolic basis and biological significance of bacterial H 2 O 2 production are largely unexplored. The presence of H 2 O 2 -producing oxidases in lactic acid bacteria has been demonstrated, and the possibility of their involvement in this phenomenon has been suggested (6,14,15,21). Notably, a homology search against the S. pyogenes genome database revealed that putative H 2 O 2 -produc...

e Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. Most of the outbreaks of leptospirosis occur after floods caused by heavy rain in countries where Leptospira spp. are endemic. It has been believed that the overflow of seawater rarely causes outbreaks of leptospirosis because the leptospires are killed by salt water. On 8 November 2013, a storm surge caused by Super Typhoon Haiyan (Yolanda) inundated the entire coastal areas of Tacloban and Palo in Leyte, Philippines. The present study was carried out in order to determine whether the environmental leptospires in soil were able to survive after the storm surge in the affected areas. We collected 23 wet soil samples along the coastal areas of Tacloban and Palo 2 months after the storm surge. The samples were suspended in HEPES buffer, and the supernatants were cultured in liquid or semisolid Korthof's medium supplemented with five antimicrobial agents to inhibit the growth of contaminants. Leptospires were isolated from primary cultures of 22 out of 23 samples. The DNA of pathogenic Leptospira species was detected in 11 samples (47.8%) by analysis of flaB by nested PCR. Eventually, two pathogenic Leptospira strains were isolated and showed the highest 16S rRNA gene sequence similarity to Leptospira kmetyi. When these isolates were experimentally mixed with soil, they were found to survive in seawater for 4 days. These results show the possibility that leptospires living in soil survived after the storm surge. Our findings may serve as a warning that when seawater inundates the land during a storm surge or a tsunami, an outbreak of leptospirosis could occur in the disaster-stricken area.

A novel combination of antimicrobial agents (sulfamethoxazole, 40 μg/mL; trimethoprim, 20 μg/mL; amphotericin B, 5 μg/mL; fosfomycin, 400 μg/mL; and 5-fluorouracil, 100 μg/mL) was developed for selective isolation of leptospires from contaminated samples. The growth of 16 microorganisms considered as possible contaminants during isolation of Leptospira were inhibited by this antimicrobial cocktail. In contrast, the growth of a smaller inoculum (10 1 cells per mL) of 25 Leptospira strains (representing 18 serovars/serogroups of 5 species) was not suppressed by this antimicrobial combination. This cocktail, after being incorporated into Leptospira growth medium (Korthof 's), successfully detected leptospires in environmental soil and water. Based on the results, this selective medium has the potential to meet the existing need for an effective selective medium for the isolation of Leptospira.

Streptococcus pneumoniae was shown to possess lactate oxidase in addition to well-documented pyruvate oxidase. The activities of both H 2 O 2 -forming oxidases in wild-type cultures were detectable even in the early exponential phase of growth and attained the highest levels in the early stationary phase. For each of these oxidases, a defective mutant was constructed and compared to the parent regarding the dynamics of pyruvate and lactate in aerobic cultures. The results obtained indicated that the energy-yielding metabolism in the wild type could be best described by the following scheme. (i) As long as glucose is available, approximately one-fourth of the pyruvate formed is converted to acetate by the sequential action of pyruvate oxidase and acetate kinase with acquisition of additional ATP; (ii) the rest of the pyruvate is reduced by lactate dehydrogenase to form lactate, with partial achievement of redox balance; (iii) the lactate is oxidized by lactate oxidase back to pyruvate, which is converted to acetate as described above; and (iv) the sequential reactions mentioned above continue to occur as long as lactate is present. As predicted by this model, exogenously added lactate was shown to increase the final growth yield in the presence of both oxidases.The gram-positive bacterium Streptococcus pneumoniae, also known as pneumococcus, is often a commensal resident of the human upper respiratory tract but also represents an important human pathogen, causing invasive diseases such as pneumonia, otitis media, and meningitis (18,20,22). Recently, a sharp rise in the incidence of drug resistance among clinical isolates of S. pneumoniae has been posing serious problems (13).A member of lactic acid bacteria, S. pneumoniae is aerotolerant but lacks the cytochromes necessary for aerobic respiration. Under anaerobic conditions, it is believed to be totally dependent on homolactic fermentation for the acquisition of energy required for growth, in which glucose is metabolized to pyruvate and then to the final product lactate. Under aerobiosis, however, pyruvate is also known to be converted to acetate, with acetyl phosphate being the intermediate capable of phosphorylating ADP to yield ATP by the action of acetate kinase (30). The H 2 O 2 -forming flavoprotein pyruvate oxidase (EC 1.2.3.3, the product of the spxB gene), which catalyzes the formation of acetyl phosphate, CO 2 and H 2 O 2 from pyruvate, orthophosphate, and O 2 , has been shown to be involved in this pathway and also to account for most of the H 2 O 2 produced by aerobically growing S. pneumoniae cells (23, 30). Massive production of H 2 O 2 has been a well-known hallmark of this bacterium since the time of Oswald Avery (2, 19), usually necessitating the addition of catalase to the culture medium to obtain full growth under aerobic conditions. S. pneumoniae possesses another H 2 O 2 -forming flavoprotein, L-lactate oxidase (the product of the lox gene; formerly EC 1.1.3.2 but now sharing the EC number 1.13.12.4 with lactate monooxigenase) catalyzing the ...