Genetic Determinants of Hydrogen Sulfide Biosynthesis in Fusobacterium nucleatum Are Required for Bacterial Fitness, Antibiotic Sensitivity, and Virulence.

The Gram-negative anaerobe Fusobacterium nucleatum is a major producer of hydrogen sulfide (H2S), a volatile sulfur compound that causes halitosis. Here, we dissected the genetic determinants of H2S production and its role in bacterial fitness and virulence in this important member of the oral microbiome. F. nucleatum possesses four enzymes, CysK1, CysK2, Hly, and MegL, that presumably metabolize l-cysteine to H2S, and CysK1 was previously shown to account for most H2S production in vitro, based on correlations of enzymatic activities with gene expression at mid-log phase. Our molecular studies showed that cysK1 and megL were highly expressed at the late exponential growth phase, concomitant with high-level H2S production, while the expression levels of the other genes remained substantially lower during all growth phases. Although the genetic deletion of cysK1 without supplementation with a CysK1-catalyzed product, lanthionine, caused cell death, the conditional ΔcysK1 mutant and a mutant lacking hly were highly proficient in H2S production. In contrast, a mutant devoid of megL showed drastically reduced H2S production, and a cysK2 mutant showed only minor deficiencies. Intriguingly, the exposure of these mutants to various antibiotics revealed that only the megL mutant displayed altered susceptibility compared to the parental strain: partial sensitivity to nalidixic acid and resistance to kanamycin. Most significantly, the megL mutant was attenuated in virulence in a mouse model of preterm birth, with considerable defects in the spread to amniotic fluid and the colonization of the placenta and fetus. Evidently, the l-methionine γ-lyase MegL is a major H2S-producing enzyme in fusobacterial cells that significantly contributes to fusobacterial virulence and antibiotic susceptibility. IMPORTANCE Fusobacterium nucleatum is a key commensal anaerobe of the human oral cavity that plays a significant role in oral biofilm development and contributes to additional pathologies at extraoral sites, such as promoting preterm birth and colorectal cancer. Although F. nucleatum is known as a major producer of hydrogen sulfide (H2S), its genetic determinants and physiological functions are not well understood. By a combination of bacterial genetics, biochemical methods, and in vivo models of infection, here, we demonstrate that the l-methionine γ-lyase MegL not only is a major H2S-producing enzyme of F. nucleatum but also significantly contributes to the antibiotic susceptibility and virulence of this organism.

A strain of Streptomyces sp. isolated from rhizospheric soil of Crataegus oxycantha producing nalidixic acid, a synthetic antibiotic.

AIM: Exploration of microbes isolated from rhizospheric soil of Crataegus oxycantha for bioactive natural products. METHODS AND RESULTS: A strain of Streptomyces sp. (C-7) was isolated from rhizospheric soil of C. oxycantha. The 16S rRNA gene sequence of strain C-7 displayed 99% sequence similarity with different Streptomyces species. The highest score was displayed for Streptomyces sp. strain Chy2-8 followed by Streptomyces violarus strain NBRC13104 and Streptomyces arenae strain ISP5293. The position of C-7 in the phylogenetic tree suggested uniqueness of the strain. Nalidixic acid (1), a quinolone antibiotic, was isolated from Streptomyces sp. strain (C-7) for the first time and characterized by NMR and chemically analysed. Compound 1 exhibited antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. The production of compound 1 was also validated by repeating fermentation of strain C-7 and compound isolation in a separate natural product laboratory with no prior information. Furthermore, Compound 1 showed a cytotoxic effect against human prostate cancer cell line PC3 with an IC50 11 µg ml-1 . CONCLUSION: To the best of our knowledge, this is the first report showing production of nalidixic acid naturally by a strain of Streptomyces sp. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we isolated a strain of Streptomyces sp. producing nalidixic acid, which was otherwise only obtained through chemical synthesis.

Modified Pseudomonas agar: new differential medium for the detection/enumeration of Pseudomonas aeruginosa in mineral water.

Pseudomonas aeruginosa has been implicated as a foodborne and waterborne pathogen and is now considered a primary infectious agent. In the present study, the survival of P. aeruginosa inoculated in mineral water was evaluated by drop counts on Pseudomonas Agar Base (PAB), PAB with CN supplement X107, PAB with cetrimide, PAB with nalidixic acid, and these media with added FeSO(4). Initial counts, before starvation, were the same in all media tested. Following this period, P. aeruginosa became sensitive to PAB with added cetrimide. The addition of FeSO(4) did not improve the recovery of stressed P. aeruginosa but gave colonies a typical dark brown colour being easily differentiated from other species that can grow at 42 degrees C. The modified Pseudomonas agar medium was also tested with several P. aeruginosa strains, other species of Pseudomonas, and other genera. Only P. aeruginosa strains (pyocyanin positive) produced the typical colonies. Our results demonstrate that Pseudomonas agar with ferrous sulphate, used for the differentiation of P. aeruginosa colonies, and nalidixic acid, used as an inhibitor of Gram-positive bacteria, might be a useful medium for the detection of injured P. aeruginosa in mineral water.

Distribution of quinoline carboxylic acid derivatives in the dog prostate.

The distribution in the prostate of norfloxacin and rosoxacin, both quinoline carboxylic acid derivatives, was investigated in an experimental dog model. The concentrations in prostatic interstitial fluid (PIF) and prostatic secretion (PS) of both antimicrobials were lower than the corresponding plasma (P) concentrations, as might be expected for acidic compounds. Norfloxacin achieved significantly higher PS/P and PIF/P ratios than rosoxacin following intravenous infusion; there were no differences between the two drugs in the various tissue/plasma ratios. The concentrations of both norfloxacin and rosoxacin in PIF, PS, and urine were in the minimal inhibitory range for most gram-negative pathogens causing bacterial prostatitis and urinary tract infections. Clinical trials may be warranted for both drugs.

[Gram negative bacillus septicemias. Results of treatment of 31 gram negative bacillus septicemias using intravenous nalidixic acid]. / Septicémies à bacilles Gram négatif. Résultats du traitement de 31 septicémies à bacilles Gram négatif per l'acide nalidixique intra-veineux

Enterobacteria multiresistant to usual antibiotics, often remain sensitive to nalidixic acid, sometimes as a result of synergy with polymixins. Use of the intravenous route makes it possible to obtain more than adequate serum levels. Pharmacological study has indicated appropriate doses in terms of renal function. Intravenous nalidixic acid has been used in the treatment of 31 cases of Gram negative septicaemia, with success in 22. These results are all the more significant given that the patients were all seriously ill, cultures having grown multiresistant organisms and that nalidixic acid was used only after other antibiotics had failed.