Morphological change of coiled bacterium Spirosoma linguale with acquisition of ß-lactam resistance.

Spirosoma linguale is a gram-negative, coiled bacterium belonging to the family Cytophagaceae. Its coiled morphology is unique in contrast to closely related bacteria belonging to the genus Spirosoma, which have a short, rod-shaped morphology. The mechanisms that generate unique cell morphology are still enigmatic. In this study, using the Spirosoma linguale ATCC33905 strain, we isolated ß-lactam (cefoperazone and amoxicillin)-resistant clones. These clones showed two different cell morphological changes: relatively loosely curved cells or small, horseshoe-shaped cells. Whole-genome resequencing analysis revealed the genetic determinants of ß-lactam resistance and changes in cell morphology. The loose-curved clones commonly had mutations in Slin_5958 genes encoding glutamyl-tRNA amidotransferase B subunit, whereas the small, horseshoe-shaped clones commonly had mutations in either Slin_5165 or Slin_5509 encoding pyruvate dehydrogenase (PDH) components. Two clones, CFP1ESL11 and CFL5ESL4, which carried only one mutation in Slin_5958, showed almost perfectly straight, rod-shaped cells in the presence of amoxicillin. This result suggests that penicillin-binding proteins targeted by amoxicillin play an important role in the formation of a coiled morphology in this bacterium. In contrast, supplementation with acetate did not rescue the growth defect and abnormal cell size of the CFP5ESL9 strain, which carried only one mutation in Slin_5509. These results suggest that PDH is involved in cell-size maintenance in this bacterium.

Anditalea andensis ANESC-ST--An Alkaliphilic Halotolerant Bacterium Capable of Electricity Generation under Alkaline-Saline Conditions.

A great challenge in wastewater bioremediation is the sustained activity of viable microorganisms, which can contribute to the breakdown of waste contaminants, especially in alkaline pH conditions. Identification of extremophiles with bioremediation capability can improve the efficiency of wastewater treatment. Here, we report the discovery of an electrochemically active alkaliphilic halotolerant bacterium, Anditalea andensis ANESC-ST (=CICC10485T=NCCB 100412T), which is capable of generating bioelectricity in alkaline-saline conditions. A. andensis ANESC-ST was shown to grow in alkaline conditions between pH 7.0-11.0 and also under high salt condition (up to 4 wt% NaCl). Electrical output was further demonstrated in microbial fuel cells (MFCs) with an average current density of ~0.5 µA/cm2, even under the harsh condition of 4 wt% NaCl and pH 9.0. Subsequent introduction of secreted extracellular metabolites into MFCs inoculated with Escherichia coli or Pseudomonas aeruginosa yielded enhanced electrical output. The ability of A. andensis ANESC-ST to generate energy under alkaline-saline conditions points towards a solution for bioelectricity recovery from alkaline-saline wastewater. This is the first report of A.andensis ANESC-ST producing bioelectricity at high salt concentration and pH.

Clinical and antimicrobial efficacy of a controlled-release device containing chlorhexidine in the treatment of chronic periodontitis.

A controlled-release device (CRD) containing chlorhexidine gluconate, such as PerioCol(™)CG (Eucare Pharmaceuticals Pvt. Ltd,, Chennai, India), for subgingival application has little reported data with clinical as well as antimicrobial efficacy. This study evaluated clinical and subgingival microbial changes on using indigenously developed PerioCol™CG as an adjunct to scaling and root planing (SRP) in the treatment of chronic periodontitis. Forty posterior first molar sites having probing pocket depth ≥ 5 mm were selected and divided into two groups, with 20 sites in each group, in a split-mouth design. Group A (test site) was treated with SRP and PerioCol(™)CG, while group B (control site) was treated with SRP alone. Subgingival microbial samples were collected at baseline and 1 month after the initial SRP, while probing depth (PD), clinical attachment level (CAL) and gingival index (GI) were recorded at baseline, after 1 month and after 3 months. Microbial detection of Porphyromonas gingivalis (P. gingivalis) and Tannerella forsythia (T. forsythia) was done by means of polymerase chain reaction (PCR). A significant improvement was observed in all clinical measures in sites treated with PerioCol(™)CG as compared to the control sites during the study period. Also, there was a statistically significant reduction in the proportion of occurrence of P. gingivalis and T. forsythia after intervention in test sites as compared to control sites. Our data suggest that SRP combined with subgingival administration of PerioCol™CG has a significantly better and prolonged effect compared to SRP alone on the PD, clinical attachment loss and elimination of periodontopathogens.

Antibiotic effects of three strains of chrysophytes (Ochromonas, Poterioochromonas) on freshwater bacterial isolates.

We investigated the antibiotic effects of extracts of freeze-dried biomass and culture supernatants from the mixotrophic chrysophyte species Ochromonas danica, Poterioochromonas sp. strain DS, and Poterioochromonas malhamensis on bacterial strains isolated from lake water. Methanolic biomass extracts inhibited the growth of all tested strains, albeit to a different extent, whereas aqueous biomass extracts only affected bacteria of the genus Flectobacillus. The antibiotic action of supernatants from flagellate cultures could be mostly attributed to lipophilic substances, but the growth of bacteria affiliated with Flectobacillus and Sphingobium was also affected by hydrophilic compounds. A comparison of biomass extracts from light- and dark-adapted cultures of Poterioochromonas sp. strain DS showed that the growth-inhibiting factor was unrelated to chlorophyll derivatives. Supernatants from a dark-adapted, phagotrophically grown flagellate culture had stronger antibiotic effects and affected more bacterial strains than the supernatant from a light-adapted culture. Significant growth reduction of a Flectobacillus isolate was already induced by extremely low concentrations of lipophilic extracts from these supernatants. Our results show that metabolites of the studied flagellates - either released actively or during cell lysis - may selectively affect the growth of some aquatic bacteria even in very small doses and thus potentially affect microbial community composition. Moreover, the antibiotic potential of mixotrophic chrysophytes may change with their nutritional mode.

[Relations of oligotrophic bacteria to oxygen].

Polluted air and the derived photochemical smog are the sources of free radicals in the atmosphere. Organic peroxides present in the smog mediate formation of peroxide radical. Oxygen species are formed by purely physical mechanisms, for instance, energy consumption converts molecular oxygen to an excited singlet state. Six active oxygen species are known: ozone, atomic oxygen, perhydroxyl, superoxide, and singlet oxygen. Singlet oxygen is the most harmful oxygen product for living cells, while hydrogen peroxide is the least harmful. Molecular oxygen is hardly toxic for prokaryotes due to an efficient protection of microbial cells by specific enzymes. This work experimentally confirms the harmlessness of molecular oxygen.

Susceptibility testing of Danish isolates of Capnocytophaga and CDC group DF-2 bacteria.

Twelve Capnocytophaga and seven DF-2 strains were tested for their susceptibility to 14 antimicrobial agents using an agar dilution and an agar diffusion method. Twenty-three other antibiotics were evaluated using the diffusion test only. All strains were fully susceptible to penicillin, ampicillin, cefuroxime, cefotaxime, erythromycin, clindamycin, chloramphenicol, doxycycline, rifamycin and ofloxacin using both methods. Clindamycin, rifamycin and cefotaxime were most active. Using agar dilution some strains were susceptible to gentamicin, but agar diffusion showed total resistance. One Capnocytophaga strain was susceptible and another moderately susceptible to metronidazole, other strains were resistant. The agar diffusion test showed that both Capnocytophaga and DF-2 were resistant to most other aminoglycosides, to fosfomycin, polymyxin and trimethoprim. All strains of both taxa were fully susceptible to piperacillin, cefoxitin, imipenem and fusidic acid and showed different susceptibilities to the other agents. Susceptibility testing by means of agar diffusion using an enriched chocolate agar and 5% CO2 atmosphere could be used to test Capnocytophaga and DF-2 strains and gives sufficient accuracy for routine use, when revised inhibition zone breakpoints are employed.

Effect of clindamycin on neutrophil killing of gram-negative periodontal bacteria.

Periodontal diseases are infections of the tissues supporting the dentition. Recognition that relatively specific microfloras are associated with distinct clinical forms of periodontal disease has prompted the use of antimicrobial agents as adjuncts in periodontal therapy. Clindamycin is one of several antibiotics known to concentrate in bioactive form in neutrophils and to potentiate phagocyte bactericidal activity against certain bacteria. Neutrophils appear to play a key role in host defense against periodontopathic gram-negative bacteria. In the present study, we evaluated the effect of preincubation of neutrophils with therapeutically achievable concentrations of clindamycin upon subsequent in vitro bactericidal activity against three species of gram-negative periodontal bacteria, including Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga ochracea. In each instance, clindamycin neither enhanced nor inhibited the kinetics of bactericidal activity at low bacterium-neutrophil multiplicities. Further, this antibiotic had no demonstrable effect upon neutrophil bactericidal capacity, as assessed at bacterium-neutrophil ratios as high as 50:1. Our results indicate that clindamycin does not potentiate neutrophil bactericidal activity against the species of gram-negative periodontal organisms tested.

Columnaris infection among cultured Nile tilapia Oreochromis niloticus.

Flexibacter columnaris was isolated from 13 cultured Oreochromis niloticus showing respiratory disorders. The isolates developed typical swarming rhizoid colonies on Cytophaga agar medium. Antibiotic sensitivity test revealed the susceptibility of F. columnaris isolated to oxytetracycline, chloramphenicol and erythromycin. A marked difference in the pathogenicity of seven tested isolates was observed: two were highly virulent, one was moderately virulent and four were avirulent. No experimental infection could be induced with the highly virulent isolates except after injuring one of the natural barriers of the fish body. The severity of the disease and the increased median death time shortened by keeping infected fishes with injured gills in water containing ammonia. In naturally infected O. niloticus, the disease became chronic as indicated by the presence of excessive proliferative and necrotic changes. On the other hand, severe dilatation of branchial blood vessel, oedema and round cell infiltration proved that, the disease among experimentally infected tilapias was acute.

In vitro susceptibility of 96 Capnocytophaga strains, including a beta-lactamase producer, to new beta-lactam antibiotics and six quinolones.

The in vitro activities of new beta-lactam antibiotics and new quinolones were studied against 96 Capnocytophaga strains, including a beta-lactamase-producing strain which was resistant to ampicillin, amoxicillin, carbenicillin, cephalothin, and cefamandole. All strains were susceptible to the combination of amoxicillin and clavulanic acid, ureidopenicillins, cefoxitin, broad-spectrum cephalosporins, and imipenem. Cephalothin and cefamandole did not show good activity against most strains. All Capnocytophaga spp. were uniformly susceptible to the five new quinolones tested.

In vitro susceptibility of Capnocytophaga species to antimicrobial agents.

A total of 33 clinical isolates of Capnocytophaga spp. were susceptible to 4-quinolone antimicrobial agents. The antipseudomonal penicillins tested were equally active against all isolates, as were cefuroxime, ceftriaxone, ceftizoxime, cefotaxime, latamoxef, and ceftazidime. Most isolates were resistant to trimethoprim, and some were resistant to aztreonam. Most regimens for the empirical treatment of septic episodes in immunocompromised patients are suitable for the treatment of Capnocytophaga spp. infections.

In vitro susceptibility of Capnocytophaga species to 29 antimicrobial agents.

A hemoglobin-supplemented medium composed of Columbia agar base supplemented with 1% hemoglobin and 1% Polyvitex was used to investigate the in vitro activity of 29 antimicrobial agents against Capnocytophaga species. Clindamycin was the most active agent, with all strains being inhibited by 0.06 microgram/ml or less. Amoxicillin-clavulanic acid and imipenem were the most active among the beta-lactam antibiotics (MIC for 90% of strains tested [MIC90], 0.50 microgram/ml); other very active drugs were BMY 28142, cefpirome, cefotaxime, ceftazidime, and ceftriaxone (MIC90, 0.06 to 0.50 micrograms/ml), although at least one strain showed resistance to each of these antibiotics (MIC, greater than or equal to 16 micrograms/ml). Ciprofloxacin was the most active among the quinolones, with all strains being inhibited by 0.50 microgram/ml. The MICs of the other four drugs ranged from 0.12 to 4 micrograms/ml. Ampicillin, penicillin G, ticarcillin, aztreonam, and temocillin were moderately active (MIC90, 1 to 8 micrograms/ml; MIC range, less than or equal to 0.03 to greater than 128 micrograms/ml). All strains were uniformly resistant to the aminoglycosides, polymyxin B, vancomycin, trimethoprim, and amphotericin B. Three strains produced beta-lactamase. No significant difference was found between the susceptibility of strains isolated from various sources or patients.

Lactic acid production by oral Streptococcus mitis inhibits the growth of oral Capnocytophaga.

Various relationships including inhibition or stimulation of growth have been demonstrated among the bacteria present in dental plaque, both in vitro and in vivo. A large number of these relationships involved oral Streptococci. An earlier study found that strains of Streptococcus mitis inhibited the growth of potential periodontopathic microorganisms, such as Actinobacillus actinomycetemcomitans, Capnocytophaga and species of Bacteroides and Fusobacterium. The present investigation showed that this inhibitory effect resulted primarily from lactic acid production.

Induction of chloramphenicol and tetracycline resistance in Flexibacter sp. strain FS-1.

The gliding bacterium Flexibacter sp. strain FS-1 exhibits inducible resistance to chloramphenicol (Cmr) and tetracycline (Tcr). Either chloramphenicol or tetracycline alone induced a Cmr Tcr phenotype. The resistance is apparently not plasmid encoded.

Lysozyme-mediated aggregation and lysis of the periodontal microorganism Capnocytophaga gingivalis 2010.

The ability of lysozyme to aggregate and lyse the gram-negative capnophilic periodontal microorganism Capnocytophaga gingivalis 2010 was monitored optically at 540 nm. Both hen egg white and chromatographically purified human lysozymes had significant but similar aggregation potentials for both logarithmic- and stationary-phase bacteria. In general, an increase in enzyme concentration resulted in a graded increase in both the initial and maximum changes in turbidity which occurred during the reaction period. The greatest change in turbidity occurred within the initial minutes of interaction of lysozyme and the cells, and the extent of aggregation paralleled a rapid depletion of lysozyme by the suspensions during the first minute of its incubation with the bacteria. Interestingly, the muramidase inhibitors N-acetyl-D-glucosamine and histamine did not block aggregation, whereas maleylation of lysozyme completely inhibited its aggregating ability. Demaleylation, however, restored aggregation activity comparable to the native enzyme, indicating that maleylated lysozyme retained its integrity and that aggregation was primarily dependent on charge. The addition of up to physiological concentrations of NaHCO3 and NaCl to cell aggregates resulted in varying degrees of deaggregation and lysis. Surprisingly, ultrastructural analysis of lysozyme-treated cells revealed morphological changes with or without the addition of salt. Damage appeared to occur at the blunted polar end of the cells where there was a large spherical outpouching bordered by a damaged cell envelope. Damaged cells uniformly contained dense granular cytoplasmic debris. In effect, the cationic enzyme lysed C. gingivalis 2010, which was not apparent in the spectrophotometric assay. The paradoxical finding that during bacterial aggregation there was lysis may be of significance to the further elucidation of lysozyme's antibacterial role in the gingival sulcus.

Altered methionyl-tRNA synthetase in a Spirulina platensis mutant resistant to ethionine.

Compared with the parental strain, a Spirulina platensis mutant that is resistant to ethionine incorporated methionine into protein at a reduced rate, whereas ethionine incorporation was practically nil. The methionyl-tRNA synthetase present in crude extracts from the resistant strain showed a reduced affinity for methionine and ethionine.

In vitro susceptibility of Capnocytophaga strains to 18 antimicrobial agents.

Twenty-seven strains of capnocytophaga were tested for their susceptibility to 18 antimicrobial agents by an agar dilution technique. All strains were susceptible to achievable blood levels of penicillin G, cefaclor, cefoxitin, cefoperazone, moxalactam, clindamycin, chloramphenicol, and tetracycline. Most were susceptible to achievable levels of cefamandole, erythromycin, and metronidazole, and more than 10% were resistant to achievable levels of cephalexin and cephradine. With antimicrobial agents used in selective media, all strains were resistant to colistin, kanamycin, and nalidixic acid at commonly recommended concentrations of bacitracin and vancomycin.

Antimicrobial susceptibility of Capnocytophaga.

Capnocytophaga (Bacteroides ochraceus, Center for Disease Control biogroup DF-1) is associated with sepsis in granulocytopenic patients and is isolated in large numbers from the affected periodontal pockets in patients with juvenile periodontosis. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of 17 antimicrobial agents for 13 strains of Capnocytophaga organisms were determined. In addition, the ratio of the MBC to the MIC for each antimicrobial agent was determined for each strain. At concentrations of 1 microgram/ml or less, penicillin, ampicillin, carbenicillin, erythromycin, and clindamycin killed 90% of the strains. At concentrations of 3.12 microgram/ml or less, tetracycline, metronidazole, cefoxitin, and chloramphenicol killed 90% of the strains. None of the aminoglycosides tested demonstrated antibacterial activity at 50 microgram/ml. Penicillin, ampicillin, carbenicillin, and cefoxitin exhibited MBC/MIC ratios of 4 or less with all strains. Erythromycin, tetracycline, and metronidazole exhibited MBC/MIC ratios of 4 or less for 12 of 13 strains. The MICs of cephalothin and cefazolin for 90% of the strains were 25 and 50 microgram/ml, respectively. The MBC/MIC ratios for these drugs were 4 or less for 12 of 13 and 7 of 13 strains, respectively. The MIC of cefamandole for 90% of the strains was 3.12 microgram/ml; however, only nine strains had an MBC/MIC ratio of 4 or less.

Association of flexing and gliding in Flexibacter.

Nongliding mutants of Flexibacter FS-1 are unable to flex; revertants regain both forms of movement. A variety of conditions and treatments reversibly inhibit both gliding and flexing.

Source of energy for gliding motility in Flexibacter polymorphus: effects of metabolic and respiratory inhibitors on gliding movement.

The effects of selected metabolic and respiratory inhibitors on the gliding motility of Flexibacter polymorphus were examined. Motility and oxygen consumption were quantitatively inhibited in a reversible manner by specific respiratory poisons, suggesting that gliding velocity was linked to electron transport activity. Arsenate had little influence on the number or rate of gliding filaments, despite a 95% decrease in the concentration of intracellular adenosine 5'-triphosphate (ATP). At concentrations of cyanide or azide that abolished gliding movement, cells possessed a level of ATP that should have been sufficient to allow motility. Proton-conducting uncouplers of oxidative phosphorylation, such as carbonylcyanide m-chlorophenylhydrazone (CCCP) and tetrachlorosalicylanilide, strongly inhibited locomotion yet did not suppress respiratory activity or intracellular ATP sufficiently to account for their effect on movement. Inhibition of motility by CCCP (but not by tetrachlorosalicylanilide) was partially reversed by sulfhydryl compounds. However, unlike CCCP, inhibition of motility by p-chloromercuribenzoate, a known sulfhydryl-blocking reagent, was associated with a corresponding reduction in respiratory activity and ATP content of cells. Protein synthesis was not blocked by concentrations of CCCP inhibitory for motility, indicating that utilization of existing ATP in this energy-requiring process was not impaired. These data suggest (but do not unequivocally prove) that ATP may not function as the sole energy donor for the gliding mechanism, but that some additional product of electron transport is required (e.g., the intermediate of oxidative phosphorylation).

Sensitivity of cellulolytic bacteria to antibiotics.

The sensitivity of eight cellulolytic bacterial strains to eight antibiotics was tested. The results showed that, in general, the strains belonging to Cytophaga, Cellvibrio, and Cellfalcicula are more sensitive to antibiotics than those strains that belong to Sporocytophaga and Cellulomonas. The inhibitory activity of the tested antibiotics, though differing with different strains, showed the following categories: tetracycline, erythromycin, and chloromycetin were most active, kanamycin, streptomycin, and neomycin were intermediate, while novobiocin and penicillin showed low activity.