Base composition and sequence homology of deoxyribonucleic acid of thermotolerantCampylobacter from human and animal sources

A total of 31Campylobacter strains were studied. Of these, 24 were thermotolerant field isolates and 7 were reference strains. Mol% G+C was estimated by thermal denaturation and DNA-DNA-relatedness by the hydroxyapatite method. The strains identified asC. jejuni orC. coli and differentiated by the hippurate hydrolysis test formed separate, homogenous DNA homology groups. The two species were genetically more related to each other than to otherCampylobacter species. Results also show that the group of hippurate-negative thermotolerant campylobacters resistant to nalidixic acid is genetically heterogenous.     

Description ofCampylobacter laridis,a new species comprising the nalidixic acid resistant thermophilicCampylobacter (NARTC) group

Ten strains of the nalidixic acid-resistant thermophilicCampylobacter (NARTC) group, of which 2 were isolated from human feces, were compared with 12 reference strains representing various species ofCampylobacter. The NARTC strains were a homogeneous group with respect to their cell morphology and 28 physiological and biochemical characters. All were microaerophilic, motile (amphitrichate), gram-negative, curved, S-shaped or helical rods, and representative strains had mean DNA base compositions of 31 to 32 mol % G+C. Distinctive features of the 10 strains were resistance to nalidixic acid and anaerobic growth in the presence of trimethylamine N-oxide hydrochloride (TMAO). The latter feature may account for the common occurrence of NARTC strains in the fecal contents of seagulls. DNA-DNA hybridizations indicated high (≥76%) base sequence relatedness within the group and low (≤15%) relatedness to other species ofCampylobacter. The 10 strains were classified in the genusCampylobacter but they could not be assigned to any previously defined species. Therefore, a new species, with the nameCampylobacter laridis, is proposed for these 10 strains; the type strain is NCTC 11352.     

ThermotolerantCampylobacter with no or weak catalase activity isolated from dogs

ThermotolerantCampylobacter strains isolated from dog feces were characterized by phenotypical tests, DNA base composition, and DNA-DNA-hybridization. Out of 98 strains, 63 were catalase negative or weakly reacting (CNW); they were found in diarrheic as well as in healthy dogs. The CNW strains were all nalidixic-acid sensitive, hippurate negative, and grew at 42°C but not at 25°C. Seven strains were further investigated. They were sensitive to 2,3,5-triphenyl-tetrazolium chloride, reduced nitrate, and produced H₂S in the lead acetate test but not in triple-sugar-iron agar. The mol% G+C for five CNW isolates ranged from 35.2–35.8, which is higher than reported for any thermotolerantCampylobacter species. The strains also formed a well-delimitated DNA homology group with 80% or more intragroup relatedness and about 40% related toC. coli andC. jejuni.     

Efficacy of Ciprofloxacin for Treatment of Cholera Associated with Diminished Susceptibility to Ciprofloxacin to Vibrio cholerae O1

Objective

We identified a poor clinical response to treatment of cholera with a single 1 g dose of ciprofloxacin, a standard treatment for cholera.

Methods

To determine reasons for the poor response and better therapeutic approaches we examined the minimal inhibitor concentration (MIC, n = 275) and disc-diffusion zone sizes (n = 205) for ciprofloxacin and nalidixic acid of V. cholerae O1 strains isolated in Bangladesh from 1994 to 2012, and reexamined data from 161patients infected with Vibrio cholerae O1 recruited in four clinical trials who received single- or multiple-dose ciprofloxacin for treatment of cholera and compared their clinical response to the V. cholerae O1 susceptibility.

Results

Although all 275 isolates of V. cholerae O1 remained susceptible to ciprofloxacin using standard MIC and disc-diffusion thresholds, the MIC⁹⁰ to ciprofloxacin increased from 0.010 in 1994 to 0.475 μgm/ml in 2012. Isolates became frankly resistant to nalidixic with the MIC⁹⁰ increasing from 21 μgm/ml in 1994 to >256 μgm/ml and 166 of 205 isolates from 1994 to 2005 being frankly resistant using disc-diffusion testing. Isolates resistant to nalidixic acid by disc-diffusion testing had a median ciprofloxacin MIC of 0.190 μgm/ml (10^th-90^th centiles 0.022 to 0.380); nalidixic acid-susceptible isolates had a median ciprofloxacin MIC of 0.002 (0.002 to 0.012).The rate of clinical success with single-dose ciprofloxacin treatment for nalidixic acid-susceptible strains was 94% (61 of 65 patients) and bacteriologic success 97% (63/65) compared to 18% (12/67) and 8% (5/67) respectively with nalidixic acid-resistant strains (P<0.001 for both comparisons). Multiple-dose treatment with ciprofloxacin had 86% and 100% clinical and bacteriologic success rates respectively in patients infected with nalidixic acid-susceptible strains of V. cholerae O1 compared to clinical success 67% and bacteriologic success 60% with nalidixic acid-resistant strains.

Conclusions

Single-dose ciprofloxacin is not effective for treating cholera caused by V. cholerae O1 with diminished susceptibility to ciprofloxacin, and nalidixic acid disc-diffusion testing effectively screens for such isolates.     

High surface hydrophobicity ofStaphylococcus aureus as revealed by hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) on Octyl Sepharose^R in a column procedure was used to compare the relative surface hydrophobicity ofStaphylococcus aureus reference strains, protein A-negative mutants, and strains isolated from bovine mastitis. High protein A-producing strains (Cowan 1 and clinical isolate SA 17970) showed a higher relative surface hydrophobicity than did strains producing a low amount of protein A. One encapsulatedS. aureus strain (Smith diffuse) did not bind to the gel, while an unencapsulated variant showed binding properties similar to weak protein A-producing strains. Studies onS. aureus strains isolated from bovine mastitis revealed a good correlation between adsorption to Octyl Sepharose and the production of protein A. Results indicate that protein A and probably other surface proteins such as fibronectin-binding protein contribute to the high relative surface hydrophobicity ofS. aureus.     

Killer behavior within the Candida parapsilosis complex

A group of 29 isolates of Candida parapsilosis sensu stricto, 29 of Candida orthopsilosis, and 4 of Candida metapsilosis were assayed for the presence of killer activity using Saccharomyces cerevisiae ATCC 26609 as a sensitive strain. All C. metapsilosis isolates showed killer activity at 25 °C while strains of C. parapsilosis sensu stricto or C. orthopsilosis did not exhibit this activity. Sensitivity to killer toxins was evaluated using a set of previously reported killer strains of clinical origin. Only 11 isolates of the C. parapsilosis complex were inhibited by at least one killer isolate without resulting in any clear pattern, except for C. parapsilosis sensu stricto ATCC 22019, which was inhibited by every killer strain with the exception of C. parapsilosis and Candida utilis. The lack of sensitivity to killer activity among isolates of the genus Candida suggests that their toxins belong to the same killer type. Differentiation of species within the C. parapsilosis complex using the killer system may be feasible if a more taxonomically diverse panel of killer strains is employed.     

Specificity among the Casuarinaceae in root nodulation byFrankia

Pure cultured isolates ofFrankia made from root nodules of plant species from among three genera of the host family Casuarinaceae were used in inoculation trials of seedlings grown in water culture. A large number of host species among the genera Allocasuarina, Casuarina and Gymnostoma from Australia, Papua New Guinea and other South Pacific Islands were tested. The most widely infectiveFrankia strains were CcI3 and AllI1; theFrankia strains with the narrowest host range within the Casuarinaceae were CcI2 and GpI1. Intrafamily cross-inoculations were uncommon. The most broadly receptive host species wasG. papuanum. For many species ofAllocasuarina tested, no infection by anyFrankia available for testing could be observed.     

Identification of catalase-producingCampylobacter species based on biochemical characteristics and on cellular fatty acid composition

A total of 50 catalase-positive campylobacters from human and animal sources were studied. The nomenclatural type strains ofCampylobacter coli, C. jejuni, C. fetus, andC. fetus subsp.venerealis, a typical strain of the nalidixic acid-resistant thermophilic group, and various clinical isolates were characterized by bacteriological tests and by gas-liquid chromatographic analysis of their cellular fatty acids. The tests most useful in the differentiation of the various catalase-positive species were growth at 25 and 42°C, H₂S production, tolerance to nalidixic acid and to 2,3,5-triphenyltetrazolium chloride, and hippurate hydrolysis. The latter test was the only reliable means to differentiate betweenC. coli andC. jejuni. Differences between.C. fetus andC. jejuni/coli were confirmed by cellular fatty acid compositions. The bacteriological results indicated thatC. fetus andC. jejuni were distinct species, although within the thermophilic campylobacters there were several related taxa that included bothC. coli andC. jejuni strains with typicalC. coli and some thermophilic strains ofC. fetus subsp.fetus at the extremes.     

The effect of Sa and MiniSa plasmids on the induction of plant crown galls and hairy roots byAgrobacterium tumefaciens andAgrobacterium rhizogenes

The Inc-W group plasmid Sa or its derivative MiniSa were introduced into two strains ofAgrobacterium tumefaciens with Ti plasmids, one strain ofA. tumefaciens with the Ri plasmid and oneA. rhizogenes strain with the Ri plasmid. The effect was similar in allAgrobacterium strains. The pSa suppressed fully the virulence ofAgrobacterium strains (i.e. their ability to induce tumor growths - crown galls or hairj⁷ roots) inKalanchoe plants and carrot root slices. The MiniSa plasmid caused only a slight decrease of the frequency and size of tumor growths induced. The mechanism of suppression of virulence by the Sa plasmid inAgrobacterium tumefaciens andAgrobacterium rhizogenes seems to be similar.     

Isolation of a newLegionella species from thermally altered waters

Legionella-like bacteria were isolated from thermally altered water in Pennsylvania and Minnesota. These organisms were isolated from tissues of infected guinea pigs after intraperitoneal inoculation of water concentrates. While the cultural and morphological characteristics indicated the isolates wereLegionella, they did not react with antibodies prepared against known species of the genus. Antisera prepared against one of the isolates reacted maximally with the other ten isolates. Fluorescent antibody analysis of water concentrates from geographically disparate sites indicated that the environmental distribution is broad and that concentrations of the Oak Ridge strain ofLegionella were similar to those of serogroups 1, 2, 3, and 4 ofLegionella pneumophila.     

A biological evaluation of five European cultures ofAnaphes flavipes [Hymenoptera: mymaridae], an egg parasite ofOulema melanopus [Coleoptera: chrysomelidae]

Comparative biological data on five European cultures ofAnaphes flavipes [Hymenoptera: Mymaridae], an egg parasite ofOulema melanopus [Coleoptera: Chrysomelidae], indicated differences in developmental rates as those individuals representing cultures from the southernmost latitudes developed the slowest at 21°C, while those from the northernmost latitudes developed the fastest at this temperature. At 15.6°C, however, the parasites from the southern latitudes developed faster than those from the northern latitudes. Variations in other biological attributes, including longevity and fecundity, were not statistically significant.     

Nonspecificity of some commonly used inhibitors of DNA synthesis in cultures ofStreptococcus faecalis

The specificity of three commonly used inhibitors of DNA synthesis were tested in the batch culture ofStreptococcus faecalis ATCC 8043 in rich broth medium. It was shown that nalidixic acid, mitomycin C and 6-(4-hydroxyphenylazo)uracil inhibit the cell mass as much as they decrease net DNA synthesis. Hence the drugs tested are highly unspecific inhibitors of DNA synthesis inS. faecalis; i.e. they all interfere with other processes as well as with DNA synthesis.     

Serologic grouping and sexual compatibility of airbornecryptococcus neoformans

Through the use of Anderson air samplers, 214 isolates ofCryptococcus neoformans were cultured from the air in a vacant tower in a large complex of buildings in Oklahoma City. The tower contained hundreds of pigeons, a massive amount of droppings, nests with eggs and young, dying and dead pigeons. All isolates were serotype A-D and self-sterile for the production of basidiospores. Among these, 193 were of the ‘alpha’ mating type, producing basidiospores when paired with ‘a’ mating type. No isolates of ‘α’ mating type were found. The remaining 21 isolates were untypable for their mating type. These findings imply that the infectious particles ofC. neoformans in nature are relatively small, nonencapsulated yeast cells andnot basidiospores.     

Host range and symptom differences between isolates of turnip mosaic virus obtained fromSisymbrium loeselii

Twenty-four isolates of turnip mosaic virus (TuMV) from spontaneously infectedSisymbriutn loeselii plants were collected in Bohemian localities. Single lesions on leaves ofNicotiana tabacum cv. Samsun served for inoculating petunia plants used as infection sources for twelve host species. In no case were two identical isolates obtained. 15 isolates could be transmitted toVicia faba, a new TuMV host. Almost all isolates infectedPhaseolus vulgaris cv. Prince locally andTrifolium incarnatum systemically. Seven isolates were transmissible toPisum sativum. No substantial differences between isolates were observed with infectedAn aranthus caudatus, Chenopodium quinoa, Datura innoxia andSinapis alba plants. Several isolates could not infectNicotiana glutinosa at all, other isolates caused in it latent systemic infection and some isolates only local infection contrary to normal local and systemic infections ofN. glutinosa. Attempts to transmit one isolate to cereals failed.     

DNA supercoiling in a thermotolerant mutant ofEscherichia coli

A spontaneously occurring, nalidixic acid-resistant (Nal^R), thermotolerant (T/r) mutant ofEscherichia coli was isolated. Bacteriophage P1-mediated transduction showed that Nal^R mapped at or neargyr A, one of the two genes encoding DNA gyrase. Expression ofgyrA ⁺ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping ingyrA. Plasmid linking number measurements, made with DNA from cells grown at 37° C or shifted to 48° C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48° C. The genetic and supercoiling data indicate that thermo-tolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when. coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.     

Aerobic endospore-forming bacteria isolated from Antarctic soils as producers of bioactive compounds of industrial interest

Spore-forming bacteria are known to produce various enzymes and bioproducts valuable to different industries and to bear the harsh conditions found in the Antarctic environment. However, aerobic or facultative spore-forming bacterial communities found in maritime Antarctic soils yet remain poorly studied. In this study, 80 spore-forming and cold-adapted bacterial strains were isolated from nine different soil samples of King George Island, in maritime Antarctica, and further clustered into amplified ribosomal DNA restriction analysis groups within each soil. Representative strains were then identified as belonging to Bacillus, Rummeliibacillus, Paenibacillus and Sporosarcina by 16S rRNA gene sequencing. The ability to produce extracellular enzymes, antimicrobial substances and biosurfactants was determined in all isolates. The enzymatic activities most frequently found among the isolates were as follows: esterase (45 %), caseinase (30 %), amylase (16.2 %) and gelatinase (15 %). Biosurfactant production was detected in 25 % of the isolates. The growth inhibition of methicillin-resistant Staphylococcus aureus was observed in 13.7 % of the strains tested, but only two strains inhibited the growth of Candida albicans. The isolated spore-forming bacterial species were also compared with the characteristics of the different Antarctic soils sampled based on their physicochemical properties, showing that pH, C and P were the main factors correlated with the distribution of this group of bacteria in the Antarctic soils studied. These Antarctic endospore-forming bacterial strains may have a potential for industrial processes occurring at low temperatures.     

Restriction fragment length polymorphism and chromosome mapping of a mouse homeo box gene,Hox-2.1

Restriction endonuclease fragment length polymorphisms (RFLPs) were found using the cDNA probe Hox-2.1 for the homeo box-2.1 gene in the mouse. Polymorphism was detected in restriction patterns generated by fragments fromHindIII digestion. The great majority of laboratory strains of mice carries theHox-2.1 ^a allele. Only two laboratory strains carry theHox-2.1 ^b allele. Among strains of wild origin, the European subspecies (Mus m. domesticus, M. m. brevirostris, andM. m. musculus) and some Asian subspecies (M. m. castaneus) carry theHox-2.1 ^a allele. The subspecies from Far Eastern countries (M. m. molossinus, Chinese mice of wild origin, andM. m. yamashinai) carry theHox-2.1 ^ballele. Using the RFLP, theHox-2.1 gene was mapped on chromosome 11. Three-point cross test data showed that the recombination frequency is 29.6% between theHba and theHox-2.1 genes and 23.5% between theHox-2.1 and theEs-3 genes. The gene order ofHba-Hox-2.1-Es-3 has been confirmed.     

Phylogenetic relationships among Phytopythium species,and re-evaluation of Phytopythium fagopyri comb. nov., recovered from damped-off buckwheat seedlings in Japan

We examined the phylogenetic relationships among Phytopythium species using the rDNA ITS region, the LSU rDNA region, and the mitochondrial coxI and coxII genes. The genus was resolved into three monophyletic clades (1–3). Clade 1 was the largest clade, composed of 12 known species. Clades 2 contained two known and one new species candidate and clade 3 contained two known species. Three isolates in clade 2 (FP1, HonMa, and a strain designated as P. helicoides CBS293.35) formed a monophyletic group with high bootstrap support. This monophyletic group was distinct from P. helicoides sensu stricto. All three isolates came from damped-off buckwheat seedlings. The isolates were morphologically identical with one another and were characterized by globose, sub-globose, or pyriform sporangia with apical papillae; internally or internally nested proliferating sporangia; simple sympodia; coiling antheridial stalks; and wavy, sessile, or clavate antheridial cells. The isolates grew at temperatures between 15 °C and 40 °C, and the optimum temperature was 30 °C, with a radial growth rate of 20 mm/24 h. The phylogenetic and morphological analyses indicated that these isolates belong to a distinct species, which was previously under the genus Pythium, named here Phytopythium fagopyri comb. nov.     

Clonal Expansion May Account for High Levels of Quinolone Resistance in Salmonella enterica Serovar Enteritidis

We have observed a high incidence of isolated nalidixic acid resistance in Salmonella enterica serovar Enteritidis isolates in Ireland, particularly isolates of phage type 1 (PT1). A group of nalidixic acid-resistant (n = 22) and nalidixic acid-susceptible (n = 28) isolates of serovar Enteritidis from multiple sites in Ireland were selected. Isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI, and the MICs for nalidixic acid and ciprofloxacin were determined. Mutations associated with nalidixic acid resistance in clinical isolates and laboratory mutants of serovar Enteritidis and 32 nalidixic acid-resistant isolates of 15 other salmonella serovars were identified. PFGE had limited discriminatory power. A specific point mutation (G246T) associated with amino acid substitution Asp87Tyr in the quinolone resistance determining region of the gyrA gene accounted for 95% of all mutations in serovar Enteritidis and for all mutations in PT1 isolates. Greater diversity of mutations was observed among all non-Enteritidis salmonella serovars studied. Rates of nalidixic acid resistance in serovar Enteritidis may predominantly reflect clonal expansion after infrequent mutation or selection events.