Viability of fastidious Phytophthora following different cryopreservation treatments

Living stock cultures with constant phenotypes and genotypes are required for a wide range of research and industrial applications; however, long-term, stable preservation of fastidious Phytophthora strains has been challenging. In this study, we systematically evaluated different cryopreservation treatments to identify and clarify freezing, thawing, and other conditions appropriate for long-term maintenance. Optimal preservation conditions were largely strain-specific, with robust strains remaining fully viable and the fastidious yielding lower recovery under all test conditions. Nevertheless, several procedures were shown to be generally applicable for effective cryopreservation of most Phytophthora organisms. Fastidious strains retained higher viability following the −1 °C min⁻¹ freezing protocol (Mr Frosty's) than either of two widely used programmed freezing procedures. Revival was higher when frozen mycelium plugs were thawed at 37 °C for 2 min or 25 °C for 5 min, while lower viability was apparent for fastidious strains thawed at 55 °C for 1.5 min. Among 15 cryoprotective solutions assessed, 5 % dimethyl sulfoxide produced the highest viability for all fastidious strains. The effect of prefreeze and postfreeze treatments on revival was mild, if any, and strain-dependent. This study has generated reliable, practical, long-term preservation solutions applicable to a majority of Phytophthora species. It also has revealed a need for in-depth physiological and morphological investigations to further enhance the preservation methods for fastidious strains.     

Comparison of methods to investigate microbial populations in soils under different agricultural management

As water is a source of nosocomial infections in hospitals, the presence of fastidious Gram-negative bacteria in water samples taken in a university hospital was investigated. Water samples were inoculated onto agar plates and into amoebal microplates for co-culture. Sixty-eight alpha proteobacteria isolates were obtained and characterized using phenotypic methods and 16S rRNA gene sequence comparison. The latter approach divided the strains into seven clusters. Of these, one corresponded to previously recognized Afipia felis and it is likely that six were closely related new species. As these bacteria are fastidious and can not be cultivated on standard microbiological media, their possible role in hospital-acquired human infections should be investigated.     

A MEDIUM FOR THE CULTIVATION OF LACTOBACILLI

SUMMARY: An improved growth medium for lactobacilli is described. It supports good growth of lactobacilli generally and also is particularly useful for a number of fastidious strains which grow only poorly in other general media. In addition, tomato juice, a highly variable material, is not required. In a slightly modified form, it can also be used as a basal medium for fermentation tests.     

Development of A Rapid Method for the Diagnosis of Citrus Greening Disease using the Polymerase Chain Reaction

The fastidious bacterium causing citrus greening disease occurs in uneven and low concentrations in the sieve tubes of host plants. A rapid and sensitive assay based on the polymerase chain reaction (PCR) has been developed using the primers derived from the sequences of the cloned DNA fragment of greening fastidious bacterium (GFB) to detect GFB infection in citrus. One set of the primer pairs (named 226-primer pair), which generates a 226 bp GFB-specific fragment from total DNA templates purified from diseased citrus plants, was tested and chosen for PCR amplification. The PCR-based assay using this 226-primer pair effectively detected GFB infection in various citrus cultivars collected from different Asian countries. This detection technique, which can be completed within 6 h, offers a rapid and efficient method for accurate diagnosis of citrus greening disease.     

Urethral syndrome: a self limiting illness.

Thirty nine adult women who were not pregnant and had the urethral syndrome in a general practice underwent detailed microbiological investigations. Patients monitored their own symptoms, and those with persisting symptoms were entered into a randomised controlled trial of treatment with doxycycline and placebo. Chlamydia trachomatis and Neisseria gonorrhoeae were not isolated and fastidious organisms were not causally associated with the urethral syndrome. Treatment with doxycycline showed no benefit; each episode of the urethral syndrome was short and self limiting and there were no recurrences in a median observation period of 12 months.     

An improved preparation method for a titanium (III) media reductant

A detailed method for the formulation of titanium(III) nitrilotriacetate, a sulfur-free, media reductant, is described. Growth of Butyribacterium methylotrophicum on methanol was not significantly inhibited by 5 mM titanium(III) nitrilotriacetate. Concentrations as low as 30 μM effectively reduced cultured media and allowed growth of this obligate anaerobe. This reductant was used to investigate the nutritional requirements for sulfur compounds and growth of fastidious anaerobes in continuous flow culture.     

Human Infection with Vibrio Fetus

Vibrio fetus, a slowly growing, microaerophilic, Gram-negative rod which causes infectious abortion in cattle, occasionally induces disease in man. Since persons receiving immunosuppressive agents are among those most susceptible to this infection, recognition of the disease may become increasingly important as transplantation operations increase. Vibrio infections may produce a spectrum of acute and chronic illnesses or may be asymptomatic. Vibriosis may develop at any age but it seems most devastating in the very young or in older debilitated patients. The organism is sensitive to many common antibiotics but has a propensity to produce chronic relapsing illness if treatment is not continued long enough. The method of transmission of the agent is uncertain. Fewer than 100 cases of human vibriosis have been reported, perhaps because of fastidious growth requirements and the initial ease of antibiotic suppression; however, the true incidence of human infection is likely much higher.     

Nitrogen Requirements and Uricolytic Activity of Cutaneous Bacteria

Uric acid, but not xanthine, was degraded by gram-positive catalase-producing cocci and diphtheroids which represented the two predominant human autochthonous skin bacteria. The proportions of uricolytic cocci and diphtheroids varied with the cutaneous site sampled. Uric acid and allantoin were not utilized by cocci or diphtheroids as sole sources of nitrogen. Uric acid appeared to act only as a secondary substrate for the gram-positive bacteria. Cutaneous cocci are known to be ureolytic but few diphtheroids had urease activity. Urea and ammonium nitrogen were not utilized as sole nitrogen sources by cocci, but some diphtheroids used these compounds for nitrogen. The majority of the cocci and diphtheroids were nutritionally fastidious and required amino-nitrogen for growth. In addition, some strains required vitamins and other unidentified metabolites found in yeast extract. These requirements were partially related to the cutaneous site from which the cocci or diphtheroids were isolated. Certain gram-negative bacilli degraded uric acid and utilized urate or its degradation products as nitrogen sources.     

A block in release of progeny virus and a high particle-to-infectious unit ratio contribute to poor growth of enteric adenovirus types 40 and 41 in cell culture.

The fastidious enteric adenovirus (FEAd) types 40 (Ad40) and 41 (Ad41) are found in stool specimens of infants and young children in association with gastroenteritis. Although they can be isolated routinely from clinical specimens by using 293 cells, they are propagated with variable success in cell lines which support the replication of other adenovirus serotypes. HeLa cells are generally considered to be nonpermissive for the replication of FEAds, but in this study, Ad40 and Ad41 grew to comparable titers in individual 293 and HeLa cells. However, virus was not efficiently released from infected HeLa cells and thus did not undergo multiple cycles of infection in HeLa cell cultures. The block in virus release was not overcome in KB18 cells which, like 293 cells, constitutively express proteins encoded by the E1B region of a subgroup C adenovirus (in this case Ad2). Moreover, it was apparent from these studies that Ad40 and Ad41 have particle-to-infectious unit ratios several orders of magnitude greater than that for Ad5, even in 293 cells which express the E1A and E1B proteins of Ad5 and are considered to be permissive for replication of the FEAds. Neither the block in release of progeny virus nor the high particle-to-infectious unit ratio is explained solely by the defect in expression of the E1B 55K protein identified by Mautner et al. (V. Mautner, N. MacKay, and V. Steinthorsdottir, Virology 171:619-622, 1989; V. Mautner, N. MacKay, and K. Morris, Virology 179:129-138, 1990).     

Bacterial Predators of Micrococcus luteus in Soil

Micrococcus luteus cells died relatively rapidly when they were added to natural soil. Microscopic observation showed that the cells were being physically destroyed by bacterial predators in the soil. Two of these predators were responsible for the initial, main attack, and they were isolated. The isolates on laboratory media lysed M. luteus cells in a manner similar to the attacks that occurred in soil. Neither predator was obligate, however, nor were they nutritionally fastidious. One of these bacteria produced mycelium and conidia. Under nutritionally poor conditions it used slender filaments of mycelium to seek out host cells. It had at least some of the characteristics of a Streptoverticillium species. The other bacterium was a short, gram-negative rod that did not easily fit into any of the known groups of gram-negative bacteria. It attached to host cells, but its mechanism of lysing these cells is not known.     

Transformation of Anaplasma phagocytophilum

Background  

Tick-borne pathogens cause emerging zoonoses, and include fastidious organisms such as Anaplasma phagocytophilum. Because of their obligate intracellular nature, methods for mutagenesis and transformation have not been available.     

Novel and potent oxazolidinone antibacterials featuring 3-indolylglyoxamide substituents

Novel oxazolidinone antibacterials bearing a variety of 3-indolylglyoxamide substituents have been explored in an effort to improve the spectrum and potency of this class of agents. A subclass of this series was also made with the diversity at C-5 terminus. These derivatives have been screened against a panel of clinically relevant Gram-positive pathogens and fastidious Gram-negative organisms. Several analogs in this series were identified with in vitro activity superior to linezolid (MIC=0.25-2 microg/mL). Compounds 10a, 10c, 10e and 10f displayed activity against linezolid resistant Gram-positive organisms (MIC=2-4 microg/mL). Selected oxazolidinones were evaluated for in vivo efficacy against a mouse systemic infection model.     

Contagious equine metritis: a review

Contagious Equine Metritis (CEM) is a highly contagious venereal disease of horses caused by a fastidious, Gram-negative coccobacillus which grows best on chocolate agar under microaerophilic conditions (5-10% CO2). Clinically, the disease is characterized by a copious watery-to-mucopurulent, vaginal discharge two to ten days after breeding by an infected stallion (11, 13). Shortened estrous cycle lengths are common and may be the only indication of endometritis in some instances (7). Inapparent carriers of the disease in both the mare and stallion make control of the disease more difficult. Outbreaks of CEM have been reported in England, Ireland, France, Australia and the United States. The current information concerning CEM is reviewed.     

Rich culture medium for the radiochemical labeling of proteins and nucleic acids.

Yeast extract was treated with tyrosine decarboxylase and used to prepare a rich, complex medium virtually free of tyrosine. The medium supported maximal growth rates for Escherichia coli prototrophs, as well as for defined and undefined auxotrophs. It has made possible the efficient radiochemical labeling of cells growing optimally in complex medium and the characterization of mutants with undefined requirements. Similarly prepared media may be useful for the study of fastidious organisms and organisms for which no defined medium has been described.     

Methanogenesis from acetate: a nonmethanogenic bacterium from an anaerobic acetate enrichment.

A methanogenic acetate enrichment was initiated by inoculation of an acetate-mineral salts medium with domestic anaerobic digestor sludge and maintained by weekly transfer for 2 years. The enrichment culture contained a Methanosarcina and several obligately anaerobic nonmethanogenic bacteria. These latter organisms formed varying degrees of association with the Methanosarcina, ranging from the nutritionally fastidious gram-negative rod called the satellite bacterium to the nutritionally nonfastidious Eubacterium limosum. The satellite bacterium had growth requirements for amino acids, a peptide, a purine base, vitamin B12, and other B vitamins. Glucose, mannitol, starch, pyruvate, cysteine, lysine, leucine, isoleucine, arginine, and asparagine stimulated growth and hydrogen production. Acetate was neither incorporated nor metabolized by the satellite organism. Since acetate was the sole organic carbon source in the enrichment culture, organism(s) which metabolize acetate (such as the Methanosarcina) must produce substrates and growth factors for associated organisms which do not metabolize acetate.     

Novel oxazolidinone-quinolone hybrid antimicrobials

Antimicrobial compounds incorporating oxazolidinone and quinolone pharmacophore substructures have been synthesized and evaluated. Representative analogues 2, 5, and 6 display an improved potency versus linezolid against gram-positive and fastidious gram-negative pathogens. The compounds are also active against linezolid- and ciprofloxacin-resistant Staphylococcus aureus and Enterococcus faecium strains. The MOA for these new antimicrobials is consistent with a combination of protein synthesis and gyrase A/topoisomerase IV inhibition, with a structure-dependent degree of the contribution from each inhibitory mechanism.     

Heme requirement for growth of fastidious atypical strains of Aeromonas salmonicida.

The growth of fastidious atypical strains of the fish pathogen Aeromonas salmonicida on both solid and liquid media was dependent specifically on a source of heme which was apparently required for initiation of growth at low inoculum densities. Thus, hemin enhanced the plating efficiencies of such strains on solid medium and significantly reduced their inoculum-size-dependent lag times in broth. The heme requirement could also be satisfied by hematoporphyrin and, less effectively, by hemoglobin. Since the requirement was a stable property of all 17 strains tested, it may prove to be another taxonomic criterion by which the atypical strains can be differentiated from the typical strains of A. salmonicida.     

MEDIA FOR THE CULTURE OF OCEANIC ULTRAPHYTOPLANKTON1,2

A medium (K) developed for culturing fastidious oceanic phytoplankton has been tested using recently isolated ultraphytoplankton clones representing at least seven different algal classes. The medium was designed to satisfy as completely as possible the nutritional requirements of this diverse group of phytoplankters. Important aspects are the addition of selenium, the inclusion of both nitrate and ammonium, an increased level of chelation and a moderate level of pH buffering. The seawater-based version of this medium has been tested on 200 clones of which 186 grew reliably. A synthetic counterpart (AK) was tested on 40 of the more difficult clones and 27 grew well; 13 grew not all. While neither medium meets the exacting nutritional needs of all the ultraphytoplankton forms tested, they are excellent for most oceanic clones and are very successful for the isolation and establishment in culture of new oceanic phytoplankton clones.     

TolC is required for pathogenicity of Xylella fastidiosa in Vitis vinifera grapevines

Xylella fastidiosa infects a wide range of hosts and causes serious diseases on some of them. The complete genomic sequences of both a citrus variegated chlorosis (CVC) and a Pierce's disease (PD) strain revealed two type I protein secretion plus two multidrug resistance efflux systems, and all evidently were dependent on a single tolC homolog. Marker exchange mutagenesis of the single tolC gene in PD strain Temecula resulted in a total loss of pathogenicity on grape. Importantly, the tolC- mutant strains were not recovered after inoculation into grape xylem, strongly indicating that multidrug efflux is critical to survival of this fastidious pathogen. Both survival and pathogenicity were restored by complementation using tolC cloned in shuttle vector pBBR1MCS-5, which was shown to replicate autonomously, without selection, for 60 days in Temecula growing in planta. These results also demonstrate the ability to complement mutations in X. fastidiosa.     

Heme requirement for growth of fastidious atypical strains of Aeromonas salmonicida

The growth of fastidious atypical strains of the fish pathogen Aeromonas salmonicida on both solid and liquid media was dependent specifically on a source of heme which was apparently required for initiation of growth at low inoculum densities. Thus, hemin enhanced the plating efficiencies of such strains on solid medium and significantly reduced their inoculum-size-dependent lag times in broth. The heme requirement could also be satisfied by hematoporphyrin and, less effectively, by hemoglobin. Since the requirement was a stable property of all 17 strains tested, it may prove to be another taxonomic criterion by which the atypical strains can be differentiated from the typical strains of A. salmonicida.