Nonselectivity of Rimler-Shotts Medium for Aeromonas hydrophila in Estuarine Environments

Thirty presumptive Aeromonas hydrophila isolates were collected from estuarine sources using Rimler-Shotts media. Many of the isolates, especially the strains from a low-salinity site, were also identified as A. hydrophila using the API 20E system and two differential techniques devised for the identification of A. hydrophila. All of the isolates, however, had deoxyribonucleic acid base compositions with guanine-plus-cytosine ratios between 40 and 49 mol%, which excludes these strains from the genus Aeromonas.     

In Vitro Susceptibility of Selected Bacteria to Cefaclor

Cefaclor is an orally absorbed cephalosporin antibiotic chemically and pharmacologically similar to cephalexin. It appears to be more active than cephalexin against susceptible strains. The in vitro sensitivity of 230 clinical bacterial isolates to cefaclor was studied. Most isolates of S. aureus, K. pneumoniae, E. coli, and indole negative Proteus species were inhibited at clinically attainable serum and urine concentrations. Like cephalexin, cefaclor was less active against isolates of Enterobacter species, indole positive Proteus species and enterococci although many of these isolates were inhibited at concentrations achievable in urine.     

Uptake of Choline and Its Conversion to Glycine Betaine by Bacteria in Estuarine Waters

The uptake and degradation of nanomolar levels of [methyl-¹⁴C]choline in estuarine water samples and in seawater filtrate cultures composed mainly of natural free-living bacteria was studied. Uptake of [¹⁴C]choline exhibited Michaelis-Menten kinetics, with K_t + S_n values of 1.7 to 2.9 nM in filtrate cultures and 1.7 to 4.1 nM in estuarine-water samples. V_max values ranged from 0.5 to 3.3 nM · h⁻¹. The uptake system for choline in natural microbial assemblages therefore displays very high affinity and appears able to scavenge this compound at the concentrations expected in seawater. Uptake of choline was inhibited by some natural structural analogs and p-chloromercuribenzoate, indicating that the transporter may be multifunctional and may involve a thiol binding site. When 11 nM [¹⁴C]choline was added to water samples, a significant fraction (>50%) of the methyl carbon was respired to CO₂ in incubations lasting 10 to 53 h. Cells taking up [¹⁴C]choline produced [¹⁴C]glycine betaine ([¹⁴C]GBT), and up to 80% of the radioactivity retained by cells was in the form of GBT, a well-known osmolyte. Alteration of the salinity in filtrate cultures affected the relative proportion of [¹⁴C]choline degraded or converted to [¹⁴C]GBT, without substantially affecting the total metabolism of choline. Increasing the salinity from 14 to 25 or 35 ppt caused more [¹⁴C]GBT to be produced from choline but less ¹⁴CO₂ to be produced than in the controls. Lowering the salinity to 7 ppt decreased [¹⁴C]GBT production and increased ¹⁴CO₂ production slightly. Intracellular accumulations of [¹⁴C]GBT in the salt-stressed cultures were osmotically significant (34 mM). Choline may be used as an energy substrate by estuarine bacteria and may also serve as a precursor of the osmoprotectant GBT, particularly as bacteria are mixed into higher-salinity waters.     

Susceptibility of Phytopathogenic Bacteria to Wheat Purothionins In Vitro

Purothionins are basic polypeptides with antimicrobial properties that are present in the endosperm of wheat and other cereal species. Susceptibility to wheat purothionins among phytopathogenic bacteria of the genera Pseudomonas, Xanthomonas, Agrobacterium, Erwinia, and Corynebacterium has been investigated. Sensitive strains have been found in all of these genera except Agrobacterium (the only strain of A. tumefaciens available proved to be resistant). Minimal inhibitory concentrations (MIC) with partially purified crude purothionins ranged from 1 μg/ml for C. sepedonicum (C.5) to 540 μg/ml for E. amylovora (E.3). Minimal bactericidal concentrations (MBC) were not higher than twice the MIC value, except for C. poinsettiae (C.4) (MBC/MIC = 8). Purothionins α and β, obtained by carboxymethyl-cellulose column chromatography, were tested against P. solanacearum (P.2) and X. phaseoli (X.2); α purothionin was more active than β against X.2, and β more active than α against P.2. This suggests a relationship between polypeptide sequence and specificity of action.     

Susceptibility of Cheese and Yoghurt Starter Bacteria to Antibiotics

Eight single-strain lactic streptococci, three commercial cheese starters, and six lactic acid bacteria isolated from yoghurt were examined for their susceptibility to penicillin, cloxacillin, tetracycline-hydrochloride and streptomycin. The ranges of the antibiotics causing 50% inhibition of the bacteria were (mug/ml): penicillin, 0.009 to 0.20; cloxacillin, 0.24 to 2.50; tetracycline, 0.09 to 0.60; and streptomycin, 0.35 to 13.0. The average concentrations required to cause 50 and 100% inhibition of the cheese starters were (mug/ml): penicillin, 0.12 and 0.26; cloxacillin, 1.91 and 3.9; tetracycline-hydrochloride, 0.13 and 0.36; and streptomycin, 0.59 and 2.06. All the cocci were about equally susceptible to tetracycline, and all organisms were more resistant to cloxacillin than penicillin. The yoghurt isolates were more resistant to streptomycin and more susceptible to penicillin than the cheese starters. The 2, 3, 5-triphenyltetrazolium chloride test, using Streptococcus thermophilus BC as assay organism, does not detect low levels of streptomycin in milk. However, it is useful in detecting cloxacillin residues.     

Efficiencies of Recovery of Bdellovibrios from Brackish- Water Environments by Using Various Bacterial Species as Prey

A total of 44 bacterial species subdivided into 10 trial experiments have been used as prey for the recovery of bdellovibrios from samples of water from a brackish tidal pond and an aquarium saltwater tank. In an initial investigation, the recovery efficiency of each of the test bacterial species was compared with that of a designated standard prey, Vibrio parahaemolyticus P-5. The results revealed that in each case strain P-5 yielded an equal or significantly greater number of plaques of bdellovibrios than the test prey with but a single exception, strain CS5. In repeat experiments, CS5 yielded fewer plaques than P-5. To determine whether the use of multiple bacterial species compared with a single species as prey would increase the number of PFU of bdellovibrios recovered, material from plaques appearing on each of the test prey in the respective trials was sequentially subcultured onto two respective agar plates, the first containing as prey V. parahaemolyticus P-5 and the second containing the initial test organism. In nearly every case, subculture of plaques from lawns of the test prey to P-5 resulted in plaque formation. On the basis of the results, the use of several test prey and P-5 did not result in the recovery of any more bdellovibrio PFU than the use of P-5 alone. In this study, V. parahaemolyticus P-5 was observed to be the most efficient prey for the recovery of bdellovibrios from moderate salt water.     

Sulfate-Reducing Bacteria: Principal Methylators of Mercury in Anoxic Estuarine Sediment

Substrate-electron acceptor combinations and specific metabolic inhibitors were applied to anoxic saltmarsh sediment spiked with mercuric ions (Hg²⁺) in an effort to identify, by a direct approach, the microorganisms responsible for the synthesis of hazardous monomethylmercury. 2-Bromoethane sulfonate (30 mM), a specific inhibitor of methanogens, increased monomethylmercury synthesis, whereas sodium molybdate (20 mM), a specific inhibitor of sulfate reducers, decreased Hg²⁺ methylation by more than 95%. Anaerobic enrichment and isolation procedures yielded a Desulfovibrio desulfuricans culture that vigorously methylated Hg²⁺ in culture solution and also in samples of presterilized sediment. The Hg²⁺ methylation activity of sulfate reducers is fully expressed only when sulfate is limiting and fermentable organic substrates are available. To date, sulfate reducers have not been suspected of Hg²⁺ methylation. Identification of these bacteria as the principal methylators of Hg²⁺ in anoxic sediments raises questions about the environmental relevance of previous pure culture-based methylation work.     

Antibiotic Susceptibility of Anaerobic Ruminal Bacteria

This study demonstrated that 15 species of ruminal bacteria with no previous history of contact with antibiotics are susceptible to bacitracin, chloramphenicol, chlortetracycline, erythromycin, novobiocin, oleandomycin, oxytetracycline, penicillin, tetracycline, tylosin, and vancomycin. A number of the species were not inhibited by kanamycin, neomycin, polymyxin, and streptomycin. The data suggest that antibiotic-resistant cells occur within susceptible cultures of these species. Streptococcus bovis FD-10 and a nonruminal anaerobe, Bacteroides melaninogenicus BE-1, showed similar antibiotic susceptibilities.     

Relative Susceptibility of Acid-Fast and Non-Acid-Fast Bacteria to Ultraviolet Light

Mycobacterium tuberculosis strain Erdman, M. bovis (BCG), and M. phlei showed a 1- to 2-log drop in viability after exposure to ultraviolet light compared to a 5-log drop over the same period for Staphylococcus albus, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis, and Serratia marcescens. L. monocytogenes showed an initial resistance to ultraviolet inactivation, but later the inactivation rate increased sharply. The significance of these findings with regard to the use of S. marcescens as a test organism for determining the bactericidal efficiency of ultraviolet lamps used to sterilize equipment contaminated with tubercle bacilli is discussed.     

Bacteria in Oligotrophic Environments: Starvation Survival Lifestyle

World Journal of Microbiology and Biotechnology -     

Effect of Salinity on Mercury-Methylating Activity of Sulfate-Reducing Bacteria in Estuarine Sediments

The biomethylation of mercury was measured in anoxic estuarine sediments that ranged in salinity from 0.03 to 2.4% with or without added molybdate, an inhibitor of sulfate reducers. Mercury methylation was inhibited by molybdate by more than 95%, regardless of sediment salinity. In the absence of inhibitor, high-salinity sediments methylated mercury at only 40% of the level observed in low-salinity sediments. In response to molybdate inhibition of sulfate reducers, methanogenesis increased up to 258% in high-salinity sediments but only up to 25% in low-salinity sediments. In contrast to an earlier low-salinity isolate, a Desulfovibrio desulfuricans strain from high-salinity sediment required 0.5 M sodium for optimal growth and mercury methylation activity. The formation of negatively charged mercuric chloride complexes at high salinity did not noticeably interfere with the methylation process. Results of these studies demonstrate that sulfate reducers are responsible for mercury methylation in anoxic estuarine sediments, regardless of the prevailing salinity.     

Detection and Enumeration of Sulphate-Reducing Bacteria in Estuarine Sediments by Competitive PCR

The distribution of sulphate-reducing bacteria (SRB) in the sediments of the Colne River estuary, Essex, UK covering different saline concentrations of sediment porewater was investigated by the use of quantitative competitive PCR. Here, we show that a new PCR primer set and a new quantitative method using PCR are useful tools for the detection and the enumeration of SRB in natural environments. A PCR primer set selective for the dissimilatory sulphite reductase gene (dsr) of SRB was designed. PCR amplification using the single set of dsr-specific primers resulted in PCR products of the expected size from all 27 SRB strains tested, including Gram-negative and positive species. Sixty clones derived from sediment DNA using the primers were sequenced and all were closely related with the predicted dsr of SRB. These results indicate that PCR using the newly designed primer set are useful for the selective detection of SRB from a natural sample. This primer set was used to estimate cell numbers by dsr selective competitive PCR using a competitor, which was about 20% shorter than the targeted region of dsr. This procedure was applied to sediment samples from the River Colne estuary, Essex, UK together with simultaneous measurement of in situ rates of sulphate reduction. High densities of SRB ranging from 0.2 ? 5.7 × 10⁸ cells ml^{? 1} wet sediment were estimated by the competitive PCR assuming that all SRB have a single copy of dsr. Using these estimates cell specific sulphate reduction rates of 10^{? 17} to 10^{? 15} mol of SO₄ ^{2 ?} cell^{? 1} day^{? 1} were calculated, which is within the range of, or lower than, those previously reported for pure cultures of SRB. Our results show that the newly developed competitive PCR technique targeted to dsr is a powerful tool for rapid and reproducible estimation of SRB numbers in situ and is superior to the use of culture-dependent techniques.     

Loss of Estuarine Bacteria by Viral Infection and Predation in Microcosm Conditions

The bacterioplankton density in Ria de Aveiro, a shallow estuarine ecosystem, varied in the broad range of 1.9-10.6 109 cells L-1. The range of values was about 2 times higher in brackish water than in marine water. At high tide bacterial abundance was 2-3 times lower than at low tide. The overall variation in virioplankton was in the range of 2.4-25.0 1010 particles L-1. Brackish water was about 2 times richer in viral particles than the marine water. Near low tide the virioplankton was 2-3 times higher that at high tide. Viral density followed the pattern of bacterial abundance (it explained 40% of virioplankton variation). The viruses to bacterium ratio varied, throughout tidal cycles, by a factor of about 10 establishing the range 4.7-55.6 (average 17.6). This ratio was rather similar in the two estuarine zones. We compared the effects of infection and predation on the control of bacterioplankton size in the two zones of the estuary. The approach to this question was conducted in experimental microcosms, set up in six combinations of plankton variables affecting the presence/absence of predators, virus-to-bacterium ratio (10-fold increase), virus-to-bacterium distance (2.2-fold increase), and bacterial growth rate. The results showed that predation was similar, in a percent basis, in marine (69%) and brackish water (73%). Viral infection was, however, higher in brackish water (59%) than in the marine water (36%). We conclude that the bacterioplankton along the salinity gradient evolves under biological pressures that are in different balance in the marine and brackish water zones. The effect of viral lysis on bacterial communities with enhanced growth (after yeast extract addition) was masked even when the initial ratio was 10-fold greater than in the natural samples. The high density of the virioplankton did not preclude the large and rapid increase in bacterial density. We suggest that the dynamics of the equilibrium between bacteria and viruses in the environment is driven to higher numerical levels during periods of intensive bacterial growth. On the contrary, at low bacterial growth rates the temporarily increased virus-to-bacterium ratio may drive the equilibrium to its lowest levels.     

Halophilic Bacteria Susceptibility to Peracetic Acid Vapor and Ethylene Oxide

Extremely to slightly halophilic bacteria were tested for susceptibility to two sterilizing agents, peracetic acid (PAA) and ethylene oxide (ETO). PAA susceptibility was explored by two methods: an agar plate (constant pH of 7.2) and a filter strip (constant incubation period of 37 days); 100% susceptibility was obtained by both methods. The dosage (0.5 ml/min) was applied to a filter pad in a petri dish cover. Glove box experiments with ETO (input 1.5 lb. [ca. 680.4 g]/24 hr, the only constant) yielded 100% susceptibility for all halophiles tested. These experiments demonstrated the efficacy of two lethal agents for extreme halophiles, PAA and ETO. Variation in pH did not affect susceptibility.