Antibiotic resistance among bacteria isolated from seawater and penguin fecal samples collected near Palmer Station, Antarctica.

Antibiotic resistance in aquatic bacteria has increased steadily as a consequence of the widespread use of antibiotics, but practice and international treaty should have limited antibiotic contamination in Antarctica. We estimated antibiotic resistance in microorganisms isolated from the Antarctic marine waters and a penguin rookery, for 2 reasons: (i) as a measure of human impact and (ii) as a potential "snapshot" of the preantibiotic world. Samples were taken at 4 established sampling sites near Palmer Station, which is situated at the southern end of the Palmer Archipelago (64 degrees 10'S, 61 degrees 50'W). Sites were chosen to provide different potentials for human contamination. Forty 50 mL samples of seawater were collected and colony-forming units (CFU)/mL were determined at 6 and 20 degrees C. For this study, presumed psychrophiles (growth at 6 degrees C) were assumed to be native to Antarctic waters, whereas presumed mesophiles (growth at 20 degrees C but not at 6 degrees C) were taken to represent introduced organisms. The 20-6 degrees C CFU/mL ratio was used as a measure of the relative impact to the ecosystem of presumably introduced organisms. This ratio was highest at the site nearest to Palmer Station and decreased with distance from it, suggesting that human presence has impacted the natural microbial flora of the site. The frequency of resistance to 5 common antibiotics was determined in each group of isolates. Overall drug resistance was higher among the presumed mesophiles than the presumed psychrophiles and increased with proximity to Palmer Station, with the presumed mesophiles showing higher frequencies of single and multiple drug resistance than the psychrophile population. The frequency of multidrug resistance followed the same pattern. It appears that multidrug resistance is low among native Antarctic bacteria but is increased by human habitation.

Influence of plant developmental stage on microbial community structure and activity in the rhizosphere of three field crops.

Seasonal shifts in rhizosphere microbial populations were investigated to follow the influence of plant developmental stage. A field study of indigenous microbial rhizosphere communities was undertaken on pea (Pisum satvium var. quincy), wheat (Triticum aestivum var. pena wawa) and sugar beet (Beta vulgaris var. amythyst). Rhizosphere community diversity and substrate utilization patterns were followed throughout a growing season, by culturing, rRNA gene density gradient gel electrophoresis and BIOLOG. Culturable bacterial and fungal rhizosphere community densities were stable in pea and wheat rhizospheres, with dynamic shifts observed in the sugar beet rhizosphere. Successional shifts in bacterial and fungal diversity as plants mature demonstrated that different plants select and define their own functional rhizosphere communities. Assessment of metabolic activity and resource utilization by bacterial community-level physiological profiling demonstrated greater similarities between different plant species rhizosphere communities at the same than at different developmental stages. Marked temporal shifts in diversity and relative activity were observed in rhizosphere bacterial communities with developmental stage for all plant species studied. Shifts in the diversity of fungal and bacterial communities were more pronounced in maturing pea and sugar beet plants. This detailed study demonstrates that plant species select for specialized microbial communities that change in response to plant growth and plant inputs.

Influence of flanking homology and insert size on the transformation frequency of Acinetobacter baylyi BD413.

RecA-mediated recombination requires regions of homology between donor and recipient DNA for successful integration. This paper investigates the effect of the relationship between the length of gene-sized inserts (434, 733, 2228 and 2400 bp) and flanking sequence homology (100 - ca. 11 000 bp) on transformation frequency in Acinetobacter baylyi strain BD413. Both insert size and size of the homologous region were varied, which improves on previous studies that kept insert size constant and varied only the homologous flank size. Transfer frequency of a non-homologous single small gene for gentamicin resistance (aac(3)I; 773 bp) was increased 18-fold when flanking homology was changed from about 2000 bp to 8000 bp, but was reduced 234-fold when two genes were inserted (nptII-gfp; 2400 bp) between similar homologous regions. To investigate the effect of smaller regions of flanking homology (100 - 2000 bp), a partial nptII-gfp deletion (434 bp) was restored. This confirmed that a minimum of 500 bp on each flank was required for transformation to be affected by flanking homology. The data obtained allowed development of a multiple regression equation to predict transformation frequency from homology, insert size and total fragment size for gene insertions. We also show that the ratio of flanking homology to insert size and not the total size of donor DNA is the most important variable determining transformation frequency. The equation developed was consistent with results previously reported by others, and so will be useful when using A. baylyi as a model for gene transfer by transformation in the laboratory, environment and for biosafety.

Transformation of Acinetobacter baylyi in non-sterile soil using recombinant plant nuclear DNA.

To provide estimates of horizontal gene transfer from transgenic crops to indigenous soil bacteria, transformation frequencies were obtained for naturally transformable Acinetobacter baylyi BD413 using a chromosomally integrated plant transgene. The transgene comprised sequences for two phenotypic markers: kanamycin resistance (npt II) and green fluorescent protein (gfp), expressed from their own bacterial promoters. Recipient bacteria carried a copy of these two genes, with deletions in their 3'-termini abolishing the marker activity, these genes were integrated into a 16S rRNA gene in the bacterial chromosomal genome or carried on a broad host range plasmid. Successful recombination between the plant transgene and the bacterial genome resulted in restoration of the markers, allowing detection through antibiotic selection and fluorescence. Transformation parameters of increasing complexity, without any enrichment steps, were used to approach the field conditions, while still obtaining measurable transformation frequencies. In pure culture filter experiments, transformation was detected using ground, chopped and whole leaves, as well as whole sterile seedlings, and ground roots. In sterile soil microcosms, transformation was detected using pure plant DNA (3.6 x 10(-8) transformants per recipient) and ground leaves (2.5 x 10(-11)). Transformation was also detected for the first time in non-sterile soil using pure plant DNA (5.5 x 10(-11)). Since the same constructs were used throughout, these data allow predictions of even more complex environmental systems where measurable frequencies are not easily obtainable.

Isolation and characterization of a bacteriophage with an unusually large genome from the Great Salt Plains National Wildlife Refuge, Oklahoma, USA.

In this study we present a bacteriophage isolated from the Great Salt Plains National Wildlife Refuge (GSP) that is shown to have a genome size of 340 kb, unusually large for a bacterial virus. Transmission electron microscopy analysis of the virion showed this to be a Myoviridae, the first reported to infect the genus Halomonas. This temperate phage, PhigspC, exhibits a broad host range, displaying the ability to infect two different Halomonas spp. also isolated from the GSP. The phage infection process demonstrates a high level of tolerance towards temperature, pH and salinity; however, free virions are rapidly inactivated in water unless supplemented with salt. We show that susceptibility to osmotic shock is correlated with the density of the packaged DNA (rho(pack)). Lysogens of Halomonas salina GSP21 were detrimental to host fitness at 10% salinity, but the lysogen was able to grow faster than the wild type at 20% salinity. From these results we propose that the extensive genome of PhigspC may encode environmentally relevant genes (ERGs); genes that are perhaps not essential for the phage life cycle but increase host and phage fitness in some environmental conditions.

Small-colony variants: a novel mechanism for triclosan resistance in methicillin-resistant Staphylococcus aureus.

OBJECTIVES: A little-understood mode of antimicrobial resistance in Staphylococcus aureus is the evolution of a sub-population of small-colony variants (SCVs). SCVs are a cause of persistent and recurring infections refractory to antimicrobial chemotherapy. Following the inadvertent isolation of suspected SCVs growing in the presence of triclosan we set out to evaluate the formation of these colonial mutants and assess their antimicrobial susceptibility. METHODS: SCVs were isolated on Mueller-Hinton agar supplemented with 1 mg/L triclosan. SCV formation frequency was calculated using a selection of both clinical methicillin-resistant S. aureus (MRSA) isolates and methicillin-susceptible S. aureus strains. Antimicrobial susceptibility was assessed and the fabI gene of SCVs was sequenced to ensure resistance was not mediated by mutation of this gene. RESULTS: We have found in vitro that triclosan can select for S. aureus colonies showing the characteristic SCV phenotype with low-level triclosan resistance and which coincidently have reduced susceptibility to penicillin and gentamicin. Additionally, triclosan-isolated SCVs were shown to have an increased tolerance to the lethal effects of triclosan. CONCLUSIONS: We propose the formation of SCVs by S. aureus is a novel mechanism of resistance to low concentrations of triclosan, which for 25 years has been used widely in the domestic setting in various consumer healthcare products. More recently it has been recommended for the control of MRSA. Consequently, our results identify the potential for treatment failure in infections already notoriously difficult to eradicate. It remains unclear to what extent isolates with decreased susceptibility to triclosan would develop and have the fitness to survive under real world conditions.

Plasmids from freshwater environments capable of IncQ retrotransfer are diverse and include pQKH54, a new IncP-1 subgroup archetype.

Nine mercury-resistance plasmids isolated from river epilithon were assessed for their ability to retrotransfer the non-conjugative IncQ plasmid, R300B, derivatives of which have commercial uses that may result in accidental or deliberate release into the environment. Retrotransfer frequencies ranging from 2.1 x 10(-4) to 1.75 x 10(-5) were obtained for five of the nine plasmids--the remaining plasmids showed low or undetectable retrotransfer ability. The majority of the retrotransfer-proficient plasmids could not be classified by the tests used. Classical incompatibility testing with RP4 identified pQKH6, pQKH54 and pQM719 as IncP-1. Hybridization to replicon probes confirmed this for pQKH6 and pQM719 and added pQKH33. PCR with primers designed to amplify trfA and korA regions of IncP-1 plasmids did not identify any other plasmids. Plasmids pQKH6 and pQM719 but not pQKH54 produced similar SphI restriction profiles to the IncP-1beta subgroup. The complete nucleotide sequence of pQKH54 was determined, revealing it to have a complete IncP-1 backbone but belonging to a new distinct subgroup which was designated IncP-1gamma. The results emphasize the ubiquity and diversity of IncP-1 plasmids in the environment but demonstrate that plasmids of as yet unknown groups are also able to retrotransfer IncQ plasmids efficiently.

Elevated abundance of bacteriophage infecting bacteria in soil.

Here we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean = 1.5 x 10(7) g(-1)), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts. Adding pure cultures of a Serratia phage to soil showed that the direct counting methods with electron microscopy developed here underestimated the added phage populations by at least eightfold. So, assuming natural phages were similarly underestimated, virus numbers in soil averaged 1.5 x 10(8) g(-1), which is equivalent to 4% of the total population of bacteria. This high abundance was to some extent confirmed by hybridizing colonies grown on Serratia and Pseudomonas selective media with cocktails of phage infecting these bacteria. This showed that 8.9 and 3.9%, respectively, hybridized with colonies from the two media and confirmed the presence of phage DNA sequences in the cultivable fraction of the natural population. Thus, soil phage, like their aquatic counterparts, are likely to be important in controlling bacterial populations and mediating gene transfer in soil.

Characterization of Serratia isolates from soil, ecological implications and transfer of Serratia proteamaculans subsp. quinovora Grimont et al. 1983 to Serratia quinivorans corrig., sp. nov.

Eleven strains of Serratia were isolated from different soils and the guts of invertebrates and characterized by their sensitivity to eight indigenous bacteriophages. They were also classified according to bacteriocin production and sensitivity, BiOLOG plate and API 20E strip profiles and 16S rRNA sequence information. One strain was thus identified as Serratia plymuthica, another as Serratia fonticola. The remaining strains were shown to be closely related to Serratia proteamaculans subsp. quinovora Grimont et al. 1983 after DNA-DNA cross-hybridization demonstrated relatedness greater than 70% with the type strain of this subspecies. From an ecological perspective, our results illustrated the wide variation in sensitivity that closely related Serratia strains have towards various indigenous soil phages and that these phages have broad host ranges within the genus. Furthermore, the phage and bacteriocin interactions within the Serratia strains examined were intricate and did not reflect phylogenetic relationships. These results together imply that complex interactions will occur in soil within the natural community of Serratia strains and their bacteriophages. DNA-DNA cross-hybridization and phenotypic characterization showed that S. proteamaculans subsp. quinovora strains formed a cohesive group at the species level. It is therefore concluded that these strains should be designated as Serratia quinivorans corrig., sp. nov.