Miniature transposable sequences are frequently mobilized in the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola

Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.661025 and 1.161026, depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria.

Warfarin susceptibility in the lesser bandicoot rat (Bandicota bengalensis)

Warfarin is a first generation anticoagulant that relies on multiple feeding events to achieve lethality in susceptible rodents. For the Bandicoot rat, warfarin susceptibility baselines were established using the lethal feeding period (LFP) test methodology. Against a 0.003% warfarin formulation, LFP50 values of 2 and 4 days, and LFP99 values of 16 and 10 days were obtained for males and females respectively. However, consumption of rodenticide was significantly reduced after the 4th and 5th days of test, at a time when animals would be expected to experience symptoms of warfarin toxicity. This would seriously compromise the Probit analysis, particularly for estimates of higher percentiles. Possible modifications to the methodology are discussed to overcome this problem. Although the majority of animals were highly susceptible to warfarin, one female animal that survived a high dose of active ingredient (79.1 mg kg-1) may bode for future resistance

Metabolism of anthocyanins by human gut microflora and their influence on gut bacterial growth

Consumption of anthocyanins has been related with beneficial health effects. However, bioavailability studies have shown low concentration of anthocyanins in plasma and urine. In this study, we have investigated the bacterial-dependent metabolism of malvidin-3-glucoside, gallic acid and a mixture of anthocyanins using a pH-controlled, stirred, batch-culture fermentation system reflective of the distal human large intestine conditions. Most anthocyanins have disappeared after 5 h incubation while gallic acid remained constant through the first 5 h and was almost completely degraded following 24 h of fermentation. Incubation of malvidin-3-glucoside with fecal bacteria mainly resulted in the formation of syringic acid, while the mixture of anthocyanins resulted in formation of gallic, syringic and p-coumaric acids. All the anthocyanins tested enhanced significantly the growth of Bif idobacterium spp. and Lactobacillus−Enterococcus spp. These results suggest that anthocyanins and their metabolites may exert a positive modulation of the intestinal bacterial population.

Theoretical insights into bacterial chemotaxis

Research into understanding bacterial chemotactic systems has become a paradigm for Systems Biology. Experimental and theoretical researchers have worked hand-in-hand for over 40 years to understand the intricate behavior driving bacterial species, in particular how such small creatures, usually not more than 5 µm in length, detect and respond to small changes in their extracellular environment. In this review we highlight the importance that theoretical modeling has played in providing new insight and understanding into bacterial chemotaxis. We begin with an overview of the bacterial chemotaxis sensory response, before reviewing the role of theoretical modeling in understanding elements of the system on the single cell scale and features underpinning multiscale extensions to population models. WIREs Syst Biol Med 2012 doi: 10.1002/wsbm.1168 For further resources related to this article, please visit the WIREs website.

A process for analysis of microarray comparative genomics hybridisation studies for bacterial genomes

Background: Microarray based comparative genomic hybridisation (CGH) experiments have been used to study numerous biological problems including understanding genome plasticity in pathogenic bacteria. Typically such experiments produce large data sets that are difficult for biologists to handle. Although there are some programmes available for interpretation of bacterial transcriptomics data and CGH microarray data for looking at genetic stability in oncogenes, there are none specifically to understand the mosaic nature of bacterial genomes. Consequently a bottle neck still persists in accurate processing and mathematical analysis of these data. To address this shortfall we have produced a simple and robust CGH microarray data analysis process that may be automated in the future to understand bacterial genomic diversity. Results: The process involves five steps: cleaning, normalisation, estimating gene presence and absence or divergence, validation, and analysis of data from test against three reference strains simultaneously. Each stage of the process is described and we have compared a number of methods available for characterising bacterial genomic diversity, for calculating the cut-off between gene presence and absence or divergence, and shown that a simple dynamic approach using a kernel density estimator performed better than both established, as well as a more sophisticated mixture modelling technique. We have also shown that current methods commonly used for CGH microarray analysis in tumour and cancer cell lines are not appropriate for analysing our data. Conclusion: After carrying out the analysis and validation for three sequenced Escherichia coli strains, CGH microarray data from 19 E. coli O157 pathogenic test strains were used to demonstrate the benefits of applying this simple and robust process to CGH microarray studies using bacterial genomes.

Piezotolerant small-colony variants with increased thermotolerance, antibiotic susceptibility, and low invasiveness in a clonal Staphylococcus aureus population

Following a pressure treatment of a clonal Staphylococcus aureus culture with 400 MPa for 30 min, piezotolerant variants were isolated. Among 21 randomly selected survivors, 9 were piezotolerant and all formed small colonies on several agar media. The majority of the isolates showed increased thermotolerance, impaired growth, and reduced antibiotic resistance compared to the wild type. However, several nonpiezotolerant isolates also demonstrated impaired growth and the small-colony phenotype. In agglutination tests for the detection of protein A and fibrinogen, the piezotolerant variants showed weaker agglutination reactions than the wild type and the other isolates. All variants also showed defective production of the typical S. aureus golden color, a characteristic which has previously been linked with virulence. They were also less able to invade intestinal epithelial cells than the wild type. These S. aureus variants showed phenotypic similarities to previously isolated Listeria monocytogenes piezotolerant mutants that contained mutations in ctsR. Because of these similarities, possible alterations in the ctsR hypermutable regions of the S. aureus variants were investigated through amplified fragment length polymorphism analysis. No mutations were identified, and subsequently we sequenced the ctsR and hrcA genes of three representative variants, finding no mutations. This work demonstrates that S. aureus probably possesses a strategy resulting in an abundance of multiple-stressresistant variants within clonal populations. This strategy, however, seems to involve genes and regulatory mechanisms different from those previously reported for L. monocytogenes. We are in the process of identifying these mechanisms.

A modified rapid enzymatic microtiter plate assay for the quantification of intracellular γ-aminobutyric acid and succinate semialdehyde in bacterial cells

The GABase assay is widely used to rapidly and accurately quantify levels of extracellular γ-aminobutyric acid (GABA). Here we demonstrate a modification of this assay that enables quantification of intracellular GABA in bacterial cells. Cells are lysed by boiling and ethanolamine-O-sulphate, a GABA transaminase inhibitor is used to distinguish between GABA and succinate semialdehyde.

Role of glutamate metabolism in bacterial responses towards acid and other stresses

Glutamate plays a central role in a wide range of metabolic processes in bacterial cells. This review focuses on the involvement of glutamate in bacterial stress responses. In particular it reviews the role of glutamate metabolism in response against acid stress and other stresses. The glutamate decarboxylase (GAD) system has been implicated in acid tolerance in several bacterial genera. This system facilitates intracellular pH homeostasis by consuming protons in a decarboxylation reaction that produces γ-aminobutyrate (GABA) from glutamate. An antiporter system is usually present to couple the uptake of glutamate to the efflux of GABA. Recent insights into the functioning of this system will be discussed. Finally the intracellular fate of GABA will also be discussed. Many bacteria are capable of metabolising GABA to succinate via the GABA shunt pathway. The role and regulation of this pathway will be addressed in the review. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Nanoscale zerovalent iron alters soil bacterial community structure and inhibits chloroaromatic biodegradation potential in Aroclor 1242-contaminated soil

Nanoscale zerovalent iron (nZVI) has potential for the remediation of organochlorine-contaminated environments. Environmental safety concerns associated with in situ deployment of nZVI include potential negative impacts on indigenous microbes whose biodegradative functions could contribute to contaminant remediation. With respect to a two-step polychlorinated biphenyl remediation scenario comprising nZVI dechlorination followed by aerobic biodegradation, we examined the effect of polyacrylic acid (PAA)-coated nZVI (mean diameter = 12.5 nm) applied at 10 g nZVI kg−1 to Aroclor-1242 contaminated and uncontaminated soil over 28 days. nZVI had a limited effect on Aroclor congener profiles, but, either directly or indirectly via changes to soil physico-chemical conditions (pH, Eh), nZVI addition caused perturbation to soil bacterial community composition, and reduced the activity of chloroaromatic mineralizing microorganisms. We conclude that nZVI addition has the potential to inhibit microbial functions that could be important for PCB remediation strategies combining nZVI treatment and biodegradation.

Spirochaetes and other bacterial species associated with bovine digital dermatitis

The 16S rRNA genes from spirochaetes associated with digital dermatitis of British cattle were amplified by polymerase chain reaction from digital dermatitis lesion biopsies using one universal and one treponeme-specific primer. Two treponemal sequences were identified both of which shared a high degree of homology with the oral pathogen Treponema denticola (98%). Two further 16S rRNA gene sequences were obtained and shared similarity to Bacteroides levii (99%) and Mycoplasma hyopharyngis (98%). Polymerase chain reaction with T. denticola-specific primers amplified a potential virulence gene from digital dermatitis lesions which shared a high degree of homology to the 46-kDa haemolysin gene of T. denticola. The significance of the presence of organisms in digital dermatitis lesions of the bovine foot which are closely related to oral pathogens is discussed.

Use of a LightCycler gyrA mutation assay for rapid identification of mutations conferring decreased susceptibility to ciprofloxacin in multiresistant Salmonella enterica serotype typhimurium DT104 isolates

A LightCycler-based PCR-hybridization gyrA mutation assay (GAMA) was developed to rapidly detect gyrA point mutations in multiresistant (MR) Salmonella enterica serotype Typhimurium DT104 with decreased susceptibility to ciprofloxacin (MIC, 0.25 to 1.0 mg/liter). Ninety-two isolates (49 human, 43 animal) were tested with three individual oligonucleotide probes directed against an Asp-87-to-Asn (GAC --> AAC) mutation, an Asp-87-to-Gly (GAC --> GGC) mutation, and a Ser-83-to-Phe (TCC --> TTC) mutation. Strains homologous to the probes could be distinguished from strains that had different mutations by their probe-target melting temperatures. Thirty-seven human and 30 animal isolates had an Asp-87-to-Asn substitution, 6 human and 6 animal isolates had a Ser-83-to-Phe substitution, and 5 human and 2 animal isolates had an Asp-87-to-Gly substitution. The remaining six strains all had mismatches with the three probes and therefore different gyrA mutations. The sequencing of gyrA from these six isolates showed that one human strain and two animal strains had an Asp-87-to-Tyr (GAC --> TAC) substitution and two animal strains had a Ser-83-to-Tyr (TCC --> TAC) substitution. One animal strain had no gyrA mutation, suggesting that this isolate had a different mechanism of resistance. Fifty-eight of the strains tested were indistinguishable by several different typing methods including antibiograms, pulsed-field gel gel electrophoresis, and plasmid profiling, although they could be further subdivided according to gyrA mutation. This study confirmed that MR DT104 with decreased susceptibility to ciprofloxacin from humans and food animals in England and Wales may have arisen independently against a background of clonal spread of MR DT104.

In vivo characterization of Lactobacillus johnsonii FI9785 for use as a defined competitive exclusion agent against bacterial pathogens in poultry

Aims: To test the efficacy of Lactobacillus johnsonii FI9785 in reducing the colonization and shedding of Salmonella enterica serotype Enteritidis, Escherichia coli O78:K80 and Clostridium perfringens in poultry. Methods and Results: Specific pathogen-free chicks (1 day old) were dosed with a single oral inoculum of 1 x 10(9) CFU. Lactobacillus johnsonii FI9785 and 24 h later were challenged in separate experiments with S. Enteritidis (S1400, nal(r)) and E. coli O78:K80 (EC34195, nal(r)). There were no significant effects against S. Enteritidis whereas colonization of the small intestine by E. coli O78:K80 was reduced significantly. Both S. Enteritidis and E. coli colonized the caeca and colon to levels equivalent to control birds and there was no reduction in shedding as assessed by a semi-quantitative cloacal swabbing technique. Specific pathogen-free chicks (20 day old) were dosed with a single oral inoculum of 1 x 10(9) CFU L. johnsonii FI9785 and 24 h later were challenged with C. perfringens. A single oral dose of L. johnsonii FI9785 was sufficient to suppress all aspects of colonization and persistence of C. perfringens. Conclusions: Lactobacillus johnsonii FI9785 may be given to poultry for use as a competitive exclusion agent to control C. perfringens. Significance and Impact of the Study: Lactobacillus johnsonii FI9785 may be a valuable tool to control the endemic disease of necrotic enteritis, thereby reducing economic losses associated with reduced use of antimicrobials in the poultry industry.

Modification of enrofloxacin treatment regimens for poultry experimentally infected with Salmonella enterica serovar Typhimurium DT104 to minimize selection of resistance

We hypothesized that higher doses of fluoroquinolones for a shorter duration could maintain efficacy (as measured by reduction in bacterial count) while reducing selection in chickens of bacteria with reduced susceptibility. Chicks were infected with Salmonella enterica serovar Typhimurium DT104 and treated 1 week later with enrofloxacin at the recommended dose for 5 days (water dose adjusted to give 10 mg/kg of body weight of birds or equivalence, i.e., water at 50 ppm) or at 2.5 or 5 times the recommended dose for 2 days or 1 day, respectively. The dose was delivered continuously (ppm) or pulsed in the water (mg/kg) or by gavage (mg/kg). In vitro in sera, increasing concentrations of 0.5 to 8 mu g/ml enrofloxacin correlated with increased activity. In vivo, the efficacy of the 1-day treatment was significantly less than that of the 2- and 5-day treatments. The 2-day treatments showed efficacy similar to that of the 5-day treatment in all but one repeat treatment group and significantly (P < 0.01) reduced the Salmonella counts. Dosing at 2.5x the recommended dose and pulsed dosing both increased the peak antibiotic concentrations in cecal contents, liver, lung, and sera as determined by high-pressure liquid chromatography. There was limited evidence that shorter treatment regimens (in particular the 1-day regimen) selected for fewer strains with reduced susceptibility. In conclusion, the 2-day treatment would overall require a shorter withholding time than the 5-day treatment and, in view of the increased peak antibiotic concentrations, may give rise to improved efficacy, in particular for treating respiratory and systemic infections. However, it would be necessary to validate the 2-day regimen in a field situation and in particular against respiratory and systemic infections to validate or refute this hypothesis.

Commonly used farm disinfectants can select for mutant Salmonella enterica serovar Typhimurium with decreased susceptibility to biocides and antibiotics without compromising virulence

Objectives: To determine if one passage of Salmonella enterica serovar Typhimurium in the presence of farm disinfectants selected for mutants with decreased susceptibility to disinfectants and/or antibiotics. Methods: Eight Salmonella Typhimurium strains including field isolates and laboratory mutants were exposed to either a tar oil phenol (PFD) disinfectant, an oxidizing compound disinfectant (OXC), an aldehyde based disinfectant (ABD) or a dairy sterilizer disinfectant (based on quaternary ammonium biocide) in agar. The susceptibility of mutants obtained after disinfectant exposure to antibiotics and disinfectants was determined as was the accumulation of norfloxacin. The proteome of SL1344 after exposure to PFD and OXC was analysed using two-dimensional liquid chromatography mass spectrometry. Results: Strains with either acrB or tolC inactivated were more susceptible to most disinfectants than other strains. The majority (3/5) of mutants recovered after disinfectant exposure required statistically significantly longer exposure times to disinfectants than their parent strains to generate a 5 log kill. Small decreases in antibiotic susceptibility were observed but no mutants were multiply antibiotic-resistant (MAR). Notably exposure to ABD decreased susceptibility to ciprofloxacin in some strains. Mutants with increased disinfectant tolerance were able to survive and persist in chicks as well as in parent strains. Analysis of proteomes revealed significantly increased expression of the AcrAB-TolC efflux system after PFD exposure. Conclusions: Data presented demonstrate that efflux pumps are required for intrinsic resistance to some disinfectants and that exposure to disinfectants can induce expression of the AcrAB-TolC efflux system, but that single exposure was insufficient to select for MAR strains.