Regulatory roles of the GacS/GacA two-component system in plant-associated and other gram-negative bacteria.

The sensor kinase GacS and the response regulator GacA are members of a two-component system that is present in a wide variety of gram-negative bacteria and has been studied mainly in enteric bacteria and fluorescent pseudomonads. The GacS/GacA system controls the production of secondary metabolites and extracellular enzymes involved in pathogenicity to plants and animals, biocontrol of soilborne plant diseases, ecological fitness, or tolerance to stress. A current model proposes that GacS senses a still-unknown signal and activates, via a phosphorelay mechanism, the GacA transcription regulator, which in turn triggers the expression of target genes. The GacS protein belongs to the unorthodox sensor kinases, characterized by an autophosphorylation, a receiver, and an output domain. The periplasmic loop domain of GacS is poorly conserved in diverse bacteria. Thus, a common signal interacting with this domain would be unexpected. Based on a comparison with the transcriptional regulator NarL, a secondary structure can be predicted for the GacA sensor kinases. Certain genes whose expression is regulated by the GacS/GacA system are regulated in parallel by the small RNA binding protein RsmA (CsrA) at a posttranscriptional level. It is suggested that the GacS/GacA system operates a switch between primary and secondary metabolism, with a major involvement of posttranscriptional control mechanisms.

Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria.

The minimal replicon of the Pseudomonas plasmid pVS1 was genetically defined and combined with the Escherichia coli p15A replicon, to provide a series of new, oligocopy cloning vectors (5.3 to 8.3 kb). Recombinant plasmids derived from these vectors were stable in growing and nongrowing cells of root-colonizing P. fluorescens strains incubated under different environmental conditions for more than 1 month.

Sensing Gram-negative bacteria: a phylogenetic perspective.

Gram-negative bacteria represent a major group of pathogens that infect all eukaryotes from plants to mammals. Gram-negative microbe-associated molecular patterns include lipopolysaccharides and peptidoglycans, major immunostimulatory determinants across phyla. Recent advances have furthered our understanding of Gram-negative detection beyond the well-defined pattern recognition receptors such as TLR4. A B-type lectin receptor for LPS and Lysine-motif containing receptors for peptidoglycans were recently added to the plant arsenal. Caspases join the ranks of mammalian cytosolic immune detectors by binding LPS, and make TLR4 redundant for septic shock. Fascinating bacterial evasion mechanisms lure the host into tolerance or promote inter-bacterial competition. Our review aims to cover recent advances on bacterial messages and host decoding systems across phyla, and highlight evolutionarily recurrent strategies.

Osmotic upshift transiently inhibits uptake via ABC transporters in gram-negative bacteria

ATP-binding cassette transporters from several rhizobia and Salmonella enterica serovar Typhimurium, but not secondarily coupled systems, were inhibited by high concentrations (100 to 500 mM) of various osmolytes, an effect reversed by the removal of the osmolyte. ABC systems were also inactivated in isolated pea bacteroids, probably due to the obligatory use of high-osmolarity isolation media. Measurement of nutrient cycling in isolated pea bacteroids is impeded by this effect.

A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram-negative bacteria

A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon). Reporter fusions can be made to gfpUV, gfprnut3.1, unstable gfpmut3.1 variants (LAA, LVA, AAV and ASV), gfp+, dsRed2, dsRedT3, dsRedT4, mRFP1, gusA or lacZ. The two vector families, pOT and pJP2, are compatible with one another and share the same polylinker for facile interchange of promoter regions. Vectors based on pJP2 have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. As a confirmation of their usefulness, the dicarboxylic acid transport system promoter (dctA(p)) was cloned into a pOT (pRU1097)- and a pJP2 (pRU1156)-based vector and shown to be expressed by Rhizobium leguminosarum in infection threads of vetch. This indicates the presence of dicarboxylates at the earliest stages of nodule formation.

Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum beta-lactamases. Validation of the array with control strains demonstrated a 99% correlation between polymerase chain reaction and array results. There was also good correlation between phenotypic and genotypic results for a large panel of Escherichia coli and Salmonella isolates. Some differences were also seen in the number and type of resistance genes harboured by E. coli and Salmonella strains. The array provides an effective, fast and simple method for detection of resistance genes in clinical isolates suitable for use in diagnostic laboratories, which in future will help to understand the epidemiology of isolates and to detect gene linkage in bacterial populations. (C) 2008 Published by Elsevier B.V. and the International Society of Chemotherapy.

Sub-MICs of Mentha piperita essential oil and menthol inhibits AHL mediated quorum sensing and biofilm of Gram-negative bacteria

Bacterial quorum sensing (QS) is a density dependent communication system that regulates the expression of certain genes including production of virulence factors in many pathogens. Bioactive plant extract/compounds inhibiting QS regulated gene expression may be a potential candidate as antipathogenic drug. In this study anti-QS activity of peppermint (Menthe piperita) oil was first tested using the Chromobacterium violaceum CVO26 biosensor. Further, the findings of the present investigation revealed that peppermint oil (PMO) at sub-Minimum Inhibitory Concentrations (sub-MICs) strongly interfered with acyl homoserine lactone (AHL) regulated virulence factors and biofilm formation in Pseudomonas aeruginosa and Aeromonas hydrophila. The result of molecular docking analysis attributed the QS inhibitory activity exhibited by PMO to menthol. Assessment of ability of menthol to interfere with QS systems of various Gram-negative pathogens comprising diverse AHL molecules revealed that it reduced the AHL dependent production of violacein, virulence factors, and biofilm formation indicating broad-spectrum anti-QS activity. Using two Escherichia colt biosensors, MG4/pKDT17 and pEAL08-2, we also confirmed that menthol inhibited both the las and pqs QS systems. Further, findings of the in vivo studies with menthol on nematode model Caenorhabditis elegans showed significantly enhanced survival of the nematode. Our data identified menthol as a novel broad spectrum QS inhibitor.

Chemical resistance of the gram-negative bacteria to different sanitizers in a water purification system

Abstract Background Purified water for pharmaceutical purposes must be free of microbial contamination and pyrogens. Even with the additional sanitary and disinfecting treatments applied to the system (sequential operational stages), Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were isolated and identified from a thirteen-stage purification system. To evaluate the efficacy of the chemical agents used in the disinfecting process along with those used to adjust chemical characteristics of the system, over the identified bacteria, the kinetic parameter of killing time (D-value) necessary to inactivate 90% of the initial bioburden (decimal reduction time) was experimentally determined. Methods Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were called in house (wild) bacteria. Pseudomonas diminuta ATCC 11568, Pseudomonas alcaligenes INCQS , Pseudomonas aeruginosa ATCC 15442, Pseudomonas fluorescens ATCC 3178, Pseudomonas picketti ATCC 5031, Bacillus subtilis ATCC 937 and Escherichia coli ATCC 25922 were used as 'standard' bacteria to evaluate resistance at 25°C against either 0.5% citric acid, 0.5% hydrochloric acid, 70% ethanol, 0.5% sodium bisulfite, 0.4% sodium hydroxide, 0.5% sodium hypochlorite, or a mixture of 2.2% hydrogen peroxide (H2O2) and 0.45% peracetic acid. Results The efficacy of the sanitizers varied with concentration and contact time to reduce decimal logarithmic (log10) population (n cycles). To kill 90% of the initial population (or one log10 cycle), the necessary time (D-value) was for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite, D = 7.9 min; (vii) mixture of hydrogen peroxide (2.2%) plus peracetic acid (0.45%), D = 5.5 min. Conclusion The contact time of 180 min of the system with the mixture of H2O2+ peracetic acid, a total theoretical reduction of 6 log10 cycles was attained in the water purified storage tank and distribution loop. The contact time between the water purification system (WPS) and the sanitary agents should be reviewed to reach sufficient bioburden reduction (over 6 log10).

Similarities between the DNA replication initiators of Gram-negative bacteria plasmids (RepA) and eukaryotes (Orc4p)/archaea (Cdc6p)

The proteins responsible for the initiation of DNA replication are thought to be essentially unrelated in bacteria and archaea/eukaryotes. Here we show that RepA, the initiator from the Pseudomonas plasmid pPS10, and the C-terminal domain of ScOrc4p, a subunit of Saccharomyces cerevisiae (Sc) origin recognition complex (ORC), share sequence similarities. Based on biochemical and spectroscopic evidence, these similarities include common structural elements, such as a winged-helix domain and a leucine-zipper dimerization motif. We have also found that ScOrc4p, as previously described for RepA-type initiators, interacts with chaperones of the Hsp70 family both in vitro and in vivo, most probably to regulate the assembly of active ORC. In evolutionary terms, our results are compatible with the recruitment of the same protein module for initiation of DNA replication by the ancestors of present-day Gram-negative bacteria plasmids, archaea, and eukaryotes.

Purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, Bombyx mori.

A 50-kDa hemolymph protein, having strong affinity to the cell wall of Gram(-) bacteria, was purified from the hemolymph of the silkworm, Bombyx mori. The cDNA encoding this Gram(-) bacteria-binding protein (GNBP) was isolated from an immunized silkworm fat body cDNA library and sequenced. Comparison of the deduced amino acid sequence with known sequences revealed that GNBP contained a region displaying significant homology to the putative catalytic region of a group of bacterial beta-1,3 glucanases and beta-1,3-1,4 glucanases. Silkworm GNBP was also shown to have amino acid sequence similarity to the vertebrate lipopolysaccharide receptor CD14 and was recognized specifically by a polygonal anti-CD14 antibody. Northern blot analysis showed that GNBP was constitutively expressed in fat body, as well as in cuticular epithelial cells of naive silkworms. Intense transcription was, however, rapidly induced following a cuticular or hemoceolien bacterial challenge. An mRNA that hybridized with GNBP cDNA was also found in the l(2)mbn immunocompetent Drosophila cell line. These observations suggest that GNBP is an inducible acute phase protein implicated in the immune response of the silkworm and perhaps other insects.

Gram-negative organisms : an approach to identification (guide to presumptive identification) /

"Reprinted August 1985."

The postantibiotic effect of the carbapenem antibiotics on gram-negative bacteria

Postantibiotic effect (PAE) describes the suppression of microbial growth occurring after a short exposure to an antimicrobial agent. PAE appears to be a property of the majority of antimicrobial agents and is demonstrated by a wide variety of microorganisms. At present, carbapenems and penems are the only members of the -lactam group of antimicrobial agents that exhibit a significant PAE on Gram-negative bacilli. A standardised method was developed to evaluate the in vitro PAE of three carbapenems; imipenem, meropenem and biapenem on Gram-negative bacteria under reproducible laboratory conditions that partially mimicked those occurring in vivo. The effects on carbapenem PAE of the method of antimicrobial removal, concentration, exposure duration, inoculum size, inoculum growth phase, multiple exposures and pooled human serum were determined. Additionally, the reproducibility, susceptibility prior to and after PAE determination and inter-strain variation of carbapenem PAE were evaluated. The method developed determined PAE by utilising viable counts and demonstrated carbapenem PAE to be reproducible, constant over successive exposures, dependent on genera, concentration, duration of exposure, inoculum size and growth phase. In addition, carbapenem PAE was not significantly effected either by agitation, the antimicrobial removal method or the viable count diluent. At present, the mechanism underlying PAE is undetermined. It is thought to be due to either the prolonged persistence of the antimicrobial at the cellular site of action or the true recovery period from non-lethal damage. Increasing the L-lysine concentration and salinity at recovery decreased and increased the carbapenem and imipenem PAE of Pseudomonas aeruginosa, respectively. In addition, no apparent change was observed in the production of virulence factors by P.aeruginosa in PAE phase. However, alterations in cell morphology were observed throughout PAE phase, and the reappearance of normal cell morphology corresponded to the duration of PAE determined by viable count. Thus, the recovery of the penicillin binding protein target enzymes appears to be the mechanism behind carbapenem PAE in P. aeruginosa.

Prevalence of AmpC β-lactamase among Gram-negative bacteria recovered from clinical specimens in Benin City, Nigeria

Purpose: Infections caused by AmpC-positive bacteria results in high patient morbidity and mortality making their detection clinically important as they cannot be detected in routine susceptibility testing. This study aim to determine the prevalence of AmpC β-lactamase among Gram negative bacteria recovered from clinical specimens in Benin City, Nigeria. Methods: A total of 256 consecutive and non-repetitive Gram negative bacteria were recovered from various clinical specimens. The prevalence of AmpC β-lactamase was determined using a combination of disc antagonism test and cefoxitin-cloxacillin inhibition test. Disc susceptibility test was performed on all isolates using standard techniques. Results: Cefoxitin-cloxacillin inhibition test detected more AmpC β-lactamase than other tests. The prevalence of AmpC β-lactamase did not differ significantly between both genders and between inpatients and out-patients (p>0.05). Isolates recovered from sputum had significantly higher prevalence of AmpC β-lactamase producers compared with isolates from other clinical specimens (p=0.0484). The prevalence of AmpC production was significantly higher among isolates of Pseudomonas aeruginosa than other isolates (p = 0.0085). Isolates that produced AmpC β-lactamase were more susceptible to the test cephalosoprins. Conclusion: An overall prevalence of AmpC β-lactamase (15.23 %) was observed in this study. Pseudomonas aeruginosa was the most prevalent producer of AmpC enzymes. Prudent use of antibiotics is advocated.

In vitro activities of tigecycline combined with other antimicrobials against multiresistant gram-positive and gram-negative pathogens.

OBJECTIVES: To test the activity of tigecycline combined with 16 antimicrobials in vitro against 22 gram-positive and 55 gram-negative clinical isolates. METHODS: Antibiotic interactions were determined by chequerboard and time-kill methods. RESULTS: By chequerboard, of 891 organism-drug interactions tested, 97 (11%) were synergistic, 793 (89%) were indifferent and 1 (0.1%) was antagonistic. Among gram-positive pathogens, most synergisms occurred against Enterococcus spp. (7/11 isolates) with the tigecycline/rifampicin combination. No antagonism was detected. Among gram-negative organisms, synergism was observed mainly with trimethoprim/sulfamethoxazole against Serratia marcescens (5/5 isolates), Proteus spp. (2/5) and Stenotrophomonas maltophilia (2/5), with aztreonam against S. maltophilia (3/5), with cefepime and imipenem against Enterobacter cloacae (3/5), with ceftazidime against Morganella morganii (3/5), and with ceftriaxone against Klebsiella pneumoniae (3/5). The only case of antagonism occurred against one S. marcescens with the tigecycline/imipenem combination. Selected time-kill assays confirmed the bacteriostatic interactions observed by the chequerboard method. Moreover, they revealed a bactericidal synergism of tigecycline with piperacillin/tazobactam against one penicillin-resistant Streptococcus pneumoniae and with amikacin against Proteus vulgaris. CONCLUSIONS: Combinations of tigecycline with other antimicrobials produce primarily an indifferent response. Specific synergisms, especially against enterococci and problematic gram-negative isolates, might be worth investigating in in vitro models and/or in animal models simulating the human environment.

Factors associated with bacteremia due to multidrug-resistant Gram-negative bacilli in hematopoietic stem cell transplant recipients

The epidemiology of bacteremia developing during neutropenia has changed in the past decade, with the re-emergence of Gram-negative (GN) bacteria and the development of multidrug resistance (MDR) among GN bacteria. We conducted a case-control study in order to identify factors associated with bacteremia due to multidrug-resistant Gram-negative (MDRGN) isolates in hematopoietic stem cell transplant recipients. Ten patients with MDRGN bacteremia were compared with 44 patients with GN bacteremia without MDR. Bacteremia due to Burkholderia or Stenotrophomonas sp was excluded from analysis (3 cases), because the possibility of intrinsical resistance. Infection due to MDRGN bacteria occurred in 2.9% of 342 hematopoietic stem cell transplant recipients. Klebsiella pneumoniae was the most frequent MDRGN (4 isolates), followed by Pseudomonas aeruginosa (3 isolates). Among non-MDRGN, P. aeruginosa was the most frequent agent (34%), followed by Escherichia coli (30%). The development of GN bacteremia during the empirical treatment of febrile neutropenia (breakthrough bacteremia) was associated with MDR (P < 0.001, odds ratio = 32, 95% confidence interval = 5_190) by multivariate analysis. Bacteremia due to MDRGN bacteria was associated with a higher death rate by univariate analysis (40 vs 9%; P = 0.03). We were unable to identify risk factors on admission or at the time of the first fever, but the occurrence of breakthrough bacteremia was strongly associated with MDRGN bacteria. An immediate change in the antibiotic regimen in such circumstances may improve the prognosis of these patients.