Association of virulence gene expression with colistin-resistance in Acinetobacter baumannii: analysis of genotype, antimicrobial susceptibility, and biofilm formation.

BACKGROUND: Acinetobacter baumannii causes difficult-to-treat nosocomial infections, which often lead to morbidity due to the development of antimicrobial drug resistance and expression of virulence genes. Data regarding the association of resistance to colistin, a last treatment option, and the virulence gene expression of A. baumannii is scarce. METHODS: We evaluated the MLVA genotype, antimicrobial resistance, and biofilm formation of 100 A. baumannii isolates from burn patients, and further compared the in vitro and in vivo expression of four virulence genes among five colistin-resistant A. baumannii (Cst-R-AB) isolates. Five Cst-R-AB isolates were tested; one from the present study, and four isolated previously. RESULTS: Our results showed that reduced expression of recA, along with increased in vivo expression of lpsB, dnaK, and blsA; are associated with colistin resistance among Cst-R-AB isolates. Differences in virulence gene expressions among Cst-R-AB isolates, may in part explain common discrepant in vitro vs. in vivo susceptibility data during treatment of infections caused by Cst-R-AB. CONCLUSIONS: Our findings highlight the intricate relationship between colistin-resistance and virulence among A. baumannii isolates, and underscore the importance of examining the interactions between virulence and antimicrobial resistance toward efforts to control the spread of multidrug-resistant A. baumannii (MDR-AB) isolates, and also to reduce disease severity in burn patients with MDR-AB infection.

Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins.

Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed "Outer Membrane Protein Biogenesis Model" (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the ß-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway.

Genes for two multicopper proteins required for Fe(III) oxide reduction in Geobacter sulfurreducens have different expression patterns both in the subsurface and on energy-harvesting electrodes.

Previous studies have shown that Geobacter sulfurreducens requires the outer-membrane, multicopper protein OmpB for Fe(III) oxide reduction. A homologue of OmpB, designated OmpC, which is 36 % similar to OmpB, has been discovered in the G. sulfurreducens genome. Deletion of ompC inhibited reduction of insoluble, but not soluble Fe(III). Analysis of multiple Geobacter and Pelobacter genomes, as well as in situ Geobacter, indicated that genes encoding multicopper proteins are conserved in Geobacter species but are not found in Pelobacter species. Levels of ompB transcripts were similar in G. sulfurreducens at different growth rates in chemostats and during growth on a microbial fuel cell anode. In contrast, ompC transcript levels increased at higher growth rates in chemostats and with increasing current production in fuel cells. Constant levels of Geobacter ompB transcripts were detected in groundwater during a field experiment in which acetate was added to the subsurface to promote in situ uranium bioremediation. In contrast, ompC transcript levels increased during the rapid phase of growth of Geobacter species following addition of acetate to the groundwater and then rapidly declined. These results demonstrate that more than one multicopper protein is required for optimal Fe(III) oxide reduction in G. sulfurreducens and suggest that, in environmental studies, quantifying OmpB/OmpC-related genes could help alleviate the problem that Pelobacter genes may be inadvertently quantified via quantitative analysis of 16S rRNA genes. Furthermore, comparison of differential expression of ompB and ompC may provide insight into the in situ metabolic state of Geobacter species in environments of interest.

Real-time characterization of virulence factor expression in Yersinia pestis using a GFP reporter system.

A real-time reporter system was developed to monitor the thermal induction of virulence factors in Yersinia pestis, the etiological agent of plague. The reporter system consists of a plasmid in Y. pestis in which the expression of green fluorescent protein (GFP) is under the control of the promoters for six virulence factors, yopE, sycE, yopK, yopT, yscN, and lcrE yopN, which are all components of the Type III secretion virulence mechanism of Y. pestis. Induction of the expression of these genes in vivo was determined by the increase in fluorescence intensity of GFP in real time, in 96-well format. Different basal levels of expression at 26 degrees C were observed for the Y. pestis promoters. Expressed as percentages of the level measured for the lac promoter (positive control), the basal expression levels before temperature shift were: yopE (15%), sycE (15%), yopK (13%), yopT (4%), lcrE (3.3%), and yscN (0.8%). Following the shift in temperature from 26 to 37 degrees C, the rates of expression of these genes increased with the yopE reporter showing the strongest degree of induction. The rates of induction of the other virulence factors after the temperature, expressed as percentages of yopE induction, were: yopK (57%), sycE (9%), yscN (3%), lcrE (3%), and yopT (2%). The thermal induction of each of these promoter fusions was repressed by calcium, and the ratios of the initial rates of thermal induction without calcium supplementation compared to the rate with calcium supplementation were: yopE (11-fold), yscN (7-fold), yopK (6-fold), lcrE (3-fold), yopT (2-fold), and sycE (1-fold). This work demonstrates a novel approach to quantify gene induction and provides a method to rapidly determine the effects of external stimuli on expression of Y. pestis virulence factors in real time, in living cells, as a means to characterize virulence determinants.

[Drug screening model acting on out-membrane protein OprM in pseudomonas aeruginosa efflux pump system].

OBJECTIVE: To establish an efflux pump inhibitor screening model with the out-membrane protein OprM in Pseudomonas aeruginosa efflux pump system as the target point. METHODS: Efflux pump out-membrane protein gene oprM was obtained from standard Pseudomonas aeruginosa PA01 strain. Expression of OprM protein was induced in E. coli strain HS151 with T-easy vector as the cloning vector, and pMMB67EH as the expression vector. In order to evaluate the function of OprM protein, we measured intracellular tetracycline concentrations with liquid scintillation counter, measured the diameters of bacteriostatic circles with paper disc, and then established a screening model accordingly. RESULTS: OprM protein was highly expressed. Using Pseudomonas aeruginosa as the main detecting bacteria, we established a drug screening model acting on OprM. A total of 1 600 microbial fermentation samples were screened with this model, among which 56 positive strains were found, with a positive rate of 3.5%. CONCLUSION: OprM plays an important role in drug efflux. The established model has good specificity and maneuverability.

The outer membrane protein X from Escherichia coli exhibits immune properties.

Outer membrane proteins (OMP) are expressed in Gram-negative bacterial cell wall. OmpA from Klebsiella pneumoniae (KpOmpA) has been shown to bind and to activate selectively antigen presenting cells (APCs), eliciting protective CTL responses. In this study, we investigated whether OmpX, another member of the OMP family and structurally related to OmpA, exhibits the same immune properties. Using recombinant OmpX from Escherichia coli (EcOmpX), we report that EcOmpX binds to and is internalized by human APCs. However, EcOmpX does not activate APCs. EcOmpX acts as an efficient carrier protein as it induces a potent and Th1/Th2 mixed anti-TNP humoral response. However, adjuvant is required to generate a protective anti-tumoral immune response in mice injected with a tumor model antigen coupled to EcOmpX. Collectively, these data show that EcOmpX is recognized by innate cells but does not activate them, suggesting that EcOmpX does not provide a signal danger to APCs. In conclusion, this study provides information on the molecular mechanisms involved in the recognition and activation of innate cells by bacterial outer membrane proteins.

DNA-encoded fetal liver tyrosine kinase 3 ligand and granulocyte macrophage-colony-stimulating factor increase dendritic cell recruitment to the inoculation site and enhance antigen-specific CD4+ T cell responses induced by DNA vaccination of outbred animals.

DNA-based immunization is a contemporary strategy for developing vaccines to prevent infectious diseases in animals and humans. Translating the efficacy of DNA immunization demonstrated in murine models to the animal species that represent the actual populations to be protected remains a significant challenge. We tested two hypotheses directed at enhancing DNA vaccine efficacy in outbred animals. The first hypothesis, that DNA-encoding fetal liver tyrosine kinase 3 ligand (Flt3L) and GM-CSF increases dendritic cell (DC) recruitment to the immunization site, was tested by intradermal inoculation of calves with plasmid DNA encoding Flt3L and GM-CSF followed by quantitation of CD1(+) DC. Peak DC recruitment was detected at 10-15 days postinoculation and was significantly greater (p < 0.05) in calves in the treatment group as compared with control calves inoculated identically, but without Flt3L and GM-CSF. The second hypothesis, that DNA encoding Flt3L and GM-CSF enhances immunity to a DNA vector-expressed Ag, was tested by analyzing the CD4(+) T lymphocyte response to Anaplasma marginale major surface protein 1a (MSP1a). Calves immunized with DNA-expressing MSP1a developed strong CD4(+) T cell responses against A. marginale, MSP1a, and specific MHC class II DR-restricted MSP1a epitopes. Administration of DNA-encoding Flt3L and GM-CSF before MSP1a DNA vaccination significantly increased the population of Ag-specific effector/memory cells in PBMC and significantly enhanced MSP1a-specific CD4(+) T cell proliferation and IFN-gamma secretion as compared with MHC class II DR-matched calves vaccinated identically but without Flt3L and GM-CSF. These results support use of these growth factors in DNA vaccination and specifically indicate their applicability for vaccine testing in outbred animals.

Inactivation of the Moraxella catarrhalis superoxide dismutase SodA induces constitutive expression of iron-repressible outer membrane proteins.

Many pathogens produce one or more superoxide dismutases (SODs), enzymes involved in the detoxification of endogenous and exogenous reactive oxygen species that are encountered during the infection process. One detectable cytoplasmic SOD was identified in the human mucosal pathogen Moraxella catarrhalis, and the gene responsible for the SOD activity, sodA, was isolated from a recent pediatric clinical isolate (strain 7169). Sequence analysis of the cloned M. catarrhalis 7169 DNA fragment revealed an open reading frame of 618 bp encoding a polypeptide of 205 amino acids with 48 to 67% identity to known bacterial manganese-cofactored SODs. An isogenic M. catarrhalis sodA mutant was constructed in strain 7169 by allelic exchange. In contrast to the wild-type 7169, the 7169::sodK20 mutant was severely attenuated for aerobic growth, even in rich medium containing supplemental amino acids, and exhibited extreme sensitivity to the redox-active agent methyl viologen. The ability of recombinant SodA to rescue the aerobic growth defects of E. coli QC774, a sodA sodB-deficient mutant, demonstrated the functional expression of SOD activity by cloned M. catarrhalis sodA. Indirect SOD detection assays were used to visualize both native and recombinant SodA activity in bacterial lysates. This study demonstrates that M. catarrhalis SodA plays a critical role in the detoxification of endogenous, metabolically produced oxygen radicals. In addition, the outer membrane protein (OMP) profile of 7169::sodK20 was consistent with iron starvation in spite of growth under iron-replete conditions. This novel observation indicates that M. catarrhalis strains lacking SodA constitutively express immunogenic OMPs previously described as iron repressible, and this potentially attenuated mutant strain may be an attractive vaccine candidate.

Physiological properties and production of siderophores detected in Yersinia enterocolitica strain 4-32.

A hydrophilic compound with siderophore activity was isolated from a culture of Yersinia enterocolitica 4-32 grown in an iron-deficient medium. It was found that the siderophore secreted did not belong to the catecholamide and hydroxamate type of siderophores and not yersiniabactin. Supplementation of cultures of Y. enterocolitica 4-32 with sodium chloride (300 mM) resulted in a decrease in the production of siderophores.

Quinolobactin, a new siderophore of Pseudomonas fluorescens ATCC 17400, the production of which is repressed by the cognate pyoverdine.

Transposon mutant strain 3G6 of Pseudomonas fluorescens ATCC 17400 which was deficient in pyoverdine production, was found to produce another iron-chelating molecule; this molecule was identified as 8-hydroxy-4-methoxy-quinaldic acid (designated quinolobactin). The pyoverdine-deficient mutant produced a supplementary 75-kDa iron-repressed outer membrane protein (IROMP) in addition to the 85-kDa IROMP present in the wild type. The mutant was also characterized by substantially increased uptake of (59)Fe-quinolobactin. The 75-kDa IROMP was produced by the wild type after induction by quinolobactin-containing culture supernatants obtained from the pyoverdine-negative mutant or by purified quinolobactin. Conversely, adding purified wild-type pyoverdine to the growth medium resulted in suppression of the 75-kDa IROMP in the pyoverdine-deficient mutant; however, suppression was not observed when Pseudomonas aeruginosa PAO1 pyoverdine, a siderophore utilized by strain 3G6, was added to the culture. Therefore, we assume that the quinolobactin receptor is the 75-kDa IROMP and that the quinolobactin-mediated iron uptake system is repressed by the cognate pyoverdine.

Bovine colostrum ameliorates diarrhea in infection with diarrheagenic Escherichia coli, shiga toxin-producing E. Coli, and E. coli expressing intimin and hemolysin.

BACKGROUND: Diarrheagenic Escherichia coli may cause serious extraintestinal complications, but there is no specific treatment. METHODS: Patients with diarrhea caused by diarrheagenic E. coli, specifically Shiga toxin-producing E. coli and E. coli-expressing intimin and enterohemorrhagic E. coli-hemolysin were treated by administration of pooled bovine colostrum, rich in antibodies to Shiga toxin and enterohemorrhagic E. coli-hemolysin, in a placebo-controlled, double-blind study. Symptom resolution and fecal excretion of infecting strains were assessed. RESULTS: No side effects were attributable to colostrum. Stool frequencies in the group treated with bovine colostrum were significantly reduced compared with those in the placebo group. No effect of therapy on the carriage of the pathogens or on complications of the infection could be demonstrated. CONCLUSIONS: Bovine colostrum is well tolerated and diminishes frequency of loose stools in children with E. coli-associated diarrhea. A prospective study should be conducted among a larger number of children with Shiga toxin-producing E. coli identified early in illness, to determine the effectiveness of colostrum therapy.

Environmental growth conditions influence the ability of Escherichia coli K1 to invade brain microvascular endothelial cells and confer serum resistance.

A major limitation to advances in prevention and therapy of neonatal meningitis is our incomplete understanding of the pathogenesis of this disease. In an effort to understand the pathogenesis of meningitis due to Escherichia coli K1, we examined whether environmental growth conditions similar to those that the bacteria might be exposed to in the blood could influence the ability of E. coli K1 to invade brain microvascular endothelial cells (BMEC) in vitro and to cross the blood-brain barrier in vivo. We found that the following bacterial growth conditions enhanced E. coli K1 invasion of BMEC 3- to 10-fold: microaerophilic growth, media buffered at pH 6.5, and media supplemented with 50% newborn bovine serum (NBS), magnesium, or iron. Growth conditions that significantly repressed invasion (i.e., 2- to 250-fold) included iron chelation, a pH of 8.5, and high osmolarity. More importantly, E. coli K1 traversal of the blood-brain barrier was significantly greater for the growth condition enhancing BMEC invasion (50% NBS) than for the condition repressing invasion (osmolarity) in newborn rats with experimental hematogenous meningitis. Of interest, bacterial growth conditions that enhanced or repressed invasion also elicited similar serum resistance phenotype patterns. This is the first demonstration that bacterial ability to enter the central nervous system can be affected by environmental growth conditions.

Choline availability modulates the expression of TGFbeta1 and cytoskeletal proteins in the hippocampus of developing rat brain.

Choline availability influences long-term memory in concert with changes in the spatial organization and morphology of septal neurons, however little is known concerning the effects of choline on the hippocampus, a region of the brain also important for memory performance. Pregnant rats on gestational day 12 were fed a choline control (CT), choline supplemented (CS), or choline deficient (CD) diet for 6 days and fetal brain slices were prepared on embryonic day 18 (E18). The hippocampus in these brain slices was studied for the immunohistochemical localization of the growth-related proteins transforming growth factor beta type 1 (TGFbeta1) and GAP43, the cytoskeletal proteins vimentin and microtubule associated protein type 1 (MAP1), and the neuronal cell marker neuron specific enolase (NSE). In control hippocampus, there was weak expression of TGFbeta1 and vimentin proteins, but moderately intense expression of MAP1 protein. These proteins were not homogeneously distributed, but were preferentially localized to cells with large cell bodies located in the central (approximately CA1-CA3) region of the hippocampus, and to the filamentous processes of small cells in the fimbria region. Feeding a choline-supplemented diet decreased, whereas a choline-deficient diet increased the intensity of immunohistochemical labeling for these proteins in E18 hippocampus. GAP43 and NSE were localized to peripheral nervous tissue but not hippocampus, indicating that the maturation of axons and neurite outgrowth in embryonic hippocampus were unaffected by the availability of choline in the diet. These data suggest that the availability of choline affects the differentiation of specific regions of developing hippocampus.

Acquisition of iron by the non-siderophore-producing Pseudomonas fragi.

The iron requirement, siderophore production and iron uptake mechanisms of the type strain Pseudomonas fragi ATCC 4973 and five P. fragi isolates from meat were analysed. The strains exhibited a high sensitivity to iron starvation: their growth was strongly inhibited in medium supplemented with the iron chelator ethylenediamine di(hydroxyphenylacetic acid) or in medium treated with 8-hydroxyquinoline to remove contaminating iron. No siderophores were detectable in the growth supernatants of iron-starved cells. Cross-feeding experiments in iron-depleted medium showed, however, that the bacterial growth could be strongly stimulated by siderophores of foreign origin including desferriferrioxamine B, enterobactin and some pyoverdines. Moreover, all the strains were capable of efficiently using the iron sources present in their natural environment, i.e., transferrin, lactoferrin and haemoglobin. Iron starvation led to the specific production of supplementary outer-membrane proteins of apparent molecular mass ranging from 80 to 88 kDa. Furthermore, growth in the presence of exogenous siderophores resulted, in some strains, in the induction of siderophore-mediated iron uptake systems. For one strain the concomitant synthesis of an iron-regulated, siderophore-inducible outer-membrane protein was observed.

Iron repressible outer membrane proteins of Moraxella bovis and demonstration of siderophore-like activity.

Moraxella bovis (strain Epp 63), grown in RPMI 1640 medium supplemented with desferrioxamine mesylate (0.05 mg/ml) resulted in cell free culture supernatants with an increased chromeazurol-S response indicating the presence of high affinity iron binding ligand(s). Supernatants of cultures where growth occurred in tryptic soy broth, RPMI 1640, or RPMI 1640-desferrioxamine supplemented with ferrous sulfate (10 micrograms/ml) were negative on the chromeazurol-S test. Growth of M. bovis in RPMI 1640 or RPMI 1640-desferrioxamine medium induced the expression of previously unrecognized outer membrane proteins whose expression was repressed when the medium was supplemented with iron and which were not produced when growth occurred in tryptic soy broth.

Influence of iron depletion on growth kinetics, siderophore production, and protein expression of Staphylococcus aureus.

Growth rates, siderophore secretion, and bacterial proteins of two clinical isolates of Staphylococcus aureus were studied over 72 h of growth in iron-supplemented and iron-restricted chemically defined media. Under iron restriction the growth rates were decreased to different extents depending on the strain. Production of siderophore was detected in the mid-exponential and stationary phases of growth. The expression of iron-regulated proteins of 81, 23, and 17 kDa was time-dependent, associated with the same stage of growth, and might be involved in siderophore efficiency.

Iron uptake by Pasteurella piscicida and its role in pathogenicity for fish.

We evaluated the iron uptake mechanisms in Pasteurella piscicida strains as well as the effect of iron overload on the virulence of these strains for fish. With this aim, the capacity of the strains to obtain iron from transferrin and heme compounds as well as their ability to overcome the inhibitory activity of fish serum was analyzed. All the P. piscicida strains grew in the presence of the iron chelator ethylene-diamine-di (O-hydroxyphenyl acetic acid) or of human transferrin, which was used by a siderophore-mediated mechanism. The chemical tests and cross-feeding assays showed that P. piscicida produced a siderophore which was neither a phenolate nor a hydroxamate. Cross-feeding assays as well as preliminary chromatographic analysis suggest that this siderophore may be chemically related to multocidin. All the P. piscicida isolates utilized hemin and hemoglobin as an iron source, since the virulence of the strains increased when the fish were preinoculated with these compounds. This effect was stronger in the avirulent strains (50% lethal dose was reduced by 4 logs when fish were pretreated with hemin or hemoglobin). Only the pathogenic P. piscicida isolates were resistant to the bactericidal action of the fresh fish serum. The nonpathogenic strains grew in fish serum only when it was heat-inactivated or when it was supplemented with ferric ammonium citrate, hemin, or hemoglobin. In all the strains, at least three iron-regulated outer membrane proteins (IROMPs) (105, 118, and 145 kDa) were increased when the strains were cultured in iron-restricted medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Immune response to Acinetobacter calcoaceticus infection in man.

After growth in an iron-depleted chemically-defined medium Acinetobacter calcoaceticus expressed four high mol. wt outer-membrane proteins (OMPs) which were repressed under iron supplementation or in a complex laboratory medium. Immunoblotting with serum from a septicaemic patient infected with A. calcoaceticus revealed antibody binding to these iron-repressible OMPs, indicating that they were expressed in vivo, and also to the 42- and 18-Kda OMPs. Although the antibody response to the OMPs did not vary significantly during convalescence, the response to the O-polysaccharide component of lipopolysaccharide decreased significantly. However, antibodies in serum from patients with A. calcoaceticus wound infections reacted with the iron repressible OMPs and a 54-Kda antigen suggesting a difference in immune recognition between local and systemic infection.

Regulatory functions of the three nodD genes of Rhizobium leguminosarum biovar phaseoli.

The three nodD genes of a strain of Rhizobium leguminosarum biovar phaseoli were cloned to study their effects on transcription of themselves and of the nodC genes of biovars phaseoli and viciae. Efficient transcription of nodD1 required nodD1 and was enhanced by exposure of the cells to bean exudate consistent with the presence of a nod-box preceding the noIE-nodD1 operon. Transcription of nodD2 and nodD3 was constitutive. nodC of R. leguminosarum biovar phaseoli was activated by each of the nodD genes of that biovar in the absence of inducers but expression was enhanced in cells grown with bean exudate or the flavonoids genistein or naringenin. A mutant of nodD2, lacking 60 bp at its 3' end, activated nodC in the presence of inducer, but was defective in regulating certain of the nodD genes. The nodC gene of R. leguminosarum biovar viciae responded differently to the various nodD genes of R. leguminosarum biovar phaseoli than did the nodC of the latter biovar.

Persistence of Pseudomonas aeruginosa during ciprofloxacin therapy of a cystic fibrosis patient: transient resistance to quinolones and protein F-deficiency.

The mechanism of persistence was characterized in Pseudomonas aeruginosa isolates obtained ten days before (4405), on the tenth day of (4419), and four days after (4478) ciprofloxacin therapy in a cystic fibrosis patient. Isolate 4419 showed a 16-fold increase in resistance to ciprofloxacin, norfloxacin and nalidixic acid. The outer membrane of 4419 had no detectable protein F. A modified lipopolysaccharide profile, a longer lag phase before growth and a slower generation time were also noted for isolate 4419. Cell surface hydrophobicity was increased by 20% in 4419 whereas uptake of [14C]ciprofloxacin was equivalent in all three isolates. Ciprofloxacin doses causing 50% inhibition of DNA synthesis were proportional to MICs for each isolate indicating that the DNA gyrase of 4419 was resistant to quinolones. A quinolone-susceptible revertant of 4419 remained deficient in protein F. Protein F-deficiency was not associated with resistance to quinolones, nor to other antibiotics, supporting the view that it plays little role in outer membrane permeability to antibiotics.