Kok-Fai Kong

Inhibition of Quorum Sensing-Controlled Virulence Factor Production in Pseudomonas aeruginosa by South Florida Plant Extracts

ABSTRACT Quorum sensing (QS) is a key regulator of virulence and biofilm formation in Pseudomonas aeruginosa and other medically relevant bacteria. Aqueous extracts of six plants, Conocarpus erectus, Chamaesyce hypericifolia, Callistemon viminalis, Bucida buceras, Tetrazygia bicolor, and Quercus virginiana, were examined in this study for their effects on P. aeruginosa virulence factors and the QS system. C. erectus, B. buceras, and C. viminalis caused a significant inhibition of LasA protease, LasB elastase, pyoverdin production, and biofilm formation. Additionally, each plant presented a distinct effect profile on the las and rhl QS genes and their respective signaling molecules, suggesting that different mechanisms are responsible for efficacy. Extracts of all plants caused the inhibition of QS genes and QS-controlled factors, with marginal effects on bacterial growth, suggesting that the quorum-quenching mechanisms are unrelated to static or cidal effects.

Beta‐lactam antibiotics: from antibiosis to resistance and bacteriology

Kong K‐F, Schneper L, Mathee K. Beta‐lactam antibiotics: from antibiosis to resistance and bacteriology. APMIS 2010; 118: 1–36.

Pseudomonas aeruginosa AmpR Is a Global Transcriptional Factor That Regulates Expression of AmpC and PoxB β-Lactamases, Proteases, Quorum Sensing, and Other Virulence Factors

ABSTRACT In members of the family Enterobacteriaceae, ampC, which encodes a β-lactamase, is regulated by an upstream, divergently transcribed gene, ampR. However, in Pseudomonas aeruginosa, the regulation of ampC is not understood. In this study, we compared the characteristics of a P. aeruginosa ampR mutant, PAOampR, with that of an isogenic ampR+ parent. The ampR mutation greatly altered AmpC production. In the absence of antibiotic, PAOampR expressed increased basal β-lactamase levels. However, this increase was not followed by a concomitant increase in the PampC promoter activity. The discrepancy in protein and transcription analyses led us to discover the presence of another chromosomal AmpR-regulated β-lactamase, PoxB. We found that the expression of P. aeruginosa ampR greatly altered the β-lactamase production from ampC and poxB in Escherichia coli: it up-regulated AmpC but down-regulated PoxB activities. In addition, the constitutive PampR promoter activity in PAOampR indicated that AmpR did not autoregulate in the absence or presence of inducers. We further demonstrated that AmpR is a global regulator because the strain carrying the ampR mutation produced higher levels of pyocyanin and LasA protease and lower levels of LasB elastase than the wild-type strain. The increase in LasA levels was positively correlated with the PlasA, PlasI, and PlasR expression. The reduction in the LasB activity was positively correlated with the PrhlR expression. Thus, AmpR plays a dual role, positively regulating the ampC, lasB, and rhlR expression levels and negatively regulating the poxB, lasA, lasI, and lasR expression levels.

Panax ginseng has anti-infective activity against opportunistic pathogen Pseudomonas aeruginosa by inhibiting quorum sensing, a bacterial communication process critical for establishing infection.

Virulent factors produced by pathogens play an important role in the infectious process, which is regulated by a cell-to-cell communication mechanism called quorum sensing (QS). Pseudomonas aeruginosa is an important opportunistic human pathogen, which causes infections in patients with compromised immune systems and cystic fibrosis. The QS systems of P. aeruginosa use N-acylated homoserine lactone (AHL) as signal molecules. Previously we have demonstrated that Panax ginseng treatment allowed the animals with P. aeruginosa pneumonia to effectively clear the bacterial infection. We postulated that the ability to impact the outcome of infections is partly due to ginseng having direct effect on the production of P. aeruginosa virulence factors. The study explores the effect of ginseng on alginate, protease and AHL production. The effect of ginseng extracts on growth and expression of QS-controlled virulence factors on the prototypic P. aeruginosa PAO1 and its isogenic mucoid variant (PAOmucA22) was determined. Ginseng did not inhibit the growth of the bacteria, enhanced the extracellular protein production and stimulated the production of alginate. However, ginseng suppressed the production of LasA and LasB and down-regulated the synthesis of the AHL molecules. Ginseng has a negative effect on the QS system of P. aeruginosa, may explain the ginseng-dependent bacterial clearance from the animal lungs in vivo in our previous animal study. It is possible that enhancing and repressing activities of ginseng are mutually exclusive as it is a complex mixture, as shown with the HPLC analysis of the hot water extract. Though ginseng is a promising natural synergetic remedy, it is important to isolate and evaluate the ginseng compounds associated with the anti-QS activity.

Pseudomonas aeruginosa β-lactamase induction requires two permeases, AmpG and AmpP

BackgroundIn Enterobacteriaceae, β-lactam antibiotic resistance involves murein recycling intermediates. Murein recycling is a complex process with discrete steps taking place in the periplasm and the cytoplasm. The AmpG permease is critical to this process as it transports N-acetylglucosamine anhydrous N-acetylmuramyl peptides across the inner membrane. In Pseudomonadaceae, this intrinsic mechanism remains to be elucidated. Since the mechanism involves two cellular compartments, the characterization of transporters is crucial to establish the link.ResultsPseudomonas aeruginosa PAO1 has two ampG paralogs, PA4218 (ampP) and PA4393 (ampG). Topology analysis using β-galactosidase and alkaline phosphatase fusions indicates ampP and ampG encode proteins which possess 10 and 14 transmembrane helices, respectively, that could potentially transport substrates. Both ampP and ampG are required for maximum expression of β-lactamase, but complementation and kinetic experiments suggest they act independently to play different roles. Mutation of ampG affects resistance to a subset of β-lactam antibiotics. Low-levels of β-lactamase induction occur independently of either ampP or ampG. Both ampG and ampP are the second members of two independent two-gene operons. Analysis of the ampG and ampP operon expression using β-galactosidase transcriptional fusions showed that in PAO1, ampG operon expression is β-lactam and ampR-independent, while ampP operon expression is β-lactam and ampR-dependent. β-lactam-dependent expression of the ampP operon and independent expression of the ampG operon is also dependent upon ampP.ConclusionsIn P. aeruginosa, β-lactamase induction occurs in at least three ways, induction at low β-lactam concentrations by an as yet uncharacterized pathway, at intermediate concentrations by an ampP and ampG dependent pathway, and at high concentrations where although both ampP and ampG play a role, ampG may be of greater importance. Both ampP and ampG are required for maximum induction. Similar to ampC, ampP expression is inducible in an ampR-dependent manner. Importantly, ampP expression is autoregulated and ampP also regulates expression of ampG. Both AmpG and AmpP have topologies consistent with functions in transport. Together, these data suggest that the mechanism of β-lactam resistance of P. aeruginosa is distinct from well characterized systems in Enterobacteriaceae and involves a highly complicated interaction between these putative permeases and known Amp proteins.

96 RACIAL DIFFERENCES IN PROSTATE-RELATED DISORDERS IN A MULTIETHNIC COMMUNITY.

Prostate cancer represents a major health problem worldwide. The aim of this study is to determine if the rate of disease screening plays a major role in prostate cancer development in different ethnic groups. Other factors, including PSA levels, smoking, and size of the prostate glands and different pathological diagnosis are also evaluated in this study. Method A retrospective study involving 190 patients, who showed either prostate-related symptoms or enlarged prostate glands in routine digital rectal examination (DRE), prostate-specific antigen (PSA) and prostate sonograms were done to evaluate for the size of the gland. Patients with the PSA levels > 4 ng/mL got transrectal prostate biopsy. The age of diagnosis, race, PSA level, and gland size were compared among patients with adenocarcinoma, benign adenomatous hyperplasia (BPH) and chronic prostatitis. Results In our study there were 35.4% Afro-Americans, 30% whites, and 17.4% Hispanics and Asians each. The mean age of diagnosing patients with prostate cancer was 65.7 years; for BPH and chronic prostatitis it was 64.4 years. Though the whites were diagnosed to have prostate carcinoma 5 years earlier than the Asians and Hispanics and 7 years earlier than the Afro-Americans, there was no statistical significance between the ethnic groups. Comparisons of the PSA levels with the overall size of the prostate gland (p = .0519), age (p = .6182), smoking (p = .025), and the duration of smoking (p = .1292) failed to show any statistical significance. There was a strong positive correlation between disease occurrence and PSA level. Only 11% of the healthy individuals had PSA level of > 10 ng/mL, 36% of patients with BPH, 38% with chronic prostatitis, and 60% of patients with adenocarcinoma (p < .05), conforming that PSA is an important diagnosis marker for prostate abnormalities. Conclusion We did not find any delay in screening among the ethnic groups, probably due to an increase in awareness of the mortality due to prostate cancer. The incidence of different pathological diagnosis also did not differ among the ethnic groups. Though PSA levels did not correlate with the age, size of the gland, or the smoking, there was a good correlation with patients with adenocarcinoma as compared to patients with BPH or chronic prostatitis.

108 ANTIBIOTIC SENSITIVITY OF PSEUDOMONAS AERUGINOSA ISOLATED FROM ENDOTRACHEAL TUBES IN A COMMUNITY-BASED HOSPITAL.

Introduction Although Pseudomonas aeruginosa is generally a commensal organism it is a major opportunistic bacterial pathogen in nosocomial infections. To ensure optimal efficiency of antibiotic treatment, antibiotic susceptibility tests must be performed and interpreted with caution before prescription. Purpose To determine the antibiotic sensitivity of P. aeruginosa isolated from endotracheal tubes and to design an optimum antibiotic regimen that could be used to effectively treat infections with Pseudomonas. Method A 3-year retrospective study was done on the tracheal aspirates in all the intubated patients. A total of 240 P. aeruginosa positive cultures were identified using Gram staining and specific culturing techniques. Subsequently, the antibiotic sensitivity of these isolates was assayed. The MIC of was checked with ceftazidime (third-generation cephalosporin), cefepime (fourth-generation cephalosporin), piperacillin, piperacillin-tazobactam, ticarcillin-clavulanate, imipenem, amikacin, gentamicin, and tobramycin. Results In our study, 64% of the P. aeruginosa isolates were sensitive to ceftazidime, and 23% were resistant However, when the MIC of a fourth-generation cephalosporin, cefepime, was determined, only 36% of the P. aeruginosa isolates were sensitive and 25% were resistant. Among the antipseudomonal beta-lactams, 50% of the P. aeruginosa isolates were sensitive to piperacillin and 45% were resistant. Among the combined regimens piperacillin-tazobactam was the most potent drug; 73% of the isolates were sensitive and 25% were resistant. P. aeruginosa was more resistant ticarcillin-clavulanate; 52% of the isolates were resistant. Fifty percent of the isolates were sensitive to imipenem. Among the aminoglycosides, P. aeruginosa isolates were most sensitive to amikacin (86%), followed by tobramycin (64%) and gentamicin (27%). Conclusion In our study, it was demonstrated that P. aeruginosa was one of the most common pathogens isolated from tracheal aspirates. Resistance against beta-lactam antibiotics varies from 23% to 50%. In our study, antipseudomonal beta-lactams were shown to be superior to the cephalosporins and carbepenem. In addition, combinatory piperacillin-tazobactam regimen and amikacin from the aminoglycoside group of antibiotics were the most effective antibiotics against P. aeruginosa.