Effect of povidone-iodine and propanol-based mecetronium ethyl sulphate on antimicrobial resistance and virulence in Staphylococcus aureus.

BACKGROUND: Reports are available on cross-resistance between antibiotics and biocides. We evaluated the effect of povidone-iodine (PVP-I) and propanol-based mecetronium ethyl sulphate (PBM) on resistance development, antibiotics cross-resistance, and virulence in Staphylococcus aureus. METHODS: The minimum inhibitory concentration (MIC) of PVP-I and PBM were determined against S. aureus ATCC 25923 using the agar-dilution method. Staphylococcus aureus ATCC 25923 was subjected to subinhibitory concentrations of the tested biocides in ten consecutive passages followed by five passages in a biocide-free medium; MIC was determined after each passage and after the fifth passage in the biocide-free medium. The developed resistant mutant was tested for cross-resistance to different antibiotics using Kirby-Bauer disk diffusion method. Antibiotic susceptibility profiles as well as biocides' MIC were determined for 97 clinical S. aureus isolates. Isolates were categorized into susceptible and resistant to the tested biocides based on MIC distribution pattern. The virulence of the biocide-resistant mutant and the effect of subinhibitory concentrations of biocides on virulence (biofilm formation, hemolysin activity, and expression of virulence-related genes) were tested. RESULTS: PVP-I and PBM MIC were 5000 µg/mL and 664 µg/mL. No resistance developed to PVP-I but a 128-fold increase in PBM MIC was recorded, by repeated exposure. The developed PBM-resistant mutant acquired resistance to penicillin, cefoxitin, and ciprofloxacin. No clinical isolates were PVP-I-resistant while 48.5% were PBM-resistant. PBM-resistant isolates were more significantly detected among multidrug-resistant isolates. PVP-I subinhibitory concentrations (» and ½ of MIC) completely inhibited biofilm formation and significantly reduced hemolysin activity (7% and 0.28%, respectively). However, subinhibitory concentrations of PBM caused moderate reduction in biofilm activity and non-significant reduction in hemolysin activity. The ½ MIC of PVP-I significantly reduced the expression of hla, ebps, eno, fib, icaA, and icaD genes. The virulence of the biocide-resistant mutant was similar to that of parent strain. CONCLUSION: PVP-I is a highly recommended antiseptic for use in healthcare settings to control the evolution of high-risk clones. Exposure to PVP-I causes no resistance-development risk in S. aureus, with virulence inhibition by subinhibitory concentrations. Also, special protocols need to be followed during PBM use in hospitals to avoid the selection of resistant strains.

Effect of spdC gene expression on virulence and antibiotic resistance in clinical Staphylococcus aureus isolates / Efecto de la expresión del gen spdC sobre la virulencia y la resistencia a los antibióticos en aislamientos clínicos de Staphylococcus aureus

Surface protein display C (SpdC) protein was described as a novel virulence factor of Staphylococcus aureus that affects biofilm formation and pathogenesis and favors resistance to antimicrobials targeting cell wall. We evaluated the possible correlation between spdC gene expression level and virulence as well as antibiotic resistance phenotypes in S. aureus clinical isolates. The antimicrobial susceptibility of S. aureus clinical isolates (n = 100) was determined by the disk diffusion method. Vancomycin susceptibility was determined by the broth microdilution method. The level of the extracellular proteases and delta-hemolysin was evaluated by measuring the proteolysis and hemolysis zone diameters in skim milk and blood agar plates, respectively. Biofilm formation was assayed using the 96-well microtiter plate method. Most of the isolates (81%) were multidrug-resistant and about half of the isolates (49%) were methicillin-resistant S. aureus. Hemolysin, protease, and biofilm production were detectable in 79%, 71%, and 96% of the isolates. No significant correlation was detectable between the level of spdC gene expression and the activity of tested virulence factors or the antimicrobial resistance phenotype. Therefore, the role of SpdC protein as a virulence regulator in S. aureus needs further evaluation together with the determination of the predominant regulators for each virulence factor.(AU)

PSORS1 Locus Genotyping Profile in Psoriasis: A Pilot Case-Control Study.

(1) Background: The psoriasis susceptibility 1 (PSORS1) locus, located within the major histocompatibility complex, is one of the main genetic determinants for psoriasis, the genotyping profile for three single-nucleotide polymorphisms (SNPs) comprising the PSORS1 locus: rs1062470 within PSORS1C1/CDSN genes, rs887466 within PSORS1C3 gene, rs10484554 within LOC105375015 gene, were investigated and correlated with psoriasis risk and severity. (2) Methods: This pilot case-controlled study involved 100 psoriatic patients and 100 healthy individuals. We investigated three SNPs and assessed the relative gene expression profile for the PSORS1C1 gene. We then correlated the results with both disease risk and severity. (3) Results: The most significantly associated SNP in PSORS1 locus with psoriasis was rs10484554 with its C/T genotype 5.63 times more likely to develop psoriasis under codominant comparison. Furthermore, C/T and T/T genotypes were 5 times more likely to develop psoriasis. The T allele was 3 times more likely to develop psoriasis under allelic comparison. The relative gene expression of PSORS1C1 for psoriatic patients showed to be under-expressed compared to normal controls. (4) Conclusions: Our study revealed the association of the three studied SNPs with psoriasis risk and severity in an Egyptian cohort, indicating that rs10484554 could be the major key player in the PSORS1 locus.

Effect of spdC gene expression on virulence and antibiotic resistance in clinical Staphylococcus aureus isolates.

Surface protein display C (SpdC) protein was described as a novel virulence factor of Staphylococcus aureus that affects biofilm formation and pathogenesis and favors resistance to antimicrobials targeting cell wall. We evaluated the possible correlation between spdC gene expression level and virulence as well as antibiotic resistance phenotypes in S. aureus clinical isolates. The antimicrobial susceptibility of S. aureus clinical isolates (n = 100) was determined by the disk diffusion method. Vancomycin susceptibility was determined by the broth microdilution method. The level of the extracellular proteases and delta-hemolysin was evaluated by measuring the proteolysis and hemolysis zone diameters in skim milk and blood agar plates, respectively. Biofilm formation was assayed using the 96-well microtiter plate method. Most of the isolates (81%) were multidrug-resistant and about half of the isolates (49%) were methicillin-resistant S. aureus. Hemolysin, protease, and biofilm production were detectable in 79%, 71%, and 96% of the isolates. No significant correlation was detectable between the level of spdC gene expression and the activity of tested virulence factors or the antimicrobial resistance phenotype. Therefore, the role of SpdC protein as a virulence regulator in S. aureus needs further evaluation together with the determination of the predominant regulators for each virulence factor.

Effect of sub-inhibitory concentrations of cefepime on biofilm formation by Pseudomonas aeruginosa.

This study investigated the effect of cefepime at sub-minimum inhibitory concentrations (sub-MICs) on in vitro biofilm formation (BF) by clinical isolates of Pseudomonas aeruginosa. The effect of cefepime at sub-MIC levels (½-1/256 MIC) on in vitro BF by six clinical isolates of P. aeruginosa was phenotypically assessed following 24 and 48 h of challenge using the tissue culture plate (TCP) assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to observe the change in expression of three biofilm-related genes, namely, a protease-encoding gene (lasA), fimbrial protein-encoding gene (cupA1), and alginate-encoding gene (algC), in a weak biofilm-producing strain of P. aeruginosa following 24 and 48 h of challenge with sub-MICs of cefepime. The BF morphology in response to cefepime was imaged using scanning electron microscopy (SEM). The TCP assay showed strain-, time-, and concentration-dependent changes in in vitro BF in P. aeruginosa following challenge with sub-MICs of cefepime, with a profound increase in strains with inherently no or weak biofilm-producing ability. RT-PCR revealed time-dependent upregulation in the expression of the investigated genes following challenge with ½ and » MIC levels, as confirmed by SEM. Cefepime at sub-MICs could upregulate the expression of BF-related genes and enhance BF by P. aeruginosa clinical isolates.

Zinc Deprivation as a Promising Approach for Combating Methicillin-Resistant Staphylococcus aureus: A Pilot Study.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are a global health burden with an urgent need for antimicrobial agents. Studies have shown that host immune responses limit essential metals such as zinc during infection, leading to the limitation of bacterial virulence. Thus, the deprivation of zinc as an important co-factor for the activity of many S. aureus enzymes can be a potential antimicrobial approach. However, the effect of zinc deprivation on S. aureus and MRSA is not fully understood. Therefore, the current study aimed to dissect the effects of zinc deprivation on S. aureus hemolytic activity and biofilm formation through employing biochemical and genetic approaches to study the effect of zinc deprivation on S. aureus growth and virulence. Chemically defined media (CDM) with and without ZnCl2, was used to assess the effect of zinc deprivation on growth, biofilm formation, and hemolytic activity in methicillin-susceptible S. aureus (MSSA) RN6390 and MRSA N315 strains. Zinc deprivation decreased the growth of RN6390 and N315 S. aureus strains significantly by 1.5-2 folds, respectively compared to the zinc physiological range encountered by the bacteria in the human body (7-20 µM) (p < 0.05). Zinc deprivation significantly reduced biofilm formation by 1.5 folds compared to physiological levels (p < 0.05). Moreover, the hemolytic activity of RN6390 and N315 S. aureus strains was significantly decreased by 20 and 30 percent, respectively compared to physiological zinc levels (p < 0.05). Expression of biofilm-associated transcripts levels at late stage of biofilm formation (20 h) murein hydrolase activator A (cidA) and cidB were downregulated by 3 and 5 folds, respectively (p < 0.05) suggested an effect on extracellular DNA production. Expression of hemolysins-associated genes (hld, hlb, hla) was downregulated by 3, 5, and 10 folds, respectively, in absence of zinc (p < 0.001). Collectively the current study showed that zinc deprivation in vitro affected growth, biofilm formation, and hemolytic activity of S. aureus. Our in vitro findings suggested that zinc deprivation can be a potential supportive anti-biofilm formation and antihemolytic approach to contain MRSA topical infections.

Expression levels of pro-inflammatory interleukin-8 and certain antimicrobial peptides in concurrent with bacterial conjunctivitis.

AIM: To detect the quantitative expression levels of the pro-inflammatory interleukin-8 (IL8), antimicrobial peptides human beta defense-2 (HBD2), and human beta defense-3 (HBD3) genes in bacterial conjunctivitis. METHODS: The human conjunctival epithelial cells were obtained using the impression cytology technique from healthy controls and patients. The genes expression levels were determined utilizing a reverse transcription quantitative polymerase chain reaction (RT-qPCR). The contribution of causative agent type, the number of isolates and severity of clinical features, in the increase of genes expression was also determined. RESULTS: The RT-qPCR showed that IL8, HBD2, and HBD3 expression increased in bacterial conjunctivitis as compared to healthy control (P<0.001). In gram-negative bacterial conjunctivitis, HBD2 was highly up-regulated (P<0.001) compared to other types of bacterial conjunctivitis. In mixed bacterial conjunctivitis, a direct correlation between HBD2 up-regulation and HBD3 up-regulation was observed (P<0.05). The severity of clinical features was related to the up-regulation of IL8 and HBD2 (P<0.05). CONCLUSION: IL8, HBD2, and HBD3 are immune-effectors in infectious conjunctivitis. HBD2 is active during different bacterial conjunctivitis but is more released with gram-negative bacteria compared to gram-positive bacteria. HBD3 is an obvious defender in different bacterial conjunctivitis.

Antimicrobial activities encountered by sulfur nanoparticles combating Staphylococcal species harboring sccmecA recovered from acne vulgaris.

Over decades, sulfur has been employed for treatment of many dermatological diseases, several skin and soft tissue, and Staphylococcus infections. Because of its abuse, resistant bacterial strains have emerged. Nanotechnology has presented a new horizon to overcome abundant problems including drug resistance. Nano-sized sulfur has proven to retain bactericidal activity. Consequently, the specific aims of this study are exclusively directed to produce various sulfur nanoparticles formulations with control of particle size and morphology and investigate the antibacterial activity response specifically classified by the category of responses of different formulations, for the treatment of acne vulgaris resistant to conventional antibiotics. In this study, we produced uncoated sulfur nanoparticles (SNPs), sulfur nano-composite with chitosan (CS-SNPs), and sulfur nanoparticles coated with polyethylene glycol (PEG-SNPs) and evaluate their bactericidal impact against Staphylococcus aureus and Staphylococcus epidermidis isolated from 173 patients clinically diagnosed acne vulgaris. Accompanied with molecular investigations of ermB and mecA resistance genes distribution among the isolates. Sulfur nanoparticles were synthesized using acid precipitation method and were characterized by scanning electron microscope (SEM), transmission electron microscopy (TEM), energy dispersed x-ray spectroscopy (EDX), and Fourier transform infrared spectroscopy (FTIR). Moreover, agar diffusion and broth micro-dilution methods were applied to determine their antibacterial activity and their minimum inhibitory concentration. PCR analysis for virulence factors detection. Results: TEM analysis showed particle size of SNPs (11.7 nm), PEG-SNPs (27 nm) and CS-SNPs (33 nm). Significant antibacterial activity from nanoparticles formulations in 100% dimethyl sulfoxide (DMSO) with inhibition zone 30 mm and MIC at 5.5 µg/mL. Furthermore, the prevalence of mecA gene was the most abundant among the isolates while ermB gene was infrequent. Conclusions: sulfur nanoparticles preparations are an effective treatment for most Staphylococcus bacteria causing acne vulgaris harboring multi-drug resistance virulence factors.

Risk factors associated with preterm labor, with special emphasis on preterm premature rupture of membranes and severe preterm labor.

BACKGROUND: Preterm labor (PTL) can lead to preterm birth, which can cause neonatal mortality and morbidity. Preterm premature rupture of membranes (PPROM) and severe PTL (SPTL) are serious PTL subtypes. Hereby, we aimed to investigate risk factors associated with PPROM and SPTL, among Egyptian women. METHODS: In this case-control study, 117 women were enrolled without any known medical risk for PTL. The control group (n = 45) had term labor (≥37 gestational weeks), while the case group (n = 72) had PTL (<37 gestational weeks). The PTL group was subdivided into those with PPROM (n = 18) and those with intact membranes (n = 54). Fifty-two PTL women, with accurate gestational age, were subdivided into SPTL (n = 31, ≤34 gestational weeks) and mild preterm labor (MPTL; n = 21, 35-36 gestational weeks). All groups were examined for different demographic characteristics, obstetrical history, clinical signs, and vaginal and urinary tract infections. Nominal logistic regression was applied to investigate significant variables associated with PPROM and intact membranes PTL, while ordinal logistic regression was used to estimate significant variables associated with SPTL and MPTL. RESULTS: The final multivariate nominal model identified abortion history, heavy vaginal bleeding history, and elevated vaginal pH as significant predictors of PPROM. The same model identified age <20 years old, abortion history, heavy growth of vaginal organisms, and any growth of Gram-negative bacilli as the significant predictors of intact membranes PTL. The final multivariate ordinal model identified age <20 years old, abortion history, vaginal pH, and heavy growth of vaginal organisms as the significant predictors of SPTL and MPTL. CONCLUSION: Age <20 years old, abortion history, heavy vaginal bleeding, vaginal pH, and heavy growth of vaginal organisms were reported as risk factors for PPROM and SPTL. Most of these factors are related to infection; therefore, proper infection control is recommended during prenatal and antenatal care.

Potential efficacy of garlic lock therapy in combating biofilm and catheter-associated infections; experimental studies on an animal model with focus on toxicological aspects.

BACKGROUND: Life-threatening central venous catheter-related infections are primarily initiated by biofilm formation on the catheter surface. Antibiotic lock therapy is recommended for eradicating intraluminal biofilm. In the era of antibiotic resistance, antibiotics of natural origins provide an effective and cheap option for combating resistant strains. Garlic especially stole the spotlight because of its impressive antimicrobial effectiveness against such superbugs. AIM: Is to estimate the potential use of fresh garlic extract (FGE) as a lock agent against multi-drug resistant (MDR) bacteria. METHODS: The agar well diffusion and broth microdilution techniques were employed to test the antimicrobial activities of FGE against five MDR strains; E. coli, Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), Serratia marscens (S. marscens) and Methicillin-resistant Staphylococcus aureus (MRSA). Then the protective and therapeutic efficiencies of FGE against bacterial biofilms were in-vitro evaluated; at concentrations of 100, 75, 50 and 25%; in tissue culture plate (TCP) and on the polyurethane (PU) sheets using the crystal violet (CV) assay and colony-forming unit (CFU), respectively. Scanning electron microscopy (SEM) was also used to confirm eradication of biofilms on PU sheets. Finally, systemic and deep tissue infections by P. aeruginosa and MRSA were induced in mice that were then treated by FGE at either 100 or 200 mg/kg for seven days. Where the antibacterial activity was assessed by tissue and blood culturing at the end of the treatment period. Biochemical, hematological and histological parameters were also investigated. RESULTS: FGE exhibited potent in-vitro and in-vivo antibacterial and antibiofilm activities against MDR strains. It not only didn't exhibit toxicological effects at the hematological and the histological levels but also provided protective effects as demonstrated by the significant drop in the biochemical parameters. CONCLUSION: FGE has the potential to be used as a prophylactic and/or therapeutic lock agent against biofilm-associated infections caused by MDR bacteria.

The increasing threat of silver-resistance in clinical isolates from wounds and burns.

PURPOSE: The widespread use of silver-containing compounds has led to emergence of silver-resistant bacteria. Few studies are available on the detectability of plasmid-mediated silver-resistance in developing countries. Therefore, we aimed to detect silver-resistance in isolates from wounds and burns, and to genetically characterize plasmid-mediated silver-resistance genes (sil genes). METHODS: One hundred and fifty clinical isolates were obtained from burns and wounds. They were identified using the suitable Analytical Profile Index and MicroScan identification systems. Their antimicrobial susceptibility was tested by the disk diffusion and broth microdilution methods. Their silver nitrate (AgNO3) minimum inhibitory concentration (MIC) was determined using the broth macrodilution method. The presence of different sil genes on plasmids extracted from silver-resistant isolates and the replicon types of the extracted plasmids were investigated using polymerase chain reaction (PCR). The ability of these plasmids to impart silver-resistance was tested by transformation. RESULTS: All except two isolates were multidrug-resistant. Nineteen silver-resistant bacterial isolates (12.6%) were detected; with AgNO3 MIC ≥512 µg/mL. They were identified as Klebsiella pneumoniae (n=7), Staphylococcus aureus (n=4), Escherichia coli (n=2), Enterobacter cloacae (n=2), Pseudomonas aeruginosa (n=2) and Acinetobacter baumannii (n=2). PCR revealed the presence of different sil genes on the extracted plasmids. Plasmid transformation resulted in the transfer of silver-resistance to the resulting transformants. The extracted plasmids had different replicon types. CONCLUSION: Plasmid-mediated silver-resistance was detected for the first time, in clinical P. aeruginosa, A. baumannii and S. aureus isolates; in addition to its detection in K. pneumoniae, E. coli and Enterobacter cloacae. Therefore, strict monitoring on the use of silver compounds in medical settings is required; with implementation of an approved standardized method for silver-resistance detection.

The use of unirradiated and γ-irradiated zinc oxide nanoparticles as a preservative in cosmetic preparations.

PURPOSE: Microbial contamination of different cosmetic preparations, as a result of preservative failure, presents a major public health threat. Also, most of the known preservatives have serious consumer side effects. The antimicrobial activity of zinc oxide nanoparticles (ZnO NP) is well documented. Therefore, we aimed to determine the possible use of unirradiated and γ-irradiated ZnO NP as a cosmetic preservative. METHODS: The possible use of ZnO NP as a preservative was tested and compared to commonly used preservatives using a challenge test. Their activity was tested in six different types of preparations. The effect of γ radiation on the antimicrobial activity of ZnO NP was tested through determination of the obtained zone diameters against different microorganisms and the total aerobic microbial count in tested preparations. The antimicrobial activity, of unirradiated and γ-irradiated ZnO NP during storage was also determined. RESULTS: ZnO NP were superior to other commonly used preservatives in all tested cosmetic preparations. They pass the challenge test in all types of tested preparations. γ irradiation enhanced their antimicrobial activity in all tested preparations. The irradiation causes a reduction in NP sizes that is directly proportional to the applied radiation dose. Upon storage, ZnO NP were effective in maintaining the microbial count of the product within the acceptable range. Their activity in stored products was enhanced by γ irradiation. CONCLUSION: Unirradiated and γ-irradiated ZnO NP can be used as effective preservatives. They are compatible with the components of all tested products. γ irradiation enhanced the antimicrobial activity of ZnO NP.

Association between preterm labor and genitourinary tract infections caused by Trichomonas vaginalis, Mycoplasma hominis, Gram-negative bacilli, and coryneforms.

BACKGROUND: Preterm labor (PTL) is responsible for most cases of neonatal death. In most of these cases, the causes of PTL have not been established although several risk factors have been described. Therefore, the aim of this study was to investigate risk factors for PTL before 37 gestational weeks among Egyptian women. METHODS: In this case-control study, 117 pregnant women without risk factors for PTL were chosen. The control group (n=45) had term labor (gestational weeks≥37 weeks), and the case group (n=72) had PTL (gestational weeks < 37 weeks). The two groups were screened for urinary and vaginal infections. The role of different demographic characteristics, patient history, and clinical signs were also investigated. RESULTS: Several risk factors were identified in this study, including age<20 years, nulliparity, previous abortion and previous preterm birth, menses vaginal bleeding, a vaginal pH>5, a positive whiff test, Trichomonas vaginalis infection, Mycoplasma hominis infection, coryneforms heavy vaginal growth, and any vaginal growth of Gram-negative bacilli. Urinary tract infection with any colony count was not associated with PTL. CONCLUSION: Our study demonstrated that the main risk factors for PTL were vaginal infection with T. vaginalis, M. hominis, coryneforms, and Gram-negative bacilli, and their determinants (vaginal pH>5, positive whiff test, heavy vaginal bleeding). Both young age (< 20 years) and poor obstetric history were also the risk factors. Therefore, screening for genitourinary tract infections is strongly recommended to be included in prenatal care.

Immune response to Vi polysaccharide, heat-killed whole cells, and outer membrane protein of Salmonella typhi.

INTRODUCTION: Salmonella typhiVi capsular polysaccharide (ViCPS) is a licensed vaccine against typhoid fever in many countries; in Egypt, the killed whole-cell vaccine is still used. In this study, mice were used as an animal model to evaluate the immune response to ViCPS and other S. typhi antigens such as heat-killed whole cells and outer membrane protein (OMP). METHODOLOGY: The three antigens were laboratory prepared, injected into mice groups, and the humoral response was evaluated using the indirect whole-cell enzyme-linked immunosorbent assay (ELISA). The sensitivity of this assay was investigated using in situ or pre-heated whole cells as coating antigens. In addition, the effect of the immunization route for ViCPS was examined. RESULTS: Immunizing doses of heat-killed whole cells as well as ViCPS, 2 and 4 µg given subcutaneously (SC) and 4 µg given intraperitoneally (IP), showed significant immune response compared to controls. However, the responses to these doses were not significantly different from each other. The OMP showed a higher significant response. The sensitivity of indirect whole-cell ELISA was enhanced significantly by in situ heat treatment of the coating antigen rather than the pre-heated coating antigen. CONCLUSIONS: The three antigens showed significant immune response. The immune response to OMP was higher. Though heat-killed whole cells and ViCPS are almost similar in immunizing level, ViCPS is recommended. The SC route was more immunizing than the IP one. Furthermore, the sensitivity of the indirect whole-cell ELISA technique could be enhanced by in situ heat inactivation of the coating cells.

TH1 cytokine response to HCV peptides in Egyptian health care workers: a pilot study.

Our objective was to elucidate the effects of different HCV peptides on TH1 cytokine synthesis (interleukin 2(IL2), gamma interferon (INFγ) and tumor necrosis factor α (TNF α)), in a proliferative response in a high risk population of HCV seronegative aviremic Egyptian healthcare workers (HCW). We studied the TH1 cytokine response to different HCV peptides among 47 HCW with and without evidence of HCV infection. Participants were classified according to the proliferation index (PI) in a CFSE proliferation assay as an indicator of previous exposure to HCV. Cytokines were analyzed using Luminex xMAP technology. Results showed that positive PI HCW produced a higher IL2 in response to all HCV peptides except NS4, a higher IFNγ response to NS3 and NS4 and no difference in TNFα response when compared to the negative PI HCWs. When compared to chronic HCV HCW, positive PI HCW showed no difference in the IL2 response, a higher IFNγ response to NS4 and NS5 HCV peptides and a higher TNFα response to all peptides. In conclusion the magnitude and type of cytokines produced in HCV infection is critical in determining the outcome of infection. NS4 & NS5 HCV peptides induce a protective TH1 response in positive PI HCW.

Detection of AmpC beta-lactamases using sodium salicylate.

AmpC ß-lactamases are enzymes that hydrolyze all ß-lactam antibiotics except cefipime and imipenem. Currently, there is no standard phenotypic method for detection of such enzymes. This study aims to report the use of sodium salicylate for AmpC ß-lactamases detection and to compare its sensitivity and specificity to other commonly known inhibitors. A total of 135 clinical isolates were used to test the effectiveness of sodium salicylate in detection of plasmid- as well as chromosomally encoded AmpC ß-lactamases. All isolates were tested by multiplex PCR testing as well as inhibitor-based methods using cloxacillin, phenylboronic acid and sodium salicylate for the detection of AmpC enzymes. Four isolates were confirmed as producers of plasmid-encoded AmpC ß-lactamase and a single isolate was confirmed to have both plasmid and chromosomal genes. Cloxacillin and phenylyboronic acid failed to detect most of the plasmid-encoded enzymes. Sodium salicylate was able to detect the Escherichia coli isolates with plasmid-encoded enzymes in addition to few other isolates that were chromosomally mediated. The sensitivity and specificity of sodium salicylate was 50% and 93%, respectively, higher than those of other known inhibitors. We thus conclude that sodium salicylate can be reliably used as an inhibitor in the detection of plasmid-encoded AmpC enzymes in E. coli.

Neutralizing activities of caprine antibodies towards conserved regions of the HCV envelope glycoprotein E2.

Anti HCV vaccine is not currently available and the present antiviral therapies fail to cure approximately half of the treated HCV patients. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 and test their neutralizing activities in a step towards developing therapeutic and/or prophylactic immunogens against HCV infection. Antibodies were generated by vaccination of goats with synthetic peptides derived from HCV E2. Viral neutralizing capacity of the generated anti E2 antibodies was tested using in vitro assays. Goats immunized with E2 synthetic peptides termed p412 [a.a 412-419], p430 [a.a 430-447] and p517 [a.a 517-531] generated high titers of antibody responses 2 to 4.5 fold higher than comparable titers of antibodies to the same epitopes in chronic HCV patients. In post infection experiments of native HCV into cultured Huh7.5 cells anti p412 and anti p 517 were proven to be neutralizing to HCV genotype 4a from patients' sera (87.5% and 75% respectively). On the contrary anti p430 exhibited weak viral neutralization capacity on the same samples (31.25%). Furthermore Ab mixes containing anti p430 exhibited reduced viral neutralization properties. From these experiments one could predict that neutralization by Abs towards different E2-epitopes varies considerably and success in the enrichment of neutralization epitope-specific antibodies may be accompanied by favorable results in combating HCV infection. Also, E2 conserved peptides p517 and p412 represent potential components of a candidate peptide vaccine against HCV infection.

WITHDRAWN: Caprine antibodies towards E2 conserved peptides interfere with hepatitis C virus infectivity in vitro.

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Circulating viral core and E1 antigen levels as supplemental markers for HCV chronic hepatitis.

The performance of polyclonal monospecific rabbit anti-sera raised against synthetic peptides derived from conserved HCV sequences of genotype 4 was evaluated for efficient detection of viral core and E1 antigens in circulating immune complexes (ICs) precipitated from 65 serum samples of HCV patients. The infection was established in those patients by the presence of HCV RNA in their sera. A novel enzyme-linked immunosorbent assay (ELISA) was developed for the detection of HCV core and E1 antigen in serum samples. Western blot analyses were used to demonstrate the presence of the core and E1 target antigen in serum samples. The mean OD readings of both core and E1 antigens were significantly higher (P < 0.05) among the viremic patients when compared to controls. Also a significant positive correlation (P < 0.05, r = 0.98) between the values of both core and E1 was recorded. Western blot analysis based on monospecific antibodies against core and E1 recognized the 38-kDa and 88 -kDa bands respectively in the sera of all infected patients. No specific reaction was observed with the sera from uninfected individuals. Interestingly the results of core and E1 antigen levels displayed no positive correlation with the HCV copy number as measured by bDNA. Liver enzymes (ALT and AST) showed a moderate positive correlation (r = 0.44 and 0.47 respectively) with the viral core antigens level. The same trend holds true for E1 (r = 0.43 and 0.64 for ALT and AST respectively). HCV load in infected patients revealed extremely poor correlation with serum ALT and AST levels (r = 0.022 and 0.002 respectively). In conclusion we present a new combination of serological tools correlating with liver enzyme levels that could be utilized as supplemental tests to viral load testing. Also, a sensitive and specific immunoassay was developed for the detection of HCV core and E1 in human serum. This test can be applied for laboratory diagnosis of HCV infection.