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)

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.

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.

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.

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.