Repurposing carvacrol, cinnamaldehyde, and eugenol as potential anti-quorum sensing agents against uropathogenic Escherichia coli isolates in Alexandria, Egypt.

BACKGROUND: Urinary tract infections represent one of the most frequent hospital and community-acquired infections with uropathogenic Escherichia coli (UPEC) being the main causative agent. The global increase in the emergence of multidrug-resistant (MDR) UPEC necessitates exploring novel approaches. Repurposing natural products as anti-quorum sensing (QS) agents to impede bacterial virulence is gaining momentum nowadays. Hence, this study investigates the anti-QS potentials of carvacrol, cinnamaldehyde, and eugenol against E. coli isolated from urine cultures of Egyptian patients. RESULTS: Antibiotic susceptibility testing was performed for 67 E. coli isolates and 94% of the isolates showed MDR phenotype. The usp gene was detected using PCR and accordingly, 45% of the isolates were categorized as UPEC. Phytochemicals, at their sub-inhibitory concentrations, inhibited the swimming and twitching motilities of UPEC isolates, with eugenol showing the highest inhibitory effect. The agents hindered the biofilm-forming ability of the tested isolates, at two temperature sets, 37 and 30 °C, where eugenol succeeded in significantly inhibiting the biofilm formation by > 50% at both investigated temperatures, as compared with untreated controls. The phytochemicals were shown to downregulate the expression of the QS gene (luxS) and critical genes related to motility, asserting their anti-QS potential. Further, the combinatory activity of the phytoproducts with five antibiotics was assessed by checkerboard assay. The addition of the phytoproducts significantly reduced the minimum inhibitory concentrations of the antibiotics and generated several synergistic or partially synergistic combinations, some of which have not been previously explored. CONCLUSIONS: Overall, carvacrol, cinnamaldehyde, and eugenol could be repurposed as potential anti-QS agents, which preferentially reduce the QS-based communication and attenuate the cascades of gene expression, thus decreasing the production of virulence factors in UPEC, and eventually, subsiding their pathogenicity. Furthermore, the synergistic combinations of these agents with antibiotics might provide a new perspective to circumvent the side effects brought about by high antibiotic doses, thereby paving the way for overcoming antibiotic resistance.

Beta-lactam Resistance Profile among Klebsiella pneumoniae Clinical Isolates from Alexandria, Egypt.

Klebsiella pneumoniae is a major drug-resistant human pathogen accountable for a wide range of infections. In this cross-sectional study, we aimed to determine the phenotypic and genotypic features of ß-lactamase-producing K. pneumoniae clinical isolates from Alexandria, Egypt. A total of 50 nonduplicated clinical isolates of K. pneumoniae were obtained from various specimens. They were identified biochemically and by biotyping using mass spectrometry. For molecular characterization, plasmid profile analysis was performed. Screening for extended spectrum ß-lactamases (ESBLs), carbapenemases and AmpC production was carried out phenotypically and genotypically. Correlation analysis was performed to assess the relationship between phenotype, genotype and resistance patterns among the studied isolates. The dendrogram demonstrated 38 distinct plasmid profiles among 62% of our isolates. According to antimicrobial susceptibility testing, 90% of isolates were multi/extensive-drug resistant. Nineteen out of 50 (38%) were resistant to cefoxitin, while only 10 (20%) were resistant to imipenem. All isolates were susceptible to colistin. Phenotypically, ESBL producers (78%) were the most common, followed by carbapenemase producers (24%). Genotypically, the most common ESBL gene was blaSHV (90%), followed by blaCTX-Mu (74%), while the most common carbapenemase genes were blaNDM (56%) and blaOXA-48 (54%). No blaKPC or blaIMP were detected. Plasmid-mediated AmpC resistance was confirmed in only two out of 19 cefoxitin-resistant isolates. Both the blaNDM and blaOXA.48 genes were significantly positive correlated (rho = 0.56, p = 0.004). Absence of blaKPC among carbapenem resistant K. pneumoniae isolates in Alexandria, Egypt. AmpC production is not the main factor behind the resistance to cefoxitin among our isolates.

Hinokitiol for hypertensive emergencies: effects on peripheral resistance, cardiac load, baroreflex sensitivity, and electrolytes balance.

Hinokitiol, a natural monoterpenoid, has been shown previously to possess a potent vasodilating activity in vitro in both control and hypertensive aortae. Here, the antihypertensive and cardioprotective effects of an intravenous hinokitiol injection were fully investigated in angiotensin II-induced hypertensive emergency in rats. Hinokitiol intravenous injection was prepared in the form of self-nanoemulsifying drug delivery system. Rat's arterial and ventricular hemodynamics were measured in real-time recordings in addition to surface electrocardiogram while slow injection of cumulative doses of hinokitiol or vehicle as well as time control. Hinokitiol at dose 10 mg/kg showed a considerable reduction in the raised systolic blood pressure (30 mmHg) within only 30 min. The decrease in blood pressure seems to be mediated through a reduction in peripheral resistance, as appears from the decreases in diastolic pressure, dicrotic notch pressure, and pulse pressure. In addition, hinokitiol injection reduced heart load due to the decrease in heart rate, increases in cycle duration (particularly the non-ejection duration) and diastolic duration, and decreases in end-diastolic pressure. An effect most likely mediated via prolongation of ventricular repolarization as appears from the increases in PR, QTc, and JT intervals. However, acute intravenous injection of hinokitiol neither affected the baroreflex sensitivity nor sodium/potassium balance. In conclusion, acute hinokitiol intravenous injection markedly reduced severe hypertension in rats. This effect seems to be mediated through decreasing peripheral resistance and decreasing cardiac load, suggesting that it is an effective treatment in hypertensive emergencies after clinical evaluation.

Prevalence and mechanisms of linezolid resistance among staphylococcal clinical isolates from Egypt.

The emergence of methicillin-resistant staphylococci necessitated the search for alternative agents as linezolid, introduced to treat infections due to multidrug-resistant bacteria. Linezolid resistance has since emerged, yet its global prevalence remains low. In Egypt, little is known about the situation. We investigated the prevalence and mechanisms of resistance among Egyptian staphylococcal clinical isolates. Linezolid resistance among 232 staphylococcal isolates obtained from Alexandria Main Hospitals between 2011 and 2016 was assessed using disc diffusion and minimum inhibitory concentration. Resistant isolates were checked for cfr presence using polymerase chain reaction. The V domain of different alleles of 23S rRNA gene was investigated for mutations. Selection for linezolid-resistant mutants was performed in vitro through serial passages in linezolid sub-inhibitory concentrations. Combinations of linezolid with imipenem or anti-inflammatory agents were investigated using time-kill and modified checkerboard assays. Three Staphylococcus haemolyticus isolates (1.3%) from 2015 to 2016 were linezolid-resistant. One isolate carried cfr which was plasmid-borne, and together with another isolate which had a G2603T point mutation in the V domain of 23S rRNA gene. Successive exposure to linezolid sub-inhibitory concentrations was selected for three resistant Staphylococcus aureus mutants out of ten susceptible isolates. These mutants were more resistant towards different antibiotic classes than their susceptible parents. Linezolid combinations with imipenem, ibuprofen, or aspirin were synergistic against the isolates and mutants. Despite unregulated use of linezolid, resistance remains fairly low among the Egyptian isolates. Strict antimicrobial stewardship guidelines are needed in hospitals and the community to guard against further evolution of resistant mutants.

Photosensitizer-eluting nanofibers for enhanced photodynamic therapy of wounds: A preclinical study in immunocompromized rats.

Electrospun nanofibers (NFs) as drug delivery/tissue regeneration template and antimicrobial photodynamic therapy (APDT) have been widely investigated as two different approaches to enhance wound healing. In the present study, the two approaches were combined in a single platform for greater healing enhancement potentials. Composite photosensitizer-eluting NFs were developed using a polyhydrohybutyrate/polyethylene glycol (60:40 PHB/PEG) polymer blend and methylene blue (MB) as antimicrobial photosensitizer (PS). NFs protected the photoactivity of entrapped MB, enhanced its photodynamic activity against two wound bacteria, Staphylococcus aureus standard strain (SAst) and MRSA and sustained MB release allowing for flexible PS dosing and irradiation schedules. This combined PS-eluting NFs/APDT approach proved effective in the treatment of SAst-inoculated excision wounds in a challenging immunocompromized rat model. This was verified by morphological, morphometric, microbiological, histopathological and RT-PCR studies. Inclusion of PS-eluting NFs as an additional active component of APDT generates a combined non-antibiotic antimicrobial/cell regeneration approach with great potentials for wound healing and other biomedical applications.

Correlation of quorum sensing and virulence factors in Pseudomonas aeruginosa isolates in Egypt.

INTRODUCTION: Pseudomonas aeruginosa is one of the most virulent nosocomial pathogens worldwide. Quorum sensing (QS) regulates the production of pathogenic virulence factors and biofilm formation in P. aeruginosa. The four genes lasR, lasI, rhlR,and rhlI were found to regulate this QS system. In this study, we aimed to assess the correlation between these four genes and QS-dependent virulence factors and to detect the inhibitory effect of clove oil on QS. METHODOLOGY: Fifty P. aeruginosa clinical isolates were collected. Susceptibility to different antibiotics was tested. Virulence factors including biofilm formation, pyocyanin production, and twitching motility were phenotypically detected. QS genes were amplified by polymerase chain reaction (PCR), and one strain subsequently underwent sequencing. The inhibitory effect of clove oil on virulence factors was also tested. RESULTS: A positive correlation was found between biofilm formation and the presence of lasR and rhlI genes. Twitching motility was positively correlated with the presence of lasR, lasI, and rhlI genes. On the other hand, no correlation was found between pyocyanin production and any of the studied genes. Only one isolate amplified all the tested QS gene primers, but it did not express any of the tested virulence factors phenotypically. Sequence analyses of this isolate showed that the four genes had point mutations. CONCLUSIONS: Results emphasize the importance of QS in P. aeruginosa virulence; however, QS-deficient clinical isolates occur and are still capable of causing clinical infections in humans. Also, clove oil has an obvious inhibitory effect on QS, which should be clinically exploited.

Development of Fed-Batch Cultivation Strategy for Efficient Oxytetracycline Production by Streptomyces rimosus at Semi-Industrial Scale

ABSTRACTOxytetracycline (OTC) production byStreptomyces rimosus was studied in batch and fed-batch cultures in shake flask and bioreactor levels using semi-defined medium. First, the effect of glucose concentration on OTC production and growth kinetics was studied intensively. The optimal glucose concentration in the medium was 15 g/L. Higher glucose concentrations supported higher biomass production by less volumetric and specific antibiotic production. Based on these data, cultivations were carried out at semi-industrial scale 15 L bioreactor in batch culture. At bioreactor level, cell growth and OTC production were higher compared to the shake flask culture by about 18 and 38%, respectively. During the bioreactor cultivation, glucose was totally consumed after only 48 h. Thus, the fed-batch experiment was designed for mono-glucose feeding and complete medium feeding to increase the OTC production by overcoming carbon limitations. The results showed that the fed-batch culture using constant glucose feeding strategy with rate of 0.33 g/L/h produced 1072 mg/L. On the other hand, feeding with complete medium resulted in 45% higher biomass but less OTC production by about 26% compared to mono-glucose fed culture. A further improvement in this process was achieved in by keeping the dissolved oxygen (DO) value at 60% saturation by cascading the glucose feeding pump with the DO controller. The later feeding strategy resulted in higher antibiotic production, reaching 1414 mg/L after 108 h.

Reversal of antibiotic resistance in Gram-positive bacteria by the antihistaminic azelastine.

Antibiotic resistance represents a serious problem that complicates microbial infection. The use of 'helper compounds' capable of enhancing the antimicrobial activity of antibiotics is being investigated. Azelastine, a new generation antihistaminic, possesses certain antibacterial activity and is capable of inducing alteration in the bacterial membrane permeability. Hence, we hypothesized that it could reverse resistance to antibiotics. Azelastine significantly increased the antibacterial activity of eight antibiotics belonging to five different classes (ß-lactams, macrolides, fluoroquinolones, aminoglycosides and tetracyclines) against nine Gram-positive clinical isolates: five Staphylococcus aureus, two Staphylococcus epidermidis and two Enterococcus faecium, seven of which were multi-drug resistant, reversing their resistance to the tested antibiotics. The synergistic effects of azelastine with the studied antibiotics increased with raising the pH from 5 to 8. Antibiotics did not affect the ability of azelastine to alter the permeability of a liposomal artificial membrane model, an effect thought to be critical for the interaction with antibiotics. The findings of this study present azelastine as a potential 'helper compound' that could reverse the resistance of multi-drug resistant Gram-positive clinical isolates to antibiotics.

In vitro antibacterial activity of some antihistaminics belonging to different groups against multi-drug resistant clinical isolates

Antihistaminics are widely used for various indications during microbial infection. Hence, this paper investigates the antimicrobial activities of 10 antihistaminics belonging to both old and new generations using multiresistant Gram-positive and Gram-negative clinical isolates. The bacteriostatic activity of antihistaminics was investigated by determining their MIC both by broth and agar dilution techniques against 29 bacterial strains. Azelastine, cyproheptadine, mequitazine and promethazine were the most active among the tested drugs. Diphenhydramine and cetirizine possessed weaker activity whereas doxylamine, fexofenadine and loratadine were inactive even at the highest tested concentration (1 mg/ml). The MIC of meclozine could not be determined as it precipitated with the used culture media. The MBC values of antihistaminics were almost identical to the corresponding MIC values. The bactericidal activity of antihistaminics was also studied by the viable count technique in sterile saline solution. Evident killing effects were exerted by mequitazine, meclozine, azelastine and cyproheptadine. Moreover, the dynamics of bactericidal activity of azelastine were studied by the viable count technique in nutrient broth. This activity was found to be concentration-dependant. This effect was reduced on increasing the inoculum size while it was increased on raising the pH. The post-antimicrobial effect of 100 fg/ml azelastine was also determined and reached up to 3.36 h.

Membrane permeability alteration of some bacterial clinical isolates by selected antihistaminics

Several antihistaminics possess antibacterial activity against a broad spectrum of bacteria. However, the exact mechanism of such activity was unclear. Hence, the aim of this study is to investigate their mechanism of antibacterial activity especially their effect upon the permeability of the bacterial cytoplasmic membrane. The effects of azelastine, cetirizine, cyproheptadine and diphenhydramine were studied using Gram-positive and Gram-negative multiresistant clinical isolates. Leakage of 260 and 280 nm UV-absorbing materials was detected upon treatment with the tested antihistaminics; indicative of membrane alteration. Using an artificial membrane model, cholesterol-free negatively-charged unilamellar liposomes, confirmed the effect of antihistaminics upon the membrane permeability both by showing an apparent membrane damage as observed microscopically and by detection of leakage of preloaded dye from the liposomes colorimatrically. Moreover, examination of the ultrastructure of cells treated with azelastine and cetirizine under the transmission electron microscope substantiated the detected abnormalities in the cell wall and membrane. Furthermore, the effect of pretreating certain isolates for both short and long periods with selected antihistaminics was followed by the viable count technique. Increased vulnerability towards further exposure to azelastine was observed in cells pretreated with azelastine for 2 days and those pretreated with azelastine or cetrizine for 30 days.

Membrane permeability alteration of some bacterial clinical isolates by selected antihistaminics.

Several antihistaminics possess antibacterial activity against a broad spectrum of bacteria. However, the exact mechanism of such activity was unclear. Hence, the aim of this study is to investigate their mechanism of antibacterial activity especially their effect upon the permeability of the bacterial cytoplasmic membrane. The effects of azelastine, cetirizine, cyproheptadine and diphenhydramine were studied using Gram-positive and Gram-negative multiresistant clinical isolates. Leakage of 260 and 280 nm UV-absorbing materials was detected upon treatment with the tested antihistaminics; indicative of membrane alteration. Using an artificial membrane model, cholesterol-free negatively-charged unilamellar liposomes, confirmed the effect of antihistaminics upon the membrane permeability both by showing an apparent membrane damage as observed microscopically and by detection of leakage of preloaded dye from the liposomes colorimatrically. Moreover, examination of the ultrastructure of cells treated with azelastine and cetirizine under the transmission electron microscope substantiated the detected abnormalities in the cell wall and membrane. Furthermore, the effect of pretreating certain isolates for both short and long periods with selected antihistaminics was followed by the viable count technique. Increased vulnerability towards further exposure to azelastine was observed in cells pretreated with azelastine for 2 days and those pretreated with azelastine or cetrizine for 30 days.

In vitro antibacterial activity of some antihistaminics belonging to different groups against multi-drug resistant clinical isolates.

Antihistaminics are widely used for various indications during microbial infection. Hence, this paper investigates the antimicrobial activities of 10 antihistaminics belonging to both old and new generations using multiresistant Gram-positive and Gram-negative clinical isolates. The bacteriostatic activity of antihistaminics was investigated by determining their MIC both by broth and agar dilution techniques against 29 bacterial strains. Azelastine, cyproheptadine, mequitazine and promethazine were the most active among the tested drugs. Diphenhydramine and cetirizine possessed weaker activity whereas doxylamine, fexofenadine and loratadine were inactive even at the highest tested concentration (1 mg/ml). The MIC of meclozine could not be determined as it precipitated with the used culture media. The MBC values of antihistaminics were almost identical to the corresponding MIC values. The bactericidal activity of antihistaminics was also studied by the viable count technique in sterile saline solution. Evident killing effects were exerted by mequitazine, meclozine, azelastine and cyproheptadine. Moreover, the dynamics of bactericidal activity of azelastine were studied by the viable count technique in nutrient broth. This activity was found to be concentration-dependant. This effect was reduced on increasing the inoculum size while it was increased on raising the pH. The post-antimicrobial effect of 100 µg/ml azelastine was also determined and reached up to 3.36 h.

Incidence analyses of bladder cancer in the Nile delta region of Egypt.

Bladder cancer is the most common malignancy among Egyptian males and previously has been attributed to Schistosoma infection, a major risk factor for squamous cell carcinoma (SCC). Recently, transitional cell carcinoma (TCC) incidence has been increasing while SCC has declined. To investigate this shift, we analyzed the geographical patterns of all bladder cancers cases recorded in Egypt's Gharbiah Population-Based Cancer Registry from 1999 through 2002. Data on tumor grade, stage, and morphology, as well as smoking, community of residence, age and sex, were collected on 1209 bladder cancer cases. Age-adjusted incidence rates were calculated for males, females, and the total population for the eight administrative Districts and 316 communities in Gharbiah. Incidence Rate Ratios (IRR) and 95% confidence intervals (CI) were computed using Poisson Regression. The male age-adjusted incidence rate (IR) in Gharbiah Province was 13.65/100,000 person years (PY). The District of Kotour had the highest age-adjusted IR 28.96/100,000 among males. The District of Kotour also had the highest IRR among all Districts, IRR=2.15 95% CI (1.72, 2.70). Kotour's capital city had the highest bladder cancer incidence among the 316 communities (IR=73.11/100,000 PY). Future studies on sources and types of environmental pollution and exposures in relation to the spatial patterns of bladder cancer, particularly in Kotour District, may improve our understating of risk factors for bladder cancer in the region.

Analysis of RhoC expression and lymphovascular emboli in inflammatory vs non-inflammatory breast cancers in Egyptian patients.

Understanding the molecular factors that distinguish inflammatory breast cancer (IBC) from non-IBC is important for IBC diagnosis. We reviewed the records of 48 IBC patients and 64 non-IBC patients from Egypt. We determined RhoC expression and tumor emboli and their relationship to demographic and reproductive characteristics. Compared with non-IBC patients, IBC patients had significantly lower parity (P=0.018) and fewer palpable tumors (P<0.0001). IBC tumors showed RhoC overexpression more frequently than non-IBC tumors (87% vs. 17%, respectively) (P<0.0001). Tumor emboli were significantly more frequent in IBC tumors than non-IBC tumors (Mean+/- SD: 14.1+/-14.0 vs. 7.0+/-12.9, respectively) (P<0.0001). This study illustrates that RhoC overexpression and tumor emboli are more frequent in tumors of IBC relative to non-IBC from Egypt. Future studies should focus on relating epidemiologic factors to molecular features of IBC in this population.

Differences in Reliability of Reproductive History Recall Among Women in North Africa.

Breast cancer is the most common cancer among women in North Africa. Women in this region have unique reproductive profiles. It is essential to obtain reliable information on reproductive histories to help better understand the relationship between reductive health and breast cancer. We tested the reliability of a reproductive history-based questionnaire. We interviewed 25 breast cancer patients and 25 non-cancer controls from hospitals in Morocco and Egypt about their reproductive history in colloquial Arabic. The questions included pregnancy history, breastfeeding practices, menstruation, contraceptive use and knowledge of breast screening and re-interviewed the same women after 2 weeks. Two-way paired t-test was used to compare observed mean changes in response, and the Fishers Exact test was used for small-cell data. Pearson's correlation test was used to estimate the correlation of subjects' responses to continuous questions between the first and second interview. For categorical questions, percentage of agreement was calculated along with Cohen's Kappa Coefficient values. Moroccan subjects showed good to excellent agreement for responses to all demographic and reproductive questions (r = 0.87 to 0.99). Egyptian subjects had excellent agreement for these questions(r = 0.87 to 0.99), except for those regarding duration of oral contraceptive pill use and reported age at menarche (r = 0.72 and 0.59, respectively). We showed highly correlated responses to most reproductive questions. Duration of contraception use and age at first pregnancy elicited slightly less than reliable responses. In Egypt, responses relating to self-reported age at menarche were less reliable than those given by Moroccan subjects. Future epidemiological studies should take these differences into account when constructing reproductive history questionnaires.

Improvement of rifemycins production by Amycolatopsis mediterranei in batch and fed-batch cultures.

The production of rifamycins B and SV using glucose as main C-source by Amycolatopsis mediterranei in batch and fed-batch culture was investigated. Fed-batch culture using glucose as mono feeding substrate either in the form of pulse addition, in case of shake flask, or with constant feeding rate, in bioreactor level, proved to be an alternative production system with a significant increase in both volumetric and specific antibiotic production. The maximal concentrations of about 1146 mg/l and 2500 mg/l of rifamycins B and SV, respectively, was obtained in fed-batch culture in bioreactor level under non-oxygen limitation. On the other hand, the rate of rifamycins production was increased from 6.58 to 12.13 mg/l x h for rifamycin B and from 9.47 to 31.83 mg/l x h for rifamycin SV on the bioprocess transfer and improvement from the conventional batch cultivation in shake flask to fed-batch cultivation in stirred tank bioreactor.