Biosurfactant from Nile Papyrus endophyte with potential antibiofilm activity against global clones of Acinetobacter baumannii.

Acinetobacter baumannii is a leading cause of biofilm-associated infections, particularly catheter-related bloodstream infections (CRBSIs) that are mostly recalcitrant to antimicrobial therapy. One approach to reducing the burden of CRBSIs is inhibiting biofilm formation on catheters. Owing to their prodigious microbial diversity, bacterial endophytes might be a valuable source of biosurfactants, which are known for their great capacity to disperse microbial biofilms. With this in mind, our study aimed to screen bacterial endophytes from plants growing on the banks of the River Nile for the production of powerful biosurfactants capable of reducing the ability of A. baumannii to form biofilms on central venous catheters (CVCs). This was tested on multidrug- and extensive drug-resistant (M/XDR) clinical isolates of A. baumannii that belong to high-risk global clones and on a standard strain of A. baumannii ATCC 19606. The drop collapse and oil dispersion assays were employed in screening the cell-free supernatants (CFS) of all endophytes for biosurfactant activity. Of the 44 bacterial endophytes recovered from 10 plants, the CFS of Bacillus amyloliquefaciens Cp24, isolated from Cyperus papyrus, showed the highest biosurfactant activity. The crude biosurfactant extract of Cp24 showed potent antibacterial activity with minimum inhibitory concentrations (MICs) ranging from 0.78 to 1.56 mg/ml. It also showed significant antibiofilm activity (p-value<0.01). Sub-MICs of the extract could reduce biofilm formation by up to 89.59%, while up to 87.3% of the preformed biofilms were eradicated by the MIC. A significant reduction in biofilm formation on CVCs impregnated with sub-MIC of the extract was demonstrated by CV assay and further confirmed by scanning electron microscopy. This was associated with three log10 reductions in adhered bacteria in the viable count assay. GC-MS analysis of the crude biosurfactant extract revealed the presence of several compounds, such as saturated, unsaturated, and epoxy fatty acids, cyclopeptides, and 3-Benzyl-hexahydro-pyrrolo [1, 2-a] pyrazine-1,4-dione, potentially implicated in the potent biosurfactant and antibiofilm activities. In the present study, we report the isolation of a B. amyloliquefaciens endophyte from the plant C. papyrus that produces a biosurfactant with potent antibiofilm activity against MDR/XDR global clones of A. baumannii. The impregnation of CVCs with the biosurfactant was demonstrated to reduce biofilms and, hence, proposed as a potential strategy for reducing CRBSIs.

Hormonal drugs: Influence on growth, biofilm formation, and adherence of selected gut microbiota.

Many studies have reported the influence of hormonal drugs on gut microbiota composition. However, the underlying mechanism of this interaction is still under study. Therefore, this study aimed to evaluate the possible in vitro changes in selected members of gut bacteria exposed to oral hormonal drugs used for years. Selected members of gut bacteria were Bifidobacterium longum, Limosilactobacillus reuteri, Bacteroides fragilis, and Escherichia coli representing the four main phyla in the gut. Selected hormonal drugs used for a long time were estradiol, progesterone, and thyroxine. The effect of intestinal concentrations of these drugs on the selected bacterial growth, biofilm formation, and adherence to Caco-2/HT-29 cell line was assessed. Short-chain fatty acids (SCFAs) have been included in host functions including the gut, immune and nervous functions; thus, the drug's effects on their production were assayed using High- Performance Liquid Chromatography. Sex steroids significantly increased the growth of all tested bacteria except B. longum, similarly, thyroxine increased the growth of tested Gram-negative bacteria however reducing that of tested Gram-positive bacteria. The effect of drugs on biofilm formation and bacterial adherence to cell lines cocultures was variable. Progesterone decreased the biofilm formation of tested Gram-positive bacteria, it nevertheless increased L. reuteri adherence to Caco-2/HT-29 cell line cell lines coculture. By contrast, progesterone increased biofilm formation by Gram-negative bacteria and increased adherence of B. fragilis to the cell lines coculture. Moreover, thyroxine and estradiol exhibited antibiofilm activity against L. reuteri, while thyroxine increased the ability of E. coli to form a biofilm. Moreover, hormones affected bacterial adherence to cell lines independently of their effect on hydrophobicity suggesting other specific binding factors might contribute to this effect. Tested drugs affected SCFAs production variably, mostly independent of their effect on bacterial growth. In conclusion, our results showed that the microbiota signature associated with some hormonal drug consumption could be the result of the direct effect of these drugs on bacterial growth, and adherence to enterocytes besides the effect of these drugs on the host tissue targets. Additionally, these drugs affect the production of SCFAs which could contribute to some of the side effects of these drugs.

State of the art in epitope mapping and opportunities in COVID-19.

The understanding of any disease calls for studying specific biological structures called epitopes. One important tool recently drawing attention and proving efficiency in both diagnosis and vaccine development is epitope mapping. Several techniques have been developed with the urge to provide precise epitope mapping for use in designing sensitive diagnostic tools and developing rpitope-based vaccines (EBVs) as well as therapeutics. In this review, we will discuss the state of the art in epitope mapping with a special emphasis on accomplishments and opportunities in combating COVID-19. These comprise SARS-CoV-2 variant analysis versus the currently available immune-based diagnostic tools and vaccines, immunological profile-based patient stratification, and finally, exploring novel epitope targets for potential prophylactic, therapeutic or diagnostic agents for COVID-19.

Epitope mapping is an important tool recently proving efficiency in both diagnosis and vaccine development. Several epitope mapping techniques have been developed for designing sensitive diagnostic tools and developing rpitope-based vaccines (EBVs) as well as therapeutics. In this review, we will discuss the state of the art in epitope mapping, emphasizing accomplishments and opportunities in combating COVID-19. These comprise SARS-CoV-2 variant analysis versus the currently available immune-based diagnostic tools and vaccines and exploring novel epitope targets for potential prophylactic, therapeutic or diagnostic agents for COVID-19.

The RND Efflux Pump Gene Expression in the Biofilm Formation of Acinetobacter baumannii.

Multidrug resistant (MDR) Acinetobacter baumannii is a critical opportunistic pathogen in healthcare-associated infections (HAI). This is attributed to several factors, including its ability to develop biofilms that can enhance antimicrobial resistance (AMR) in addition to creating an environment for horizontal transfer of antibiotic resistance genes. The role of the efflux pump in biofilm formation is important for studies on alternative treatments for biofilms. One of the significant efflux pump families is the RND efflux pump family, which is common in Gram negative bacteria. The aim is to study the role of the RND efflux pump in biofilm formation by A. baumannii. The biofilm formation potential of thirty-four MDR A. baumannii isolates was evaluated by crystal violet assays. The effect of efflux pump inhibition and activation was studied using the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the RND efflux pump substrate levofloxacin (at sub-MIC), respectively. The isolates were genotypically grouped by enterobacterial repetitive intergenic consensus (ERIC) typing and the expression of adeABC, adeFGH, and adeIJK efflux pump genes was measured by qPCR. Overall, 88.2% (30/34) of isolates were biofilm producers (the phenotype was variable including strong and weak producers). Efflux pump inhibition by CCCP reduced the biofilm formation significantly (p < 0.05) in 17.6% (6/34) of some isolates, whereas sub-MICs of the substrate levofloxacin increased biofilm formation in 20.5% (7/34) of other isolates. Overexpression of the three RND efflux pump genes was detected in five out of eleven selected isolates for qPCR with remarkable overexpression in the adeJ gene. No correlation was detected between the biofilm phenotype pattern and the RND efflux pump gene expression in biofilm cells relative to planktonic cells. In conclusion, the role of the RND efflux pumps AdeABC, AdeFGH, and AdeIJK in biofilm formation does not appear to be pivotal and the expression differs according to the genetic background of each strain. Thus, these pumps may not be a promising target for biofilm inhibition.

Novel linezolid loaded bio-composite films as dressings for effective wound healing: experimental design, development, optimization, and antimicrobial activity.

Biphasic release bio-composite films of the low water-soluble drug, linezolid (LNZ), were formulated using the solvent casting technique. Different polymers and plasticizers (gelatin, Tween 80, polyethylene glycol 400, and glycerol) were assessed for the preparation of bio-composite films. An I-optimal design was applied for the optimization and to study the impact of polymer concentration (X1), plasticizer concentration (X2), polymer type (X3), and plasticizer type (X4) on different LNZ-loaded bio-composite films. The film thickness, moisture content, mechanical properties, swelling index, and percentage of drug release at fixed times opted as dependent variables. Results demonstrated a significant effect of all independent variables on the drug release from the prepared bio-composite films. The plasticizer concentration significantly increased the thickness, moisture content, elongation at break, swelling index, and in vitro drug release and significantly reduced the tensile strength. The optimized LNZ-loaded bio-composite film comprised of 15% Tween 80 and 30% PEG 400 was highly swellable, elastic, acceptable tensile properties, safe, maintained a moist environment, and indicated great antimicrobial activity against both Staphylococcus aureus (ATCC® 25922) and methicillin-resistant Staphylococcus aureus (MRSA), which are common wound infectious bacteria. The present study concludes that the optimized LNZ-loaded bio-composite film was successfully designed with fast drug release kinetics and it could be regarded as a promising novel antimicrobial wound dressing formulation.

Silicone Foley catheters impregnated with microbial indole derivatives inhibit crystalline biofilm formation by Proteus mirabilis.

Proteus mirabilis is a common causative agent for catheter-associated urinary tract infections (CAUTI). The crystalline biofilm formation by P. mirabilis causes catheter encrustation and blockage leading to antibiotic treatment resistance. Thus, biofilm formation inhibition on catheters becomes a promising alternative for conventional antimicrobial-based treatment that is associated with rapid resistance development. Our previous work has demonstrated the in vitro antibiofilm activity of microbial indole derivatives against clinical isolates of P. mirabilis. Accordingly, we aim to evaluate the capacity of silicone Foley catheters (SFC) impregnated with these indole derivatives to resist biofilm formation by P. mirabilis both phenotypically and on the gene expression level. Silicon Foley catheter was impregnated with indole extract recovered from the supernatant of the rhizobacterium Enterobacter sp. Zch127 and the antibiofilm activity was determined against P. mirabilis (ATCC 12435) and clinical isolate P8 cultured in artificial urine. The indole extract at sub-minimum inhibitory concentration (sub-MIC=0.5X MIC) caused a reduction in biofilm formation as exhibited by a 60-70% reduction in biomass and three log10 in adhered bacteria. Results were confirmed by visualization by scanning electron microscope. Moreover, changes in the relative gene expression of the virulence genes confirmed the antibiofilm activity of the indole extract against P. mirabilis. Differential gene expression analysis showed that extract Zch127 at its sub-MIC concentration significantly down-regulated genes associated with swarming activity: umoC, flhC, flhD, flhDC, and mrpA (p< 0.001). In addition, Zch127 extract significantly down-regulated genes associated with polyamine synthesis: speB and glnA (p< 0.001), as well as the luxS gene associated with quorum sensing. Regulatory genes for capsular polysaccharide formation; rcsB and rcsD were not significantly affected by the presence of the indole derivatives. Furthermore, the impregnated catheters and the indole extract showed minimal or no cytotoxic effect against human fibroblast cell lines indicating the safety of this intervention. Thus, the indole-impregnated catheter is proposed to act as a suitable and safe strategy for reducing P. mirabilis CAUTIs.

High Counts and Anthracene Degradation Ability of Streptococcus mutans and Veillonella parvula Isolated From the Oral Cavity of Cigarette Smokers and Non-smokers.

The composition and metabolic functions of oral microbiota are affected by many factors including smoking leading to several health problems. Cigarette smoking is associated with changes in oral microbiota composition and function. However, it is not known if the depletion of certain bacterial genera and species is due to specific toxins in cigarette smoke, or indirectly due to competition for colonization with smoking-enriched bacteria. Therefore, the aim of this study was to determine the effect of cigarette smoking on the microbial prevalence and polycyclic aromatic hydrocarbons (PAHs) biodegradation of selected enriched and depleted oral bacteria from oral microbiota of smokers compared to that in non-smokers. Samples of oral rinse from smokers and non-smokers were collected (n = 23, 12 smokers and 11 non-smokers) and screened for oral bacterial strains of Streptococcus mutans, Lactobacillus spp., and Veillonella spp. Comparing counts, S. mutans, V. tobetsuensis, and V. dispar showed higher counts in smokers compared to non-smokers while the Lactobacillus spp. were higher in non-smokers. Lactobacillus fermentum was prevalent in smokers, representing 91.67% of the total Lactobacillus spp. isolates. The biodegradation potential of anthracene; a representative of PAHs of collected isolates, in single and mixed cultures, was assayed with anthracene as the sole source of carbon using 2,6-dichlorophenol indophenol (2,6-DCPIP) as indicator. S. mutans isolates recovered from smokers showed higher degradation of anthracene compared to those recovered from non-smokers. The anaerobic anthracene biodegradation activity of V. parvula isolates from non-smokers was the highest among all isolates of the three recovered genera from the same subject. The anthracene biodegradation potential of Lactobacillus spp. was variable. Combinations of isolated bacteria in co-cultures showed that Lactobacillus spp. interfered with anthracene biodegradation ability along with the viable counts of S. mutans and Veillonella spp. In conclusion, oral dysbiosis due to cigarette smoking was observed not only due to changes in oral bacterial relative abundance but also extended to bacterial functions such as anthracene biodegradation tested in this study. Microbe-microbe interactions changed the anthracene biodegradation potential and growth of the microbial mixture compared to their corresponding single isolates, and these changes differ according to the constituting bacteria.

Indole Derivatives Obtained from Egyptian Enterobacter sp. Soil Isolates Exhibit Antivirulence Activities against Uropathogenic Proteus mirabilis.

Proteus mirabilis is a frequent cause of catheter associated urinary tract infections (CAUTIs). Several virulence factors contribute to its pathogenesis, but swarming motility, biofilm formation, and urease activity are considered the hallmarks. The increased prevalence in antibiotic resistance among uropathogens is alarming and requires searching for new treatment alternatives. With this in mind, our study aims to investigate antivirulence activity of indole derivatives against multidrug resistant P. mirabilis isolates. Ethyl acetate (EtOAc) extracts from Enterobacter sp. (rhizobacterium), isolated from Egyptian soil samples were tested for their ability to antagonize the virulence capacity and biofilm activity of P. mirabilis uropathogens. Extracts of two Enterobacter sp. isolates (coded Zch127 and Cbg70) showed the highest antivirulence activities against P. mirabilis. The two promising rhizobacteria Zch127 and Cbg70 were isolated from soil surrounding: Cucurbita pepo (Zucchini) and Brassica oleracea var. capitata L. (Cabbage), respectively. Sub-minimum inhibitory concentrations (Sub-MICs) of the two extracts showed potent antibiofilm activity with significant biofilm reduction of ten P. mirabilis clinical isolates (p-value < 0.05) in a dose-dependent manner. Interestingly, the Zch127 extract showed anti-urease, anti-swarming and anti-swimming activity against the tested strains. Indole derivatives identified represented key components of indole pyruvate, indole acetamide pathways; involved in the synthesis of indole acetic acid. Additional compounds for indole acetonitrile pathway were detected in the Zch127 extract which showed higher antivirulence activity. Accordingly, the findings of the current study model the feasibility of using these extracts as promising antivirulence agent against the P. mirabilis uropathogens and as potential therapy for treatment of urinary tract infections (UTIs).

Co-Existence of Carbapenemase-Encoding Genes in Acinetobacter baumannii from Cancer Patients.

INTRODUCTION: Acinetobacter baumannii is an opportunistic pathogen, which can acquire new resistance genes. Infections by carbapenem-resistant A. baumannii (CRAB) in cancer patients cause high mortality. METHODS: CRAB isolates from cancer patients were screened for carbapenemase-encoding genes that belong to Ambler classes (A), (B), and (D), followed by genotypic characterization by enterobacterial-repetitive-Intergenic-consensus-polymerase chain reaction (ERIC-PCR) and multilocus-sequence-typing (MLST). RESULTS: A total of 94.1% of CRAB isolates co-harbored more than one carbapenemase-encoding gene. The genes blaNDM, blaOXA-23-like, and blaKPC showed the highest prevalence, with rates of 23 (67.7%), 19 (55.9%), and 17 (50%), respectively. ERIC-PCR revealed 19 patterns (grouped into 9 clusters). MLST analysis identified different sequence types (STs) (ST-268, ST-195, ST-1114, and ST-1632) that belong to the highly resistant easily spreadable International clone II (IC II). Genotype diversity indicated the dissemination of carbapenem-hydrolyzing, ß-lactamase-encoding genes among genetically unrelated isolates. We observed a high prevalence of metallo-ß-lactamase (MBL)-encoding genes (including the highly-resistant blaNDM gene that is capable of horizontal gene transfer) and of isolates harboring multiple carbapenemase-encoding genes from different classes. CONCLUSION: The findings are alarming and call for measures to prevent and control the spread of MBL-encoding genes among bacteria causing infections in cancer patients and other immunocompromised patient populations.

Proteus mirabilis Biofilm: Development and Therapeutic Strategies.

Proteus mirabilis is a Gram negative bacterium that is a frequent cause of catheter-associated urinary tract infections (CAUTIs). Its ability to cause such infections is mostly related to the formation of biofilms on catheter surfaces. In order to form biofilms, P. mirabilis expresses a number of virulence factors. Such factors may include adhesion proteins, quorum sensing molecules, lipopolysaccharides, efflux pumps, and urease enzyme. A unique feature of P. mirabilis biofilms that build up on catheter surfaces is their crystalline nature owing to their ureolytic biomineralization. This leads to catheter encrustation and blockage and, in most cases, is accompanied by urine retention and ascending UTIs. Bacteria embedded in crystalline biofilms become highly resistant to conventional antimicrobials as well as the immune system. Being refractory to antimicrobial treatment, alternative approaches for eradicating P. mirabilis biofilms have been sought by many studies. The current review focuses on the mechanism by which P. mirabilis biofilms are formed, and a state of the art update on preventing biofilm formation and reduction of mature biofilms. These treatment approaches include natural, and synthetic compounds targeting virulence factors and quorum sensing, beside other strategies that include carrier-mediated diffusion of antimicrobials into biofilm matrix. Bacteriophage therapy has also shown successful results in vitro for combating P. mirabilis biofilms either merely through their lytic effect or by acting as facilitators for antimicrobials diffusion.

First-time characterization of viable but non-culturable Proteus mirabilis: Induction and resuscitation.

Pathogenic bacteria can enter into a viable but non-culturable (VBNC) state under unfavourable conditions. Proteus mirabilis is responsible for dire clinical consequences including septicaemia, urinary tract infections and pneumonia, but is not a species previously known to enter VBNC state. We suggested that stress-induced P. mirabilis can enter a VBNC state in which it retains virulence. P. mirabilis isolates were incubated in extreme osmotic pressure, starvation, low temperature and low pH to induce a VBNC state. Resuscitation was induced by temperature upshift and inoculation in tryptone soy broth with Tween 20 and brain heart infusion broth. Cellular ultrastructure and gene expression were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR), respectively. High osmotic pressure and low acidity caused rapid entry into VBNC state. Temperature upshift caused the highest percentage of resuscitation (93%) under different induction conditions. In the VBNC state, cells showed aberrant and dwarf morphology, virulence genes and stress response genes (envZ and rpoS) were expressed (levels varied depending on strain and inducing factors). This is the first-time characterization of VBNC P. mirabilis. The ability of P. mirabilis pathogenic strains to enter a stress-induced VBNC state can be a serious public health threat.

Comparative lyophilized platelet-rich plasma wafer and powder for wound-healing enhancement: formulation, in vitro and in vivo studies.

Platelet-rich plasma (PRP) accelerates wound healing, as it is an excellent source of growth factors. PRP was separated from whole human blood by centrifugation. PRP powder and wafers were prepared by lyophilization, with the wafers prepared using sodium carboxymethylcellulose (Na CMC). The PRP wafers showed porous structures, as indicated by scanning electron microscopy (SEM) images, and the ability of the wafer to absorb exudates and thus promote wound healing was tested with the hydration capacity test. The platelet count was tested and indicated that the presence of PRP in the wafers had no effect on the platelet count. An antimicrobial activity test was carried out, showing that PRP had antibacterial activity against Gram-negative bacteria. Compared with lyophilized PRP powder and PRP-free wafers, PRP wafers showed the highest percent of wound size reduction on induced wounds in rats. Histopathological examination of rat skin showed that the PRP wafers achieved the shortest healing time, followed by the lyophilized PRP powder and finally the PRP-free wafers. The present study revealed that PRP can be formulated as a wafer, which is a promising pharmaceutical delivery system that can be used for enhanced wound-healing activity and improved the ease of application compared to lyophilized PRP powder.

Probiotic Lactobacillus sp. inhibit growth, biofilm formation and gene expression of caries-inducing Streptococcus mutans.

Streptococcus mutans contributes significantly to dental caries, which arises from homoeostasic imbalance between host and microbiota. We hypothesized that Lactobacillus sp. inhibits growth, biofilm formation and gene expression of Streptococcus mutans. Antibacterial (agar diffusion method) and antibiofilm (crystal violet assay) characteristics of probiotic Lactobacillus sp. against Streptococcus mutans (ATCC 25175) were evaluated. We investigated whether Lactobacillus casei (ATCC 393), Lactobacillus reuteri (ATCC 23272), Lactobacillus plantarum (ATCC 14917) or Lactobacillus salivarius (ATCC 11741) inhibit expression of Streptococcus mutans genes involved in biofilm formation, quorum sensing or stress survival using quantitative real-time polymerase chain reaction (qPCR). Growth changes (OD600) in the presence of pH-neutralized, catalase-treated or trypsin-treated Lactobacillus sp. supernatants were assessed to identify roles of organic acids, peroxides and bacteriocin. Susceptibility testing indicated antibacterial (pH-dependent) and antibiofilm activities of Lactobacillus sp. against Streptococcus mutans. Scanning electron microscopy revealed reduction in microcolony formation and exopolysaccharide structural changes. Of the oral normal flora, L. salivarius exhibited the highest antibiofilm and peroxide-dependent antimicrobial activities. All biofilm-forming cells treated with Lactobacillus sp. supernatants showed reduced expression of genes involved in exopolysaccharide production, acid tolerance and quorum sensing. Thus, Lactobacillus sp. can inhibit tooth decay by limiting growth and virulence properties of Streptococcus mutans.

Carriage frequency, phenotypic, and genotypic characteristics of methicillin-resistant Staphylococcus aureus isolated from dental health-care personnel, patients, and environment.

There is limited data on methicillin-resistant Staphylococcus aureus (MRSA) carriage in dental clinics. 1300 specimens from patients, health personnel, and environmental surfaces of a dental clinic in Egypt were tested for MRSA. Antibiotic susceptibility, biofilm formation, Staphylococcal protein A (spa) typing, SCCmec typing, and PCR-based assays were used to detect mecA, mecC, vanA, Panton-Valentine Leukocidin toxin (PVL), and toxic shock syndrome toxin-1 (tst) genes. Among 34 mecA-positive MRSA isolates, five (14.7%) were PVL-positive, seventeen (50%) were tst-positive, ten (29.4%) were vanA-positive, while none harboured mecC. MRSA hand carriage rates in patients, nurses, and dentists were 9.8%, 6.6%, and 5%. The respective nasal colonization rates were 11.1%, 6.7%, and 9.7%. 1.3% of the environmental isolates were MRSA-positive. Strong and moderate biofilm-forming isolates represented 23.5% and 29.4% of MRSA isolates. 24 MRSA isolates (70.6%) were multi-resistant and 18 (52.9%) harboured SCCmec IV. Among eight spa types, t223 (26.5%), t267 (23.5%), and t14339 (23.5%) were predominant. We noted an alarming genetic relatedness between 7 (20.6%) MRSA isolates and the epidemic EMRSA-15 clone, as well as a combined occurrence of tst and PVL in 3 (8.8%) isolates. Results suggest high MRSA pathogenicity in dental wards highlighting the need for more efficient surveillance/infection control strategies.

Molecular typing and virulence analysis of multidrug resistant Klebsiella pneumoniae clinical isolates recovered from Egyptian hospitals.

Klebsiella pneumonia infection rates have increased dramatically. Molecular typing and virulence analysis are powerful tools that can shed light on Klebsiella pneumonia infections. Whereas 77.7% (28/36) of clinical isolates indicated multidrug resistant (MDR) patterns, 50% (18/36) indicated carpabenem resistance. Gene prevalence for the AcrAB efflux pump (82.14%) was more than that of the mdtK efflux pump (32.14%) in the MDR isolates. FimH-1 and mrkD genes were prevalent in wound and blood isolates. FimH-1 gene was prevalent in sputum while mrkD gene was prevalent in urine. Serum resistance associated with outer membrane protein coding gene (traT) was found in all blood isolates. IucC, entB, and Irp-1 were detected in 32.14%, 78.5% and 10.7% of MDR isolates, respectively. We used two Polymerase Chain Reaction (PCR) analyses: Enterobacterial Repetitive Intergenic Consensus (ERIC) and Random Amplified Polymorphic DNA (RAPD). ERIC-PCR revealed 21 and RAPD-PCR revealed 18 distinct patterns of isolates with similarity ≥80%. ERIC genotyping significantly correlated with resistance patterns and virulence determinants. RAPD genotyping significantly correlated with resistance patterns but not with virulence determinants. Both RAPD and ERIC genotyping methods had no correlation with the capsule types. These findings can help up better predict MDR Klebsiella pneumoniae outbreaks associated with specific genotyping patterns.

Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli.

The purpose of this study was to: (i) evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram) against Escherichia coli; (ii) investigate the effects of these honeys on bacterial ultrastructure; and (iii) assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism. The minimum inhibitory concentration (MIC) of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR) analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed "ghost" cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets.

Phenotypic and molecular characterization of plasmid mediated AmpC ß-lactamases among Escherichia coli, Klebsiella spp., and Proteus mirabilis isolated from urinary tract infections in Egyptian hospitals.

The incidence of resistance by Enterobacteriaceae to ß-lactam/ß-lactamase inhibitors combination is increasing in Egypt. Three phenotypic techniques, comprising AmpC disk diffusion and inhibition dependent methods using phenylboronic acid (PBA) and cloxacillin, were compared to PCR based method for detection of plasmid mediated AmpC ß-lactamase in common urinary tract isolates. A total of 143 isolates, including E. coli, Klebsiella pneumonia, and Proteus mirabilis, were collected from urinary tract infections cases in Egyptian hospitals. Plasmid encoded AmpC genes were detected by PCR in 88.46% of cefoxitin resistant isolates. The most prevalent AmpC gene family was CIT including CMY-2, CMY-4, and two CMY-2 variants. The second prevalent gene was DHA-1 which was detected in E. coli and Klebsiella pneumonia. The genes EBC, FOX, and MOX were also detected but in small percentage. Some isolates were identified as having more than one pAmpC gene. The overall sensitivity and specificity of phenotypic tests for detection of AmpC ß-lactamase showed that AmpC disk diffusion and inhibition dependent method by cloxacillin were the most sensitive and the most specific disk tests. PCR remains the gold standard for detection of AmpC ß-lactamases. This study represents the first report of CMY-2 variants of CMY-42 and CMY-102 ß-lactamase-producing E. coli, Klebsiella pneumonia, and Proteus mirabilis isolates in Egypt.