Influence of selected non-antibiotic pharmaceuticals on antibiotic resistance gene transfer in Escherichia coli.

BACKGROUND: Antibiotic resistance genes (ARGs) transfer rapidly among bacterial species all over the world contributing to the aggravation of antibiotic resistance crisis. Antibiotics at sub-inhibitory concentration induce horizontal gene transfer (HRT) between bacteria, especially through conjugation. The role of common non-antibiotic pharmaceuticals in the market in disseminating antibiotic resistance is not well studied. OBJECTIVES: In this work, we indicated the effect of some commonly used non-antibiotic pharmaceuticals including antiemetic (metoclopramide HCl) and antispasmodics (hyoscine butyl bromide and tiemonium methyl sulfate) on the plasmid-mediated conjugal transfer of antibiotic resistance genes between pathogenic E. coli in the gastric intestinal tract (GIT). METHODS: Broth microdilution assay was used to test the antibacterial activity of the tested non-antibiotic pharmaceuticals. A conjugation mating system was applied in presence of the studied non-antibiotic pharmaceuticals to test their effect on conjugal transfer frequency. Plasmid extraction and PCR were performed to confirm the conjugation process. Transmission electron microscopy (TEM) was used for imaging the effect of non-antibiotic pharmaceuticals on bacterial cells. RESULTS: No antibacterial activity was reported for the used non-antibiotic pharmaceuticals. Plasmid-mediated conjugal transfer between isolates was induced by metoclopramide HCl but suppressed by hyoscine butyl bromide. Tiemonium methylsulfate slightly promoted conjugal transfer. Aggregation between cells and periplasmic bridges was clear in the case of metoclopramide HCl while in presence of hyoscine butyl bromide little affinity was observed. CONCLUSION: This study indicates the contribution of non-antibiotic pharmaceuticals to the dissemination and evolution of antibiotic resistance at the community level. Metoclopramide HCl showed an important role in the spread of antibiotic resistance.

COVID-19 associated Mucormycosis among ICU patients: risk factors, control, and challenges.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic is still difficult to be controlled. The spread of this virus and the emergence of new variants are considered a great challenge worldwide. Disturbance in infection control guidelines implementation, use of steroids, antibiotics, hospital crowdedness, and repeated use of oxygen masks during the management of critically ill COVID-19 patients lead to an increase in the rate of opportunistic infections. So, patients need to fight both the virus with its different variants and opportunistic pathogens including bacteria and fungi especially patients with diabetes mellitus, malignancy, or those who undergo hemodialysis and receive deferoxamine. During the pandemic, many cases of Mucormycosis associated with COVID-19 infection were observed in many countries. In this review, we discuss risk factors that increase the chance of infection by opportunistic pathogens, especially fungal pathogens, recent challenges, and control measures.

Prevalence of Quorum Sensing and Virulence Factor Genes Among Pseudomonas aeruginosa Isolated from Patients Suffering from Different Infections and Their Association with Antimicrobial Resistance.

Purpose: Antimicrobial resistance and virulence genes play important roles in increasing the severity of Pseudomonas aeruginosa infections, especially in hospitalized patients with high antibiotic pressure. Most genes that encode Pseudomonas aeruginosa virulence factors are controlled and regulated by the quorum sensing (QS) system. The aim of this study was to investigate the frequency of some virulence genes (rhlR, rhlI, lasR, lasI, lasB, toxA, aprA, algD, ExoS, and plcH genes) and their association with antibiotic resistance. Methods: Antimicrobial susceptibility was determined by Kirby-Bauer agar disk diffusion method. A total of 125 clinical isolates of P. aeruginosa were tested for some virulence genes using polymerase chain reaction (PCR). Results: The highest resistance was observed against cefepime (92.8%). Multi-drug resistant (MDR) P. aeruginosa represented 63.2% of total isolates with high distribution among wound isolates (21/79, 26.3% of MDR isolates). LasB was the most prevalent virulence gene among the tested isolates (89.6%) followed by aprA (85.6%), exoS (84%), algD (80%), toxA (76.8%), and plcH (75.2). Furthermore, a significant association (P < 0.05) among most of the tested virulence genes and MDR isolates was found. The presence of more than 5 virulence genes was highly observed among wound infections, otitis media, and respiratory tract infection isolates. Conclusion: The complex association of virulence genes including QS system regulating genes with antibiotic resistance indicates the importance of the tested factors in the progression of infections, which is considered a great challenge for the health-care team with the need for specific studies for each area having different antibiotic resistance profiles and the development of effective treatment strategies such as anti-virulent and quorum sensing inhibiting drugs against P. aeruginosa infections.

Efficacy and durability of bovine virus diarrhea (BVD) virus killed vaccine adjuvanted with monolaurin.

The bovine virus diarrhea virus (BVDV) causes reproductive, enteric, and respiratory diseases. Vaccination is essential in increasing herd resistance to BVDV spread. The selection of an adjuvant is an important factor in the success of the vaccination process. Monolaurin or glycerol monolaurate is a safe compound with an immunomodulatory effect. This study aimed to evaluate the efficacy of monolaurin as a novel adjuvant. This was examined through the preparation of an inactivated BVDV (NADL strain) vaccine adjuvanted with different concentrations of monolaurin and compared with the registered available locally prepared polyvalent vaccine (Pneumo-4) containing BVD (NADL strain), BoHV-1 (Abou Hammad strain), BPI3 (strain 45), and BRSV (strain 375L), and adjuvanted with aluminum hydroxide gel. The inactivated BVDV vaccine was prepared using three concentrations, 0.5%, 1%, and 2%, from monolaurin as adjuvants. A potency test was performed on five groups of animals. The first group, which did not receive vaccination, served as a control group while three other groups were vaccinated using the prepared vaccines. The fifth group received the Pneumo-4 vaccine. Vaccination response was monitored by measuring viral neutralizing antibodies using enzyme-linked immunosorbent assay (ELISA). It was found that the BVD inactivated vaccine with 1% and 2% monolaurin elicited higher neutralizing antibodies that have longer-lasting effects (nine months) with no reaction at the injection site in comparison to the commercial vaccine adjuvanted by aluminum hydroxide gel.

Effect of Imipenem and Amikacin Combination against Multi-Drug Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen associated with high morbidity and mortality rates. Combination of antibiotics has been found to combat multi-drug resistant or extensively drug resistance P. aeruginosa. In this study we investigate the in vitro and in vivo effect of amikacin and imipenem combination against resistant P. aeruginosa. The checkerboard technique and time-killing curve have been performed for in vitro studies showed synergistic effect for combination. A peritonitis mouse model has been used for evaluation of the therapeutic efficacy of this combination which confirmed this synergistic effect. The in vitro and in vivo techniques showed synergistic interaction between tested drugs with fractional inhibitory concentration indices (FICIs) of ≤0.5. Conventional PCR and quantitative real-time PCR techniques were used in molecular detection of bla IMP and aac(6')-Ib as 35.5% and 42.2% of P. aeruginosa harbored bla IMP and aac(6')-Ib respectively. Drug combination viewed statistically significant reduction in bacterial counts (p value < 0.5). The lowest bla IMP and aac(6')-Ib expression was observed after treatment with 0.25 MIC of imipenem + 0.5 MIC of amikacin. Morphological changes in P. aeruginosa isolates were detected by scanning electron microscope (SEM) showing cell shrinkage and disruption in the outer membrane of P. aeruginosa that were more prominent with combination therapy than with monotherapy.

Co-Existence of Certain ESBLs, MBLs and Plasmid Mediated Quinolone Resistance Genes among MDR E. coli Isolated from Different Clinical Specimens in Egypt.

The emergence of multi-drug resistant (MDR) strains and even pan drug resistant (PDR) strains is alarming. In this study, we studied the resistance pattern of E. coli pathogens recovered from patients with different infections in different hospitals in Minia, Egypt and the co-existence of different resistance determinants. E. coli was the most prevalent among patients suffering from urinary tract infections (62%), while they were the least isolated from eye infections (10%). High prevalence of MDR isolates was found (73%) associated with high ESBLs and MBLs production (89.4% and 64.8%, respectively). blaTEM (80%) and blaNDM (43%) were the most frequent ESBL and MBL, respectively. None of the isolates harbored blaKPC and blaOXA-48 carbapenemase like genes. Also, the fluoroquinolone modifying enzyme gene aac-(6')-Ib-cr was detected in 25.2% of the isolates. More than one gene was found in 81% of the isolates. Azithromycin was one of the most effective antibiotics against MDR E. coli pathogens. The high MAR index of the isolates and the high prevalence of resistance genes, indicates an important public health concern and high-risk communities where antibiotics are abused.

Effect of Titanium Dioxide Nanoparticles on the Expression of Efflux Pump and Quorum-Sensing Genes in MDR Pseudomonas aeruginosa Isolates.

Most of the infections caused by multi-drug resistant (MDR) P. aeruginosa strains are extremely difficult to be treated with conventional antibiotics. Biofilm formation and efflux pumps are recognized as the major antibiotic resistance mechanisms in MDR P. aeruginosa. Biofilm formation by P. aeruginosa depends mainly on the cell-to-cell communication quorum-sensing (QS) systems. Titanium dioxide nanoparticles (TDN) have been used as antimicrobial agents against several microorganisms but have not been reported as an anti-QS agent. This study aims to evaluate the impact of titanium dioxide nanoparticles (TDN) on QS and efflux pump genes expression in MDR P. aeruginosa isolates. The antimicrobial susceptibility of 25 P. aeruginosa isolates were performed by Kirby-Bauer disc diffusion. Titanium dioxide nanoparticles (TDN) were prepared by the sol gel method and characterized by different techniques (DLS, HR-TEM, XRD, and FTIR). The expression of efflux pumps in the MDR isolates was detected by the determination of MICs of different antibiotics in the presence and absence of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Biofilm formation and the antibiofilm activity of TDN were determined using the tissue culture plate method. The effects of TDN on the expression of QS genes and efflux pump genes were tested using real-time polymerase chain reaction (RT-PCR). The average size of the TDNs was 64.77 nm. It was found that TDN showed a significant reduction in biofilm formation (96%) and represented superior antibacterial activity against P. aeruginosa strains in comparison to titanium dioxide powder. In addition, the use of TDN alone or in combination with antibiotics resulted in significant downregulation of the efflux pump genes (MexY, MexB, MexA) and QS-regulated genes (lasR, lasI, rhll, rhlR, pqsA, pqsR) in comparison to the untreated isolate. TDN can increase the therapeutic efficacy of traditional antibiotics by affecting efflux pump expression and quorum-sensing genes controlling biofilm production.

Distribution of naturally -occurring NS5B resistance-associated substitutions in Egyptian patients with chronic Hepatitis C.

BACKGROUND: NS5B polymerase inhibitors represent the cornerstone of the present treatment of Hepatitis C virus infection (HCV). Naturally occurring substitution mutations to NS5B inhibitors have been recorded. The current study intended to demonstrate possible natural direct acting antiviral (DAA)-mutations of the HCV NS5B region in HCV patients in Minia governorate, Egypt. METHODS: Samples were collected from 27 treatment-naïve HCV patients and 8 non-responders. Out of 27 treatment-naïve patients, 17 NS5B sequences (amino acids 221-345) from treatment-naïve patients and one sample of non-responders were successfully amplified. Nucleotide sequences have been aligned, translated into amino acids, and compared to drug resistance mutations reported in the literature. RESULTS: NS5B amino acid sequence analysis ensures several novel NS5B mutations existence (more than 40 substitution mutations) that have not been previously documented to be correlated with a resistant phenotype. It was found that K304R (82.4%), E327D and P300T (76.5% each) substitutions were the most distributed in the tested samples, respectively. S282T, the major resistance mutation that induces high sofosbuvir-resistance level in addition to other reported mutations (L320F/C) and (C316Y/N) were not recognized. Q309R mutation is a ribavirin-associated resistance, which was recognized in one strain (5.9%) of genotype 1g sequences. Besides, one substitution mutation (E237G) was identified in the successfully amplified non-responder sample. CONCLUSION: Our study showed various combinations of mutations in the analyzed NS5B genes which could enhance the possibility of therapy failure in patients administered regimens including multiple DAA.

Virulence profiles of some Pseudomonas aeruginosa clinical isolates and their association with the suppression of Candida growth in polymicrobial infections.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause a variety of diseases especially in the hospital environment. However, this pathogen also exhibits antimicrobial activity against Gram-positive bacteria and fungi. This study aimed to characterize different virulence factors, secreted metabolites and to study their role in the suppression of Candida growth. Fifteen P. aeruginosa isolates were tested for their anticandidal activity against 3 different Candida spp. by the cross-streak method. The effect on hyphae production was tested microscopically using light and scanning electron microscopy (SEM). Polymerase chain reaction was used in the detection of some virulence genes. Lipopolysaccharide profile was performed using SDS-polyacrylamide gel stained with silver. Fatty acids were analyzed by GC-MS as methyl ester derivatives. It was found that 5 P. aeruginosa isolates inhibited all tested Candida spp. (50-100% inhibition), one isolate inhibited C. glabrata only and 3 isolates showed no activity against the tested Candida spp. The P. aeruginosa isolates inhibiting all Candida spp. were positive for all virulence genes. GC-Ms analysis revealed that isolates with high anticandidal activity showed spectra for several compounds, each known for their antifungal activity in comparison to those with low or no anticandidal activity. Hence, clinical isolates of P. aeruginosa showed Candida species-specific interactions by different means, giving rise to the importance of studying microbial interaction in polymicrobial infections and their contribution to causing disease.

Prevalence of Virulence Genes and Their Association with Antimicrobial Resistance Among Pathogenic E. coli Isolated from Egyptian Patients with Different Clinical Infections.

INTRODUCTION: Escherichia (E.) coli can cause intestinal and extra-intestinal infections which ranged from mild to life-threatening infections. The severity of infection is a product of many factors including virulence properties and antimicrobial resistance. OBJECTIVES: To determine the antibiotic resistance pattern, the distribution of virulence factors and their association with one another and with some selected resistance genes. METHODS: Virulence properties were analyzed phenotypically while antimicrobial susceptibility was tested by Kirby-Bauer agar disc diffusion method. In addition, 64 E. coli isolates were tested for 6 colicin genes, fimH, hlyA, traT, csgA, crl virulence genes and bla-CTX-M-15, bla-oxa-2 , and bla-oxa-10 resistance genes by polymerase chain reaction (PCR). RESULTS: Extra-intestinal pathogenic E. coli isolated from urine and blood samples represented a battery of virulence factors and resistance genes with a great ability to produce biofilm. Also, a significant association (P<0.05) among most of the tested colicin, virulence and resistance genes was observed. The observed associations indicate the importance and contribution of the tested factors in the establishment and the progress of infection especially with Extra-intestinal E. coli (ExPEC) which is considered a great challenging health problem. CONCLUSION: There is a need for studying how to control these factors to decrease the rate and the severity of infections. The relationship between virulence factors and resistance genes is complex and needs more studies that should be specific for each area.

Antimicrobial Activity of Silver-Treated Bacteria against other Multi-Drug Resistant Pathogens in Their Environment.

Silver is a potent antimicrobial agent against a variety of microorganisms and once the element has entered the bacterial cell, it accumulates as silver nanoparticles with large surface area causing cell death. At the same time, the bacterial cell becomes a reservoir for silver. This study aims to test the microcidal effect of silver-killed E. coli O104: H4 and its supernatant against fresh viable cells of the same bacterium and some other species, including E. coli O157: H7, Multidrug Resistant (MDR) Pseudomonas aeruginosa and Methicillin Resistant Staphylococcus aureus (MRSA). Silver-killed bacteria were examined by Transmission Electron Microscopy (TEM). Agar well diffusion assay was used to test the antimicrobial efficacy and durability of both pellet suspension and supernatant of silver-killed E. coli O104:H4 against other bacteria. Both silver-killed bacteria and supernatant showed prolonged antimicrobial activity against the tested strains that extended to 40 days. The presence of adsorbed silver nanoparticles on the bacterial cell and inside the cells was verified by TEM. Silver-killed bacteria serve as an efficient sustained release reservoir for exporting the lethal silver cations. This promotes its use as a powerful disinfectant for polluted water and as an effective antibacterial which can be included in wound and burn dressings to overcome the problem of wound contamination.

Isolation and evaluation of cocktail phages for the control of multidrug-resistant Escherichia coli serotype O104: H4 and E. coli O157: H7 isolates causing diarrhea.

Escherichia coli serotype O157: H7 and E. coli O104: H4 are well known foodborne pathogens causing sever enteric illness. Using bacteriophages as biocontrol agents of some foodborne pathogens and multidrug-resistant (MDR) bacteria has a great attention nowadays. This study aims to test the effect of cocktail phages on the growth of some foodborne pathogens and MDR E. coli. Routine conventional PCR was used to confirm the identification of E. coli isolates. Double-layered culture technique was used to isolate phages from sewage water. Morphology of bacteriophage was described using transmission electron microscopy, and spot test was performed to determine host range of the phage cocktail. Phage cocktail of Siphoviridae and Podoviridae family infecting E. coli O157: H7, E. coli O104: H4 and untypeable E. coli (neither O157 nor O104) has been isolated from sewage water. Phage cocktail showed both lytic and lysogenic activity. Lytic activity was observed against E. coli O157: H7, E. coli O104: H4 isolates, Staphylococcus. aureus ATCC6538 and Pseudomonas aeruginosa ATCC 10145, while the lysogenic activity was observed against the untypeable strain. The tested phage cocktail showed a promising inhibitory action on E. coli O157: H7 and O104: H4, S. aureus ATCC6538 and P. aeruginosa ATCC 10145, suggesting the possibility of its use as a biocontrol tool or as natural food preservatives for many food products.