Multiple Drug-resistant Bacteria Research Articles

COMPARATIVE STUDY OF QUERCETIN & LEMON PEEL EXTRACT ON MULTIPLE DRUG RESISTANT BACTERIA

The current investigation explores the antibacterial properties of Quercetin, a flavonoid found in plants, and lemon peel extract, renowned for its rich bioactive content, against multiple drug resistant bacteria. The study method involved the identification of multidrug-resistant bacteria through antimicrobial susceptibility testing (AST). Nine standard bacterial strains exhibited resistance to different antibiotics, with a MAR index exceeding 0.10, including Ampicillin, Vancomycin, Polymyxin B, Nitrofurantoin, Trimethoprim, and penicillins.In the present study bioactive compounds from lemon peel were extracted using two solvents, methanol and water, and their efficacy was assessed against drug-resistant bacteria. Minimum Inhibitory Concentration (MIC) values, determined by using the well plate method, revealed lower MIC values for the water extract (LPWE) compared to the methanol extract (LPME) and quercetin. For instance, the MIC for LPWE against E. hermanniensis ATCC 700323 was 140 mg/ml, whereas it was 50 mg/ml for quercetin & 1 mg/ml for LPME. From the results, it can be interpreted that Methanol extract was most effective against drug-resistant bacteria when compared to water extract and quercetin.Furthermore, GC-MS analysis was performed to analyze the chemical entities present in each extract. Given the activity of LPME, LPWE, and Quercetin against both Gram-negative bacteria and Gram-positive bacteria, these extracts hold promise as broad-spectrum antibacterial agents applicable in various fields such as medicine, food, and cosmetics.

Biosynthesized silver nanoparticles prevent bacterial infection in chicken egg model and mitigate biofilm formation on medical catheters.

Investigating the application of innovative antimicrobial surface coatings on medical devices is an important field of research. Many of these coatings have significant drawbacks, including biocompatibility, coating stability and the inability to effectively combat multiple drug-resistant bacteria. In this research, we developed an antibiofilm surface coating for medical catheters using biosynthesized silver nanoparticles (b-Cs-AgNPs) developed using leaves extract of Calliandra surinamensis. Various characterization techniques were employed to thoroughly characterize the synthesized b-Cs-AgNPs and c-AgNPs. b-Cs-AgNPs were compatible with human normal kidney cells and chicken embryos. It did not trigger any skin inflammatory response in in vivo rat model. b-Cs-AgNPs demonstrated potent zone of inhibition of 19.09mm when subjected to the disc diffusion method in E. coli confirming strong antibacterial property. Different anti-bacterial assays including liquid growth curve, colony counting assay, biofilm formation assay supported the potent antimicrobial efficacy of b-Cs-AgNPs alone and when coated to medical grade catheters. Mechanistic studies reveal the presence of ferulic acid, that was important for the synthesis of b-AgNPs along with enhanced antibacterial effects of b-Cs-AgNPs compared to c-AgNPs, supported by molecular docking analysis. These results together demonstrated the effective role b-Cs-AgNPs in combating infections and mitigating biofilm formations, highlighting their need for further study in the field of biomedical applications.

Enhanced bactericidal, antibiofilm and antioxidative response of Lawsonia inermis leaf extract synthesized ZnO NPs loaded with commercial antibiotic.

Globally, antibiotic resistance is a challenging issue in healthcare sector. The emergence of multiple drug-resistant bacteria has forced us to modify existing medicines and or formulate newer medicines that are effective and inexpensive. In this perspective, this study involves theformation of zinc oxide nanoparticles (ZnO NPs) by utilizing the Lawsonia inermis (Li) leaf extract. The prepared L. inermis leaf extract mediated ZnO NPs (Li-ZnO NPs) were bio-physically characterized. The antibacterial and radical scavenging effects of Li-ZnO NPs were evaluated. In addition, ZnO NPs were conjugated with standard antibiotic (ciprofloxacin) and its drug loading efficiency, drug release and antibacterial efficacy were tested and compared with non-drugloaded ZnO NPs. An absorbance peak at 340nm was noted for Li-ZnO NPs. After conjugation with the drug, two absorbance peaks- one at 242nm characteristic of ciprofloxacin and the other at 350nm characteristics of ZnO NPs were observed. The crystallite size was 18.7nm as determined by XRD. The antibacterial effect was higher on Gram-positive (S. aureus and S. pyogenes) than the Gram-negative pathogens (E. coli and K. pneumoniae). Inhibition of S. aureus and S. pyogenes biofilm at 100μgmL-1were, respectively, 97.5 and 92.6%. H2O2 free radicals was inhibited to 90% compared to the standard ascorbic acid at 100μgmL-1. After drug loading, the FTIR spectrum confirmed the existence of ciprofloxacin peaks at 965cm-1 and Zn-O bond at 492cm-1. The drug loading capacity of 15nm sized ZnO NPs was higher (58, 75, 90 and 95% at 1, 2.5, 5 and 10% drug concentrations, respectively) compared to 20nm. Similarly, the percentage of drug (ciprofloxacin) released from 15nm ZnO NPs were increased to 90% at 10% drug-loaded samples, respectively. Also, the antibiotic loaded ZnO NPs had significant antibacterial effects against tested bacteria compared to Li-ZnO NPs and ciprofloxacin alone. This revealed that the antibiotic loaded ZnO NPs offer a sustainable route to treat multi-drug-resistant bacterial infections.

Exploitation of mangliculous marine fungi, Amarenographium solium, for the green synthesis of silver nanoparticles and their activity against multiple drug-resistant bacteria

Abstract The green synthesis pathway for silver nanoparticles (AgNPs) used in bacterial treatment is regarded as crucial because of its cost-effectiveness, nontoxicity, and eco-friendliness. During the present work, the mangliculous marine fungi Amarenographium solium isolated from the Arabian Gulf Coast of Saudi Arabia were utilized for the synthesis of AgNP, through the bio-reduction of aqueous silver nitrate (AgNO3) solution. The success in AgNP synthesis was visually identified by the development of dark brown color in the cell-free filtrate and was further confirmed by ultraviolet–visible spectroscopy, which showed a peak at 425 nm. The AgNPs produced were further characterized using X-ray diffraction data analysis that proved the bioreduction of silver to 20 nm, and transmission electron microscopy revealed the formation of well-dispersed spherical nanoparticles with an average mean size of 12 nm. The optimization reaction parameters of temperature, pH, and metal salt concentration were carried out and resulted in a combination of 30°C, 7 and 1.5 mM, respectively, for rapid and maximum yield production. The antibacterial activity of the produced nanoparticles was evaluated using the two-fold microdilution method and showed a minimum inhibitory concentration of 9.375 μg/mL of AgNP against multiple drug-resistant bacterial strains.

Durable antibacterial cotton fabrics based on synergy of bacterial repellence and bactericidal action

The development of textiles with durable antifouling and antibacterial properties is essential to inhibit many healthcare-associated infections and fulfill the increasing demand for public health. However, it is a challengeable task to build high-performance antibacterial surfaces on fabrics without compromise of their intrinsic serviceability in an eco-friendly way. Herein, we reported the fabrication of durable antibacterial cotton fabrics through a one-bath pad−dry−cure technology by using eco-friendly quaternized short perfluoroalkyl polymer/Ag nanocomposite particles (qf-polymer/Ag NCPs) as the finishing agents. Each fiber of the finished cotton fabrics was covered by a low surface energy thin qf-polymer/Ag composite film (CF), allowing the finished fabrics to keep the intrinsic serviceability of fabrics for a comfortable wearing feeling. The qf-polymer/Ag CF-coated cotton fabrics demonstrated a superior long-term broad-spectrum antibacterial ability against normal and multiple drug resistant bacteria. An antibacterial mechanism of the qf-polymer/Ag CF-coated cotton fabrics based on the synergism of qf-polymers, Ag NPs, and textile structure was proposed. This work provides an inspiring insight into the design and fabrication of advanced durable antibacterial surfaces on fabrics.

Molecular characterization and detection of multidrug-resistant gene in bacterial strains in a health care centre located in Iwo, Osun State, Nigeria

The importance of bacterial isolates from hospital wastewater as a reservoir of antibiotic resistance and a potential source of resistance genes to clinical pathogens is underestimated. This study is aimed to isolate and molecularly characterize bacteria from healthcare wastewater in addition to evaluating the distribution of multiple drug-resistant bacteria in the study area. A study was conducted in a Healthcare Centre, in Iwo, Osun State Nigeria. The wastewater was taken from four sections of the wastewater discharge point. Collection of samples was aseptically carried out, transported, and, analyzed immediately following standard procedures. Identification was carried out using biochemical tests and 16S rRNA sequencing. Antibiotic susceptibility was carried out on Mueller Hinton agar following the Kirby-Bauer disk diffusion method. Detection of resistant genes was done using Polymerase Chain Reaction (PCR). A total of 86 bacterial isolates were obtained from wastewater discharge locations. Twenty-five isolates were picked for molecular identification and six different bacterial genera belonging to Bacillus (68%) the most occurring, followed by Cronobacter, Acinetobacter, Enterobacter, with 8%, Klebsiella, and Enterococcus (4%) were identified. Out of 30 randomly picked PCR amplicons, the result revealed 19 isolates (63%) and 10 isolates (33%) were positive for blaTEM and blaSHV respectively, while 9 isolates (30%) harbored both the two genes concurrently. The contamination of wastewater by antibiotics may lead to the rise of antibiotic resistance in the environment, so there is a need to monitor and treat the hospital wastewater that is discharged into the environment.

Treatment of Macrophages with Gram-Negative and -Positive Bacterial Secretomes Induce Distinct Metabolic Signatures

Infectious diseases represent major health and economic challenges globally. Emergence of multiple drug-resistant bacteria in the community and hospital has become a worldwide concern that requires novel approaches for rapid diagnosis and treatment. Metabolomics approach is a powerful tool providing important chemical information about the cellular phenotype of living systems, and the changes in their metabolic pathways in response to various perturbations. Metabolomics has become an important tool to study host-pathogen interactions and to discover potential novel therapeutic targets. In this study, untargeted LC-MS metabolic profiling was applied to differentiate between the impact of the secretome of the Gram-positive S. aureus SH1000 and Gram-negative P. aeruginosa PAO1 bacterial pathogens on THP-1 macrophages. The results showed that S. aureus and P. aeruginosa secretome affected alanine, aspartate and glutamate metabolism; sphingolipid metabolism; glycine and serine metabolism; GL metabolism; and tryptophan metabolism with different trends in THP-1 macrophages. However, the impact of both bacterial secretome on arginine and proline metabolism was similar. These data could contribute to a better understanding of pathogenesis and resistance of these bacteria and could pave the way for developing new therapeutics that selectively targeting Gram-positive or Gram-negative bacteria.

Antimicrobial activity of essential oils of Curcuma longa and Syzygium aromaticum against multiple drug-resistant pathogenic bacteria.

The present study was conducted to investigate the antimicrobial potential of essential oils of Curcuma longa and Syzygium aromaticum against multidrug-resistant pathogenic bacteria. Four identified bacterial isolates including Methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii were selected and their antibiotic sensitivity was checked by disc diffusion assay. C. longa and S. aromaticum were subjected to steam distillation to obtain their essential oils. The crude essential oils were fractioned by employing column chromatography. Crude essential oils and their fractions were evaluated for their antibacterial activity by agar well diffusion assay and minimum inhibitory concentrations were calculated. All the selected bacterial isolates showed resistance to three or more than three antibiotic groups and were declared as multidrugresistant (MDRs). Crude essential oils of C. longa and S. aromaticum exhibited antimicrobial activity against all selected isolates but S. aromaticum activity was better than the C. longa with a maximum 19.3±1.50 mm zone of inhibition against A. baumannii at 1.04 µL/mL MIC. GC/MS analysis revealed the abundance of components including eugenol, eugenyl acetate, b- caryophyllene, and a- Humulene in both crude oil and fractions of S. aromaticum. While the main components of C. longa essential oil were Ar-tumerone, a-tumerone, b- Tumerone, I-Phellandrene, a-zingibirene, b- sesquiphellandrene, and p- Cymene. This study highlights that plant-based essential oils could be a promising alternative to antibiotics for which pathogens have developed resistance. C. longa and S. aromaticum carry compounds that have antimicrobial potential against multiple drug-resistant bacteria including MRSA. E. coli, K. pneumoniae and A. baumannii.

Antimicrobial and Antioxidant Evaluation of Various Parts of Gossypium Herbacium (Linn) Plant

The methanol extracts of the various plant parts were screened for anti-oxidant activity by thin layer chromatography using 2,2-diphenyl-1-dipicrylhydrazyl (DPPH) while in vitro antimicrobial evaluation was determined by agar diffusion method and bioautographic technique using Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Pseudomonas aeruginosa as test organisms; aqueous solvent served as control and ciprofloxacin served as standard drug. The result showed that methanol extracts of the stem, leaf, root and seed of the plant were found to be stronger against the entire test organisms and the stem and leaf of the plant were also found to possess strong antioxidant activity. The antimicrobial property shown by the leaf is an evidence of the ethno medicinal uses of the plant. G. herbaceum leaf, seed, epicarp, stem bark, root bark, may provide novel plant-derived therapeutic agents, effective in treating infectious diseases arising from multiple drug-resistant bacteria and a target in the management of oxidative stress that could easily lead to aging.

Antibacterial Effect of Raw Honey on Clinical Bacterial Isolates of Pseudomonas aeruginosa, Staphylococcus aureus, And Escherichia coli

The development of multiple drug resistant bacteria, causing human infection in clinical practice unfold the use of natural sources like honey, medicinal plants of non-antibiotic drugs, having antibacterial potentiality. Beside the medicinal plants, the antibacterial effects of raw honey on clinical bacterial isolates of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli were studied. The raw honey samples were collected from the beekeepers at Umugwu Ezedike in Udi Local Government Area Enugu State Nigeria. The honey samples were prepared by diluting the honey with distilled water to produce honey of various concentrations, i.e. 20% v/v, 40% v/v, 60% v/v, 80% v/v and 100% v/v. The clinical isolates from Accident and Emergency Department University of Nigeria Teaching Hospital Ituku-Ozalla Enugu were cultured in Nutrient agar, Cetrimide agar, Macconkey agar, and Methylene Blue agar (EMB) for identification and confirmation. The antibacterial effects of raw honey on the clinical bacterial isolates were evaluated in-vitro using agar well diffusion methods with Gentamicin as control. The results showed that raw honey demonstrated antibacterial effect against the test isolates with higher effect in Staphylococcus aureus (with zone of inhibition of 21mm) followed by Pseudomonas aeruginosa (19mm) and Escherichia coli (18mm). The minimum inhibitory concentration (MIC) of the extract from this study ranges from 12.5-50mg/ml while the minimum bactericidal concentration (MBC) of the extracts ranges from 25-50mg/ ml. Hence, raw honey has antibacterial properties and can also serve as a therapeutic substance for the treatment of bed sores, and other infections resulting from burns.

Antibacterial effect of rose bengal against colistin-resistant gram-negative bacteria.

Increasing drug resistance in Gram-negative bacteria presents significant health problems worldwide. Despite notable advances in the development of a new generation of β-lactams, aminoglycosides, and fluoroquinolones, it remains challenging to treat multi-drug resistant Gram-negative bacterial infections. Colistin (polymyxin E) is one of the most efficacious antibiotics for the treatment of multiple drug-resistant Gram-negative bacteria and has been used clinically as a last-resort option. However, the rapid spread of the transferable gene, mcr-1 which confers colistin resistance by encoding a phosphoethanolamine transferase that modifies lipid A of the bacterial membrane, threatens the efficacy of colistin for the treatment of drug-resistant bacterial infections. Colistin-resistant strains of Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae often reduce their susceptibility to other anti-Gram-negative bacterial agents. Thus, drugs effective against colistin-resistant strains or methods to prevent the acquisition of colistin-resistance during treatment are urgently needed. To perform cell-based screenings of the collected small molecules, we have generated colistin-resistant strains of E. coli, A. baumannii, K. pneumoniae, P. aeruginosa, and S. enterica Typhimurium. In-house MIC assay screenings, we have identified that rose bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) is the only molecule that displays unique bactericidal activity against these strains at low concentrations under illumination conditions. This article reports the antibacterial activity of a pharmaceutical-grade rose bengal against colistin-resistant Gram-negative bacteria.

Prevalence and Potential Risk Factors for the Acquisition of Antibiotic-Resistant Staphylococcus spp. Bacteria Among Pastoralist Farmers in Kajiado Central Subcounty, Kenya.

Antimicrobial resistance (AMR) is a growing health problem globally. To address this challenge, there is a need to generate baseline data on the prevalence and AMR profile of the main disease-causing bacteria. Here, we interrogated the prevalence of bacteria in the nasal cavity of healthy pastoralists in Kajiado Central Subcounty, Kenya, and the occurrence of AMR in Staphylococcus isolates among the study subjects. Nasal swabs from 176 pastoralists were cultured, and the bacteria isolates identified using standard phenotypic and biochemical bacteriological methods. Among the obtained 195 isolates, the most prevalent isolates were coagulase-negative Staphylococcus (CoNS) (44.9%), followed by Enterococci spp. (43.2%) while Staphylococcus aureus prevalence was 8%. Antimicrobial sensitivity of the Staphylococcus spp. isolates to 14 antibiotics representing six antibiotic groups was undertaken using the Kirby-Bauer disk diffusion method. Among the CoNS, the highest resistance was reported in amoxicillin (78.7%) and ceftazidime (76%), while the most resistance for S. aureus was reported in ceftazidime (100%), amoxicillin (71.4%), and streptomycin (71.4%). From an administered questionnaire looking at gender, animal contact frequency, history of hospital visitation and antibiotic usage, and habitual intake of raw milk, the study showed that male participants had a higher risk of carrying multiple drug resistant (MDR) bacteria than females (p = 0.02, OR = 1.3). Likewise, habitual intake of raw milk was significantly associated MDR acquisition (p = 0.02, OR = 1.82). This study reveals a high prevalence of AMR Staphylococcus isolates in the study area laying a foundation for further analysis of molecular characterization of the observed resistance as well as the development of interventions that can reduce the occurrence of AMR in the study area.

Isolation, characterization, therapeutic potency, and genomic analysis of a novel bacteriophage vB_KshKPC-M against carbapenemase-producing Klebsiella pneumoniae strains (CRKP) isolated from Ventilator-associated pneumoniae (VAP) infection of COVID-19 patients

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant clinical problem, given the lack of therapeutic options. The CRKP strains have emerged as an essential worldwide healthcare issue during the last 10 years. Global expansion of the CRKP has made it a significant public health hazard. We must consider to novel therapeutic techniques. Bacteriophages are potent restorative cases against infections with multiple drug-resistant bacteria. The Phages offer promising prospects for the treatment of CRKP infections.ObjectiveIn this study, a novel K. pneumoniae phage vB_KshKPC-M was isolated, characterized, and sequenced, which was able to infect and lyse Carbapenem-resistant K. pneumoniae host specifically.MethodsOne hundred clinical isolates of K. pneumoniae were collected from patients with COVID-19 associated with ventilator-associated acute pneumonia hospitalized at Shahid Beheshti Hospital, Kashan, Iran, from 2020 to 2021. Initially, all samples were cultured, and bacterial isolates identified by conventional biochemical tests, and then the ureD gene was used by PCR to confirm the isolates. The Antibiotic susceptibility test in the disc diffusion method and Minimum inhibitory concentrations for Colistin was done and interpreted according to guidelines. Phenotypic and molecular methods determined the Carbapenem resistance of isolates. The blaKPC, blaNDM, and blaOXA-23 genes were amplified for this detection. Biofilm determination of CRKP isolates was performed using a quantitative microtiter plate (MTP) method. The phage was isolated from wastewater during the summer season at a specific position from Beheshti Hospital (Kashan, Iran). The sample was processed and purified against the bacterial host, a CRKP strain isolated from a patient suffering from COVID-19 pneumoniae and resistance to Colistin with high potency for biofilm production. This isolate is called Kp100. The separated phages were diluted and titration by the double overlay agar plaque assay. The separate Phage is concentrated with 10% PEG and stored at −80 °C until use. The phage host range was identified by the spot test method. The purified phage morphology was determined using a transmission electron microscope. The phage stability tests (pH and temperature) were analyzed. The effect of cationic ions on phage adsorption was evaluated. The optimal titer of bacteriophage was determined to reduce the concentration of the CRKP strain. One-step growth assays were performed to identify the purified phage burst’s latent cycle and size. The SDS-PAGE was used for phage proteins analysis. Phage DNA was extracted by chloroform technique, and the whole genome of lytic phage was sequenced using Illumina HiSeq technology (Illumina, San Diego, CA). For quality assurance and preprocessing, such as trimming, Geneious Prime 2021.2.2 and Spades 3.9.0. The whole genome sequence of the lytic phage is linked to the GenBank database accession number. RASTtk—v1.073 was used to predict and annotate the ORFs. Prediction of ORF was performed using PHASTER software. ResFinder is used to assess the presence of antimicrobial resistance and virulence genes in the genome. The tRNAs can-SE v2.0.6 is used to determine the presence of tRNA in the genome. Linear genome comparisons of phages and visualization of coding regions were performed using Easyfig 2.2.3 and Mauve 2.4.0. Phage lifestyles were predicted using the program PHACTS. Phylogenetic analysis and amino acid sequences of phage core proteins, such as the major capsid protein. Phylogenies were reconstructed using the Neighbor-Joining method with 1000 bootstrap repeat. HHpred software was used to predict depolymerase. In this study, GraphPad Prism version 9.1 was used for the statistical analysis. Student’s t-test was used to compare the sets and the control sets, and the significance level was set at P ≤ 0.05.ResultsPhage vB_KshKPC-M is assigned to the Siphoviridae, order Caudovirales. It was identified as a linear double-stranded DNA phage of 54,378 bp with 50.08% G + C content, had a relatively broad host range (97.7%), a short latency of 20 min, and a high burst size of 260 PFU/cell, and was maintained stable at different pH (3–11) and temperature (45–65 °C). The vB_KshKPC-M genome contains 91 open-reading frames. No tRNA, antibiotic resistance, toxin, virulence-related genes, or lysogen-forming gene clusters were detected in the phage genome.Comparative genomic analysis revealed that phage vB_KshKPC-M has sequence similarity to the Klebsiella phages, phage 13 (NC_049844.1), phage Sushi (NC_028774.1), phage vB_KpnD_PeteCarol (OL539448.1) and phage PWKp14 (MZ634345.1).ConclusionThe broad host range and antibacterial activity make it a promising candidate for future phage therapy applications. The isolated phage was able to lyse most of the antibiotic-resistant clinical isolates. Therefore, this phage can be used alone or as a phage mixture in future studies to control and inhibit respiratory infections caused by these bacteria, especially in treating respiratory infections caused by resistant strains in sick patients.

Hyaluronic acid-modified curcumin-copper complex nano delivery system for rapid healing of bacterial prostatitis

Bacterial prostatitis is a bacterial infection of the prostate gland presenting with lower quadrant abdominal pain, urination disorders and poor fertility. In recent years, reports have emerged on the significantly reduced efficacy of fluoroquinolone drugs attributed to multiple drug-resistant bacteria, emphasizing the need for new drugs. In this study, we designed a targeting drug delivery system via curcumin copper complex grafted with hyaluronic acid. Subsequently, the prepared system was characterized using FT-IR, XRD, SEM, XPS and 1H NMR methods. In addition to the substantial improvement in the solubility of the carrier, its antibacterial performance and targeting ability were improved. Interestingly, the grafting of hyaluronic acid endowed the carrier with excellent CD44 receptor targeting function and good water solubility, and the complexation of copper ions greatly enhanced its antibacterial capability, especially the inhibitory effect on E. coli. The anti-prostatitis effect of the drug was evaluated comprehensively by establishing a bacterial prostatitis model infected by E. coli. Assessment of the anti-prostatitis effects in vivo indicated that the Cur-Cu@HA delivery system could effectively promote recovery from bacterial prostatitis by downregulating inflammation. In conclusion, our Cur-Cu@HA delivery system has great potential for treating bacterial prostatitis.

AmiP from hyperthermophilic Thermus parvatiensis prophage is a thermoactive and ultrathermostable peptidoglycan lytic amidase.

Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple-drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8Å displays a compact α/β-fold with multiple secondary structure elements omitted or shortened compared with protein structures of similar proteins. The functional characterization of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity toward thermophilic and mesophilic bacteria strains containing Orn-type or DAP-type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterized with a temperature optimum at 85°C. The extraordinary high melting temperature Tm 102.6°C confirms fold stability up to approximately 100°C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology.

The combination of violet light and infra-red as well as violet light only effectively suppress the survival of multiple-drug resistant bacteria

Since recent global pandemic started, there has been a high demand for establishing an inexpensive but effective method to interfere with the spread of infectious diseases. Here, we have tested several combinations of violet light (V, 405 nm) with infra-red (IR, 850 nm) to identify an optimal light for suppressing pathogens. Our results demonstrate that both violet only (4 V) and 3V-1IR (3:1 ratio in combination of violet and infra-red) effectively suppressed all the bacterial growth tested, including Gram-negative and -positive multi-drug resistant (MDR) strains. Both 4 V and 3V-1IR equally terminated standard strains of Escherichia coli and Staphylococcus aureus, as well as MDR-strains (E. coli, Salmonella enterica and S. aureus from ATCC) effectively. In mechanism, the violet light enhanced the level of reactive oxygen species (ROS) for bactericidal effects, however, we have observed a slightly higher potency from 3V-1IR at a shorter distance, probably due to mild heat-derived dehydration by IR. Therefore, we suggest to expose 3V-1IR for short distance applications (≤1 meter) and both 4 V and 3V-1IR for longer distance (≥1 m). Notably, our results strongly suggest that the exposure of safe violet light or with infra-red can be an effective method to suppress the potential spread of bacteria-derived infectious diseases.

Combined Effect of Isolated Bacteriophage and Neem Extract on Isolated Multiple Drug-Resistant Pathogenic Escherichia coli E1 From Well Water.

Multiple drug-resistant Escherichia coli (E. coli) is a serious cause of concern, and they can be observed in hospital settings, natural environment, and animals. Dissemination of multiple drug-resistant (MDR) E. coli can pose a high risk to public health. Moreover, they are hard to control with commercial antibiotics, since they have acquired resistance against most of them. Therefore, to control multiple drug-resistant bacteria, alternative strategies have been adopted such as phage therapy, herbal remedies, nanoparticles etc. In the current study, the combined application of neem leaf extract and bacteriophage is used to control an isolated multiple drug-resistant E. coli E1. We have applied 0.1 mg/ml concentration of neem extract in combination with an isolated phage vB_EcoM_C2 of 1011 titer and found that the combinatorial treatment approach significantly controls the growth of E. coli E1 as compared to a single non-combinatorial treatment. In this study, every E. coli cell is targeted by 2 antimicrobials (phage and neem extract) at the same time, which is more effective as compared to the sole treatment. Implementation of the neem extract with phage opens a new alternative approach to the chemotherapeutics for the control of multiple drug-resistant bacterial pathogens. This approach may be effective, economical, and eco-friendly to combat MDR.

Prevalence of Multiple Drug-Resistant Bacteria in the Main Campus Wastewater Treatment Plant of Wolaita Sodo University, Southern Ethiopia.

Wastewater treatment plants (WWTPs) are important reservoirs for the development of drug resistance and a potential route for the dissemination of antibiotic resistance genes (ARGs) in the environment. One of the most serious challenges in Ethiopia is the widespread emergence of antibiotic resistance among bacterial pathogens. The bacteria were isolated between September 2018 and May 2019 from the main campus of Wolaita Sodo University in Southern Ethiopia. Using an enrichment process and selective media isolation, 380 wastewater treatment plant samples were collected and screened for the presence of various bacterial isolates. Of a total of 380 wastewater treatment samples, 136 were isolated. Positive prevalence was documented in 136 sample isolates of bacteria from six genera. Escherichia coli 34 (8.94%), Salmonella spp. 15 (3.94%), Shigella spp. 32 (8.42%), Staphylococcus aureus 23 (6.05%), Pseudomonas aeruginosa 21 (5.52%), and Proteus spp. 11 (2.89%). The general prevalence of bacterial isolates was assessed, and 136 (37.58%) samples tested positive for culture. Furthermore, isolates were used to determine sensitivity/resistance patterns using the Kirby-Bauer disc diffusion method and the agar well diffusion technique, respectively. Multiple drug resistance isolates and multiple values of the antibiotic resistance index were evaluated and recorded according to the resistant pattern. Some organisms were sensitive to sparfloxacin and tobramycin, while Staphylococcus aureus was sensitive to methicillin and others showed the highest resistance. At least four of the seven antibiotic classes were found to be resistant to multiple drug resistance isolates, and some classes of antibiotics were found to be highly sensitive to these isolates. Multiple antibiotic resistance index values ranged from 0.37 to 0.75, with Salmonella spp., Shigella spp., and Staphylococcus aureus having the highest score values. The current study has shown that some of the bacterial isolates were resistant to common antibiotics. Therefore, it is recommended that the emergence of multiple drug resistance increased rapidly, pathogenic bacteria inappropriate treated wastewater treatment plant systems were continuously contaminated, and bacterial resistance increased day by day as a result of environmental factors. As a result, due to the serious challenges facing the community's health, multiple drug-resistant prevention and control strategies must be implemented.

Incidence and Susceptibility Patterns of Urine Bacterial Flora in Young Saudi Females

It has been established that the urinary tract is not sterile; however, research related to the study of urinary bacteria is limited. This study aimed to investigate the frequency and patterns of resistance of normal urinary aerobic bacterial flora and clean catch midstream urine specimens collected from 120 young healthy females and cultured. Bacterial identification and antimicrobial susceptibility were performed using the Biomérieux VITEK® 2 automated system. Participants who had undergone antimicrobial treatment within one month were not included. The incidence of positive bacterial cultures was 54.2%, of which 21.5% were polymicrobial. Approximately 107 bacterial isolates that encompass 12 genera and 27 species that were predominated by gram-positive bacteria (72%) were cultivated. Staphylococcaceae (46.1%) and Enterobacteriaceae (17.8%) were the most frequent isolates among gram-positive and gram-negative bacteria, respectively, of which 36 species have been identified as β-lactamase producers. The top four frequently isolated bacteria were Micrococcus spp. (16%), Staphylococcus haemolyticus (13.2%), Staphylococcus aureus (10%), and Klebsiella pneumoniae (10%). Twenty-two bacterial species were subjected to antimicrobial susceptibility testing using broad- and narrow-spectrum antibiotics and antimicrobials, which showed the lowest susceptibility rate against gram-positive bacteria, followed by erythromycin and azithromycin. A lower antimicrobial susceptibility potential among gram-negative bacteria was observed against ampicillin, followed by piperacillin and cefotaxime. Our findings emphasize the importance of highlighting urine bacterial flora in studies, especially those related to susceptibility patterns, by employing more advanced culture methods as multiple drug-resistant bacteria were isolated.

Enhancing activity of β-lactam and fluoroquinolones antibiotics by artemisinin and its derivatives against MDR Escherichia coli.

Artemisinin and its derivatives had played a biocidal role in biomedical remedies, while they were expected to enhance the activity of antibiotics against multiple drug-resistant (MDR) bacteria. The current study evaluated the interaction of artemisinin (ART), dihydroartemisinin (DHA), artesunate (AS), and artemisinic acid (AA) with β-lactam and fluoroquinolones antibiotics against Escherichia coli. Antibiotic strip test (E-test), Kirby Bauer's disc test (KB method), and broth microdilution method were adopted for susceptibility analysis, while the checkerboard method was applied to assess synergisms. ART, DHA, AS, and AA showed significantly enhanced antibacterial effects of β-lactam antibiotics against different strains of E. coli. The study showed ciprofloxacin to be most effective by presenting the least MIC (0.017125 ± 0.0022 μg/ml), while oxacillin was least effective (MIC 256 μg/ml) against E. coli. Synergism between AA and penicillin G (75%), ampicillin (25%), and oxacillin (50%) was observed in all isolates tested. AA and AS significantly decreased the MIC of ampicillin (-0.912 ± 0.908 μg/ml) and ciprofloxacin (-0.901 ± 0.893 g/ml), respectively. Artemisinin and its derivatives increased antibiotic accumulation within E. coli in a dose-dependent manner. The time-kill assay significantly reduced the bacterial number within 24 h of incubation. The study thus concludes greater room for improvement in enhancing the efficacy of antibiotics if used with artemisinin and its derivatives.