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Maha Alzayer,1 Manal M Alkhulaifi,2 Ahmed Alyami,3 Mohammed S Aldosary,3 Abdulaziz Alageel,3 Ghada Garaween,1 Nada Alsalloum,1 Atef Shibl,1 Arif M Al-Hamad,4 Michel Doumith5 1Department of Microbiology and Immunology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia; 2Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia; 3Pathology and Clinical Laboratory, Medicine Administration, King Fahad Medical City, Riyadh, Saudi Arabia; 4Division of Clinical Microbiology, Pathology and Laboratory Medicine, Qatif Central Hospital, Qatif, Saudi Arabia; 5Infectious Diseases Research Department, King Abdullah International Medical Research Center, Riyadh, Saudi ArabiaCorrespondence: Maha Alzayer, College of Medicine, Microbiology Department, Al Faisal University, PO Box 50927, Riyadh, 11533, Saudi Arabia, Email maha.alzayer180@gmail.com; maalzayer@alfaisal.eduAbstract: Published data on the molecular mechanisms underlying antimicrobial resistance in Group B Streptococcus (GBS) isolates from Saudi Arabia are lacking. Here, we aimed to determine the genetic basis of resistance to relevant antibiotics in a collection of GBS clinical isolates (n = 204) recovered from colonized adults or infected patients and expressing serotypes Ia, Ib, II, III, V, and VI. Initial susceptibility testing revealed resistance to tetracycline (76.47%, n = 156/204), erythromycin (36.76%, n = 75/204), clindamycin (25.49%, n = 52/204), levofloxacin (6.37%, n = 13/204), and gentamicin (2.45%, n = 5/204). Primers designed for the detection of known resistance determinants in GBS identified the presence of erm(A), erm(B), mef(A), and/or lsa(C) genes at the origin of resistance to macrolides and/or clindamycin. Of these, erm(B) and erm(A) were associated with the cMLSB (n = 46) and iMLSB (n = 28) phenotypes, respectively, while mef(A) was linked to the M phenotype (n = 1) and lsa(C) was present in isolates with the L phenotype (n = 8). Resistance to tetracycline was mainly mediated by tet(M) alone (n = 112) or in combination with tet(O) (n = 10); the remaining isolates carried tet(O) (n = 29), tet(L) (n = 2), or both (n = 3). Isolates resistant to gentamicin (n = 5) carried aac(6′)-Ie-aph(2′)-Ia, and those exhibiting resistance to levofloxacin (n = 13) had alterations in GyrA and/or ParC. Most isolates with the erm gene (93.24%, n = 69/74) also had the tet gene and were therefore resistant to erythromycin, clindamycin, and tetracycline. Overall, there were no clear associations between serotypes and resistance genotypes except for the presence of erm(B) in serotype Ib isolates. Dissemination of antibiotic resistance genes across different serotypes represents a public health concern that requires further surveillance and appropriate antibiotic use in clinical practice.Keywords: antibiotic resistance, gene resistance, macrolides, levofloxacin, gentamicin, Group B Streptococcus (GBS)

F Mohamad,1,* Raghad R Alzahrani,2,3,* Ahlam Alsaadi,1 Bahauddeen M Alrfaei,4 Alaa Eldeen B Yassin,5 Manal M Alkhulaifi,3 Majed Halwani2 1Infectious Diseases Research Department, King Abdullah International Medical Research Center, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia; 2Nanomedicine Department, King Abdullah International Medical Research Center, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia; 3Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia; 4Stem Cells and Regenerative Medicine, King Abdullah International Medical Research Center, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia; 5College of Pharmacy, King Saud bin Abdulaziz University for Health Sciences, King Abdullah International Medical Research Center, Riyadh, Saudi Arabia*These authors contributed equally to this workCorrespondence: Majed Halwani, P.O. Box 3660, Mail Code 1515 (KAIMRC), Riyadh, 11481, Tel +966 (11) 429-4433, Fax +966 (11) 429-4440, Email majed.halwani@gmail.com; halawanima@ngha.med.sa Manal M Alkhulaifi, P.O. Box 55670, Riyadh, 11544, Tel +966 (11) 805-1685, Email manalk@ksu.edu.saAbstract: The continuous emergence of multidrug-resistant pathogens evoked the development of innovative approaches targeting virulence factors unique to their pathogenic cascade. These approaches aimed to explore anti-virulence or anti-infective therapies. There are evident concerns regarding the bacterial ability to create a superstructure, the biofilm. Biofilm formation is a crucial virulence factor causing difficult-to-treat, localized, and systemic infections. The microenvironments of bacterial biofilm reduce the efficacy of antibiotics and evade the host’s immunity. Producing a biofilm is not limited to a specific group of bacteria; however, Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus biofilms are exemplary models. This review discusses biofilm formation as a virulence factor and the link to antimicrobial resistance. In addition, it explores insights into innovative multi-targeted approaches and their physiological mechanisms to combat biofilms, including natural compounds, phages, antimicrobial photodynamic therapy (aPDT), CRISPR-Cas gene editing, and nano-mediated techniques.Keywords: bacterial biofilms, natural compounds, phages, aPDT, CRISPR, nanotechnology

Background and Purpose: Gentamicin (GEN) is a broad-spectrum antibiotic that cannot be prescribed freely because of its toxicity. Thymoquinone (THQ), a phytochemical, has antibacterial, antioxidant, and toxicity-reducing properties. However, its hydrophobicity and light sensitivity make it challenging to utilize. This incited the idea of co-encapsulating GEN and THQ in liposomes (Lipo-GEN-THQ)., Method: Lipo-GEN-THQ were characterized using the zeta-potential, dynamic light scattering, Fourier transform infrared spectroscopy, and transmission electron microscope (TEM). The liposomes' stability was evaluated under different storage and biological conditions. Lipo-GEN-THQ's efficacy was investigated by the minimum inhibitory/bactericidal concentrations (MICs-MBCs), time-kill curves, and antibiofilm and antiadhesion assays. Bacterial interactions with the empty and GEN-THQ-loaded liposomes were evaluated using TEM., Results: The Lipo-GEN-THQ were spherical, monodispersed, and negatively charged. The Lipo-GEN-THQ were relatively stable and released GEN sustainably over 24 h. The liposomes exhibited significantly higher antibacterial activity than free GEN, as evidenced by the four-fold lower MIC and biofilm eradication in resistant E. coli strain (EC-219). TEM images display how the empty liposomes fused closely to the tested bacteria and how the loaded liposomes caused ultrastructure damage and intracellular component release. An antiadhesion assay showed that the Lipo-GEN-THQ and free GEN (0.125 mg/L) similarly inhibited Escherichia coli (EC-157) adhesion to the A549 cells (68% vs. 64%)., Conclusions: The Lipo-THQ-GEN enhanced GEN by combining it with THQ within the liposomes, reducing the effective dose. The reduction in the GEN dose after adding THQ may indirectly reduce the toxicity and aid in developing an enhanced and safer form of GEN.

In Saudi Arabia, the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in food and livestock represents a major public health hazard. The emergence of livestock-associated MRSA has heightened the risk of human infection with comparable virulence traits. The lack of information about MRSA transmission in our region hinders accurate risk assessment, despite its detection in food animals and retail foods. Adopting a One Health approach is essential for effectively combating MRSA in Saudi Arabia. This method unites actions in the human, animal, and environmental spheres. To combat MRSA contamination, surveillance measures need strengthening; interdisciplinary collaboration among healthcare professionals, veterinarians, and environmental scientists is crucial, and targeted interventions must be implemented in local food chains and animal populations. Through a holistic strategy, public health and sustainable food production in the region are protected. This review aims to improve public health interventions by increasing understanding of MRSA prevalence and related risks in local food chains and animal populations., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Alkuraythi, et al.)

Crohn's disease (CD) entails intricate interactions with gut microbiome diversity, richness, and composition. The relationship between CD and gut microbiome is not clearly understood and has not been previously characterized in Saudi Arabia. We performed statistical analysis about various factors influencing CD activity and microbiota dysbiosis, including diagnosis, treatment, and its impact on their quality of life as well as high-throughput metagenomic V3-V4 16S rRNA encoding gene hypervariable region of a total of eighty patients with CD, both in its active and inactive state with healthy controls. The results were correlated with the demographic and lifestyle information, which the participants provided via a questionnaire. α-diversity measures indicated lower bacterial diversity and richness in the active and inactive CD groups compared to the control group. Greater dysbiosis was observed in the active CD patients compared to the inactive form of the disease, showed by a reduction in microbial diversity. Specific pathogenic bacteria such as Filifactor, Peptoniphilus, and Sellimonas were identified as characteristic of CD groups. In contrast, anti-inflammatory bacteria like Defluviitalea, Papillibacter, and Petroclostridium were associated with the control group. Among the various factors influencing disease activity and microbiota dysbiosis, smoking emerged as the most significant, with reduced α-diversity and richness for the smokers in all groups, and proinflammatory Fusobacteria was more present (p<0.05). Opposite to the control group, microbial diversity and richness were lower in CD participants of older age compared to younger ones, and male CD participants showed less diversity compared to women participants from the same groups. Our results describe the first report on the relationship between microbiota and Crohn's disease progress in Saudi Arabia, which may provide a theoretical basis for the application of therapeutic methods to regulate gut microbes in CD., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Alahdal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Detailed assessment of the population structure of group B Streptococcus (GBS) among adults is still lacking in Saudi Arabia. Here we characterized a representative collection of isolates from colonized and infected adults., Methods: GBS isolates (n=89) were sequenced by Illumina and screened for virulence and antimicrobial resistance determinants. Genetic diversity was assessed by single nucleotide polymorphisms and core-genome MLST analyses., Results: Genome sequences revealed 28 sequence types (STs) and nine distinct serotypes, including uncommon serotypes VII and VIII. Majority of these STs (n=76) belonged to the human-associated clonal complexes (CCs) CC1 (33.71%), CC19 (25.84%), CC17 (11.24%), CC10/CC12 (7.87%), and CC452 (6.74%). Major CCs exhibited intra-lineage serotype diversity, except for the hypervirulent CC17, which exclusively expressed serotype III. Virulence profiling revealed that nearly all isolates (94.38%) carried at least one of the four alpha family protein genes (i.e., alphaC , alp1 , alp2/3 , and rib ), and 92.13% expressed one of the two serine-rich repeat surface proteins Srr1 or Srr2. In addition, most isolates harbored the pilus island (PI)-2a alone (15.73%) or in combination with PI-1 (62.92%), and those carrying PI-2b alone (10.11%) belonged to CC17. Phylogenetic analysis grouped the sequenced isolates according to CCs and further subdivided them along with their serotypes. Overall, isolates across all CC1 phylogenetic clusters expressed Srr1 and carried the PI-1 and PI-2a loci, but differed in genes encoding the alpha-like proteins. CC19 clusters were dominated by the III/ rib / srr1 /PI-1+PI-2a (43.48%, 10/23) and V/ alp1 / srr1 /PI-1+PI-2a (34.78%, 8/23) lineages, whereas most CC17 isolates (90%, 9/10) had the same III/ rib / srr2 /P1-2b genetic background. Interestingly, genes encoding the CC17-specific adhesins HvgA and Srr2 were detected in phylogenetically distant isolates belonging to ST1212, suggesting that other highly virulent strains might be circulating within the species. Resistance to macrolides and/or lincosamides across all major CCs (n=48) was associated with the acquisition of erm(B) (62.5%, 30/48), erm(A) (27.1%, 13/48), lsa(C) (8.3%, 4/48), and mef(A) (2.1%, 1/48) genes, whereas resistance to tetracycline was mainly mediated by presence of tet(M) (64.18%, 43/67) and tet(O) (20.9%, 14/67) alone or in combination (13.43%, 9/67)., Discussion: These findings underscore the necessity for more rigorous characterization of GBS isolates causing infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Alzayer, Alkhulaifi, Alyami, Aldosary, Alageel, Garaween, Shibl, Al-Hamad and Doumith.)

Lactoferrin is a multifunctional glycoprotein present in mammalian milk. It possesses antimicrobial, antioxidant, immunomodulatory, and several biological functions. Owing to the current trend of increasing antibiotic resistance, our study was designed to purify lactoferrin from camel milk colostrum using cation exchange chromatography on the SP-Sepharose high-performance column. The purity and molecular weight of lactoferrin were checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The chromatogram of the purification procedure illustrated a single peak corresponding to lactoferrin, while the SDS-PAGE revealed 78 kDa molecular weight protein. Furthermore, lactoferrin protein and its hydrolysate form were assessed for its antimicrobial potential. The highest inhibitory effect of whole lactoferrin at the concentration (4 mg/ml) was observed against methicillin-resistant S. aureus (MRSA) and S. aureus, while 10 mg/ml concentration was effective against K. pneumonia, and 27 mg/ml was potent against multidrug-resistant (MDR) bacteria, P. aeruginosa. Likewise, MRSA was more sensitive toward iron-free lactoferrin (2 mg/ml) and hydrolyzed lactoferrin (6 mg/ml). The tested lactoferrin forms showed variability in minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) among tested bacteria. The scanning electron microscopy (SEM) analysis images revealed distortions of the bacterial cells exposed to lactoferrin. The antibiofilm effect differed depending on the concentration and the type of the bacteria; biofilm inhibition ranged from 12.5 to 91.3% in the tested pathogenic bacteria. Moreover, the anticancer activity of lactoferrin forms exhibited a dose-dependent cytotoxicity against human lung cancer cell line (A549)., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Staphylococcus aureus is a significant human pathogen that poses a threat to public health due to its association with foodborne contamination and a variety of infections. The factors contributing to the pathogenicity of S. aureus include virulence, drug resistance, and toxin production, making it essential to monitor their prevalence and genetic profiles. This study investigated and compared the genomic characteristics of S. aureus isolates from retail meat and patients in Saudi Arabia., Methods: A total of 136 S. aureus isolates were obtained between October 2021 and June 2022:84 from patients and 53 from meat samples in Riyadh, Saudi Arabia. S. aureus isolates were identified using conventional methods and MALDI-TOF MS, and methicillin-resistant S. aureus (MRSA) was identified using VITEK2 and BD Phoenix systems. MRSA was confirmed phenotypically using chromogenic agar, and genotypically by detecting mec A. Genomic data were analyzed using BactopiaV2 pipeline, local BLAST, and MLST databases., Results: Antibiotic resistance genes were prevalent in both meat and patient S. aureus isolates, with high prevalence of tet 38, bla Z, and fos B. Notably, all S. aureus isolates from patients carried multidrug-resistant (MDR) genes, and a high percentage of S. aureus isolates from meat also harbored MDR genes. Phenotypically, 43% of the S. aureus isolates from meat and 100% of the patients' isolates were MDR. Enterotoxin genes, including sel X, sem , and sei , exhibited high compatibility between meat and patient S. aureus isolates. Virulence genes such as cap, hly/hla, sbi , and isd were found in all S. aureus isolates from both sources., Conclusion: Our study established a genetic connection between S. aureus isolates from meat and patients, showing shared antibiotic resistance and virulence genes. The presence of these genes in meat derived isolates underscores its role as a reservoir. Genomic relatedness also suggests potential transmission of resistance between different settings. These findings emphasize the necessity for a comprehensive approach to monitor and control S. aureus infections in both animals and humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Alkuraythi, Alkhulaifi, Binjomah, Alarwi, Mujallad, Alharbi, Alshomrani, Gojobori and Alajel.)

In this study, we investigated both meat-derived and methicillin-resistant Staphylococcus aureus (MRSA), exploring their genetic relatedness to patient-derived MRSA isolates in Saudi Arabia. We collected 250 meat samples and identified 53 S. aureus isolates, with 79% being methicillin-sensitive Staphylococcus aureus (MSSA) and 21% being MRSA. Moreover, we included 80 clinically confirmed patient-derived MRSA isolates. We identified the most common S. aureus clone in both patients and retail meat. In meat, ST6 and ST97 were the most common clones in 55% of the MRSA isolates, and ST1153 and ST672 were the most common in 21% and 17% of the MSSA isolates. In patients, ST5 and ST6 were the predominant clones in 46% of the S. aureus isolates. CC5/ST5-SCCmecVc-t311 and CC361/ST672-SCCmecV-t3841 were common MRSA clones in both meat and patients. CC97 and CC361 clones were the second most prevalent S. aureus clones in meat and were relatively common in patients. Furthermore, we sequenced and characterized novel S. aureus strains ST8109, ST8110, and ST8111. The genomic similarities between meat- and patient-derived S. aureus isolates suggest that retail meat might be a reservoir for S.aureus and MRSA transmission. Therefore, a structured One Health approach is recommended for S. aureus dissemination, genetic characterization, antibiotic resistance, and impact on human health.

Objectives: Group B Streptococcus (GBS) has emerged as an important cause of severe infections in adults. However, limited data are available regarding the epidemiology of GBS in Saudi Arabia., Methods: Isolates were collected over a period of eight months from colonized (n = 104) and infected adults (n = 95). Serotypes and virulence determinants were detected by polymerase chain reactions (PCRs). Genetic relatedness was assessed using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Antimicrobial susceptibilities were determined by disk diffusion., Results: Serotypes III and V (25% each) were the most prevalent, followed by serotypes II (16.18%), Ia (13.24%), VI (9.31%), and Ib (8.82%), while five isolates remained non-typeable (2.45%). Hypervirulent serotype III/CC17 clone (n = 21) accounted for 41.18% of the serotype III isolates. Most isolates (53.92%) harboured pilus island (PI) 1 and 2a types, while PI-2b was predominantly detected in the hypervirulent clone. Isolates were variably resistant to tetracycline (76.47%), erythromycin (36.76%), clindamycin (25.49%), and levofloxacin (6.37%), but remained susceptible to penicillin. Macrolide resistant isolates exhibited constitutive (55.42%) and inducible macrolide-lincosamide-streptogramin B resistance phenotypes (33.74%), while a few had L (9.64%) or M (1.2%) phenotypes. MLVA patterns of dominant serotypes III and V revealed 40 different types divided into 12 clusters and 28 singletons. Interestingly, macrolide resistance was significantly associated with two major MLVA types., Conclusions: GBS isolates belonged predominantly to serotypes III and V, but there were no clear associations between serotypes and patient groups. The studied isolates exhibited high levels of resistance to erythromycin and clindamycin that need further surveillance., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Being one of the leading industries worldwide, the textile industry has been consuming large quantities of groundwater and discharging huge volumes of dye-contaminated effluents into our aquatic environment. Augmentation of water sources via reuse of treated effluents is therefore highly necessary. In the present study, the decolorization and degradation of synthetic toxic dye from an aqueous solution were investigated through an electro-biological route. Initially, decolorization of synthetic dye solutions (100, 500, and 1000 mg L -1 ) was carried out by electrooxidation process using mixed metal oxide and titanium as anode and cathode, respectively. The electrooxidation solutions were further treated using bacteria (Pseudomonas aeruginosa) that were isolated from petroleum-transporting pipelines. UV-Vis, TOC, chemical oxygen demand, and NMR analyses revealed that the biodegradation process with electrooxidation enhanced the mineralization of the synthetic dye solutions. An optimum NaCl electrolyte concentration of 3 g L -1 was sufficient to produce reactive species viz., free chlorine and hypochlorite, which are responsible for the Reactive Blue 19 (RB-19) decolorization. Among the three RB-19 concentrations, the highest removal percentage was noticed at 100 mg L -1 (100%) with energy consumption and energy costs equal to 5.44 kWh m -3 and 0.65 USD m -3 , respectively., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The antibacterial activity and biofilm reduction capability of liposome formulations encapsulating tobramycin (TL), and Tobramycin- N -acetylcysteine (TNL) were tested against tobramycin-resistant strains of E. coli , K. pneumoniae and A. baumannii in the presence of several resistant genes. All antibacterial activity were assessed against tobramycin-resistant bacterial clinical isolate strains, which were fully characterized by whole-genome sequencing (WGS). All isolates acquired one or more of AMEs genes, efflux pump genes, OMP genes, and biofilm formation genes. TL formulation inhibited the growth of EC&#95;089 and KP&#95;002 isolates from 64 mg/L and 1024 mg/L to 8 mg/L. TNL formulation reduced the MIC of the same isolates to 16 mg/L. TNL formulation was the only effective formulation against all A. baumannii strains compared with TL and conventional tobramycin (in the plektonic environment). Biofilm reduction was significantly observed when TL and TNL formulations were used against E. coli and K. pneumoniae strains. TNL formulation reduced biofilm formation at a low concentration of 16 mg/L compared with TL and conventional tobramycin. In conclusion, TL and TNL formulations particularly need to be tested on animal models, where they may pave the way to considering drug delivery for the treatment of serious infectious diseases.

Currently, the mortality rate in Saudi Arabia's ICUs is increasing due to the spread of Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria. This study was carried out to evaluate the ability of biologically synthesized zinc oxide nanoparticles (ZnO-NPs) using Aspergillus niger to overcome carbapenem-resistant K. pneumoniae (KPC) in vitro and in vivo . ZnO-NPs were synthesized via a biological method and characterized using UV-Vis spectroscopy, Zetasizer and zeta potential analyses, x-ray diffraction spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy (SEM), and energy-dispersive x-ray spectroscopy (EDX). In vitro sensitivity of KPC to ZnO-NPs was identified using the well diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by a macro-dilution method. The morphological alteration of KPC cells after ZnO-NPs treatment was observed by SEM. The in vivo susceptibility of KPC cells to ZnO-NPs ointment was evaluated using wound healing in experimental rats. The chemical characterization findings showed the formation, stability, shape, and size of the synthesized nanoparticles. The MIC and MBC were 0.7 and 1.8 mg/ml, respectively. The in vivo results displayed reduced inflammation and wound re-epithelialization of KPC-infected rats. These findings demonstrated that ZnO-NPs have great potential to be developed as antibacterial agents., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rasha, Alkhulaifi, AlOthman, Khalid, Doaa, Alaa, Awad and Abdalla.)

The release of untreated tannery effluents comprising biotoxic heavy metal (HM) compounds into the ecosystem is one of our society's most serious environmental and health issues. After discharge, HM-containing industrial effluents reach agricultural soils and thus negatively affect the soil microbial diversity. Considering these, we assessed the effect of HMs on identified soil beneficial bacteria. Here, the effects of four heavy metals (HMs), viz., chromium (Cr), cadmium (Cd), nickel (Ni), and lead (Pb), on cellular growth, physiology, cell permeability, and biofilm formation of Enterobacter cloacae MC9 (accession no.: MT672587) were evaluated. HMs in a concentration range of 25-200 μg mL -1 were used throughout the study. Among HMs, Cd in general had the maximum detrimental effect on bacterial physiology. With increasing concentrations of HMs, bacterial activities consistently decreased. For instance, 200 μgCr mL -1 concentration greatly and significantly ( p ≤ 0.05) reduced the synthesis of indole-3-acetic acid (IAA) by 70% over control. Furthermore, 200 μg mL -1 Cd maximally and significantly ( p ≤ 0.05) reduced the synthesis of 2,3-dihydroxybenzoic acid (2,3-DHBA), salicylic acid (SA), 1-aminocyclopropane 1-carboxylate (ACC) deaminase, and extra polymeric substances (EPSs) of E. cloacae MC9 by 80, 81, 77, and 59%, respectively, over control. While assessing the toxic effect of HMs on the P-solubilizing activity of E. cloacae , the toxicity pattern followed the order Cr (mean value = 94.6 μg mL -1 ) > Cd (mean value = 127.2 μg mL -1 ) > Pb (mean value = 132.4 μg mL -1 ) > Ni (mean value = 140.4 μg mL -1 ). Furthermore, the colony-forming unit (CFU) count (Log 10 ) of strain MC9 was completely inhibited at 150, 175, and 200 μg mL -1 concentrations of Cr and Cd. The confocal laser scanning microscopic (CLSM) analysis of HM-treated bacterial cells showed an increased number of red-colored dead cells as the concentration of HMs increased from 25 to 200 μg mL -1 . Likewise, the biofilm formation ability of strain MC9 was maximally ( p ≤ 0.05) inhibited at higher concentrations of Cd. In summary, the present investigation undoubtedly suggests that E. cloacae strain MC9 recovered from the HM-contaminated rhizosphere endowed with multiple activities could play an important role in agricultural practices to augment crop productivity in soils contaminated with HMs. Also, there is an urgent need to control the direct discharge of industrial waste into running water to minimize heavy metal pollution. Furthermore, before the application of HMs in agricultural fields, their appropriate field dosages must be carefully monitored., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Background and Objectives : The multidrug resistant (MDR) bacterial pathogenic infection is one of the chief worldwide public health threat to humanity. The development of novel antibiotics against MDR Gram negative bacteria has reduced over the last half century. Research is in progress regarding the treatment strategies that could be engaged in combination with antibiotics to extend the duration of these life-saving antibacterial agents. The current study was therefore planned to assess the synergistic effects of bovine lactoferrin (bLF) in combination with different antibiotics that are conventionally used. This synergism would provide a newer therapeutic choice against MDR pathogens. LF is present in mucosal secretions, vastly in milk. LF is considered an important constituent in host defense. In previous reports, LF has been co-administered as a combination antibiotic therapy. Materials and Methods : This study included synergistic (LF + appropriate antibiotic) exposure against 147 locally encountered bacterial pathogens, which were completely characterized strains. The anti-biofilm effects and the outcome of bLF on minimum inhibitory concentrations (MICs) of antibacterials on clinical MDR bacterial pathogens were determined by standard techniques. Results : In our study, synergism of bLF with antibacterial agents were reproducible and found to be significant. LF on its own had an important effect of inhibiting the biofilm production of some significant bacterial pathogens. Conclusion : The results of this study provides useful data on the antibacterial potential of the combination of LF with antibiotics against drug resistant pathogens.

The present work aimed to synthesis silver nanoparticles (AgNPs) using biological waste products Citrus limon peels, its characterization, antimicrobial activities and the cytotoxic effect of the synthesized green AgNPs. Characterization of the prepared AgNPs showed the formation of spherical, and few agglomerated AgNPs forms as measured by UV-visible spectrophotometer. The average size of the prepared AgNPs was 59.74 nm as measured by DLS technique. The spectrum of the synthesized AgNPs was observed at 3 KeV using the EDX. On the other hand, FTIR analysis of the green synthesized AgNPs showed the presence of alcohols, phenolics, mono-substituted alkynes, aliphatic primary amines, sodium salt, amino acid, or SiOH alcohol groups. The antimicrobial studies of the formed AgNPs showed positive activity against most of the studied human pathogenic bacteria with varying degrees. Finally, the evaluation of the cytotoxic effect of the green synthesized AgNPs were done using two types of cell lines, human breast cancer cell line (MCF-7) and human colon carcinoma cell line (HCT-116). The results revealed the concentration has a direct correlation with cell viability. The 50% inhibitory concentration (IC 50 ) of MCF-7 cell line was in of 23.5 ± 0.97 µL/100 µL, whereas the HCT-116 cell line was in 37.48 ± 5.93 µL/100 µL., (© 2020 The Author(s).)

In the animal production industry, plant-derived antimicrobial phytobiotics are used as an alternative to antibiotics. Here we investigated the role sanguinarine-based phytobiotic in broiler recovery from Necrotic Enteritis (NE) infection. A total of 100 one-day-old broiler chicks (Ross 308) were randomly allocated to four treatments: negative control CTR (no challenge, no phytobiotic supplementation); positive control NE (NE challenged); phytobiotic SG (sanguinarine phytobiotic, 0.12 g/kg); and SG + NE, (sanguinarine phytobiotic, 0.12 g/kg and NE challenge). Sanguinarine-based phytobiotic supplementation caused significant changes between the groups in performance, livability and histological measurements, however, these changes were not significantly different between SG + NE and NE groups. Significant improvement was detected in NE lesion score of the duodenum and ileum of SG + NE birds compared to NE challenged birds at the end of the production cycle at 40 days old, indicating improved post-NE recovery with the addition of phytobiotic. Sanguinarine-based phytobiotic supplementation in NE challenged birds significantly compensated for a NE associated reduction of Firmicutes and an increase in Bacteroidetes. Functional profile of sanguinarine-based phytobiotic supplemented birds microbiota was distinct from CTR functional profile. NE challenge was associated with a significant increase in cecal propionic acid, while sanguinarine-based phytobiotic supplementation resulted in an increase in cecal acetic acid., (© 2020 The Authors.)

In poultry production, birds are raised under intensive conditions, which can enable rapid spread of infections, with Clostridium perfringens-caused necrotic enteritis (NE) being one of the most devastating for the industry. The current investigation was conducted to evaluate the effectiveness of Bacillus subtilis PB6 probiotic supplementation on bird's post NE recovery, based on chicken performance, cecal microbiota composition, ileum histomorphometric measurements, and short-chain fatty acid production in the cecum of the birds that were challenged with NE mid-production. Birds were split into four groups, including a negative control, positive control challenged with C. perfringens, group supplemented with B. subtilis probiotic, and NE challenged birds supplemented with B. subtilis probiotic. Following NE challenge birds were allowed to reach the end of production time at 40 days, and samples were collected to estimate if probiotic supplementation resulted in better post-NE recovery. Intestinal lesion score across the duodenum, jejunum, and ileum indicated that at the end of production timeline NE challenged birds supplemented with B. subtilis probiotic had lower intestinal lesion scores compared to NE challenged birds without probiotic supplementation implying improved recovery. Probiotic supplementation improved performance of NE challenged birds only in the post-NE recovery stage. NE challenged birds had a significant increase in cecal propionic acid, which was not observed in NE challenged birds supplemented with B.subtilus. Both B. subtilis supplemented groups (challenged and unchanged) were characterized by a significant rise in cecal acetic and butyric acid. Our results demonstrate that B. subtilis supplementation can assist the birds in dealing with NE outbreak and long term recovery., Competing Interests: The authors have declared that no competing interests exist.

The burden of enteric pathogens in poultry is growing after the ban of antibiotic use in animal production. Organic acids gained attention as a possible alternative to antibiotics due to their antimicrobial activities, improved nutrient metabolism and performance. The current study was conducted to evaluate the effectiveness of organic acid blend on broilers cecal microbiota, histomorphometric measurements, and short-chain fatty acid production in Salmonella enterica serovar Typhimurium challenge model. Birds were divided into four treatments, including a negative control, positive control challenged with S. Typhimurium, group supplemented with an organic acid blend, and birds supplemented with organic acid blend and Salmonella challenged. Results illustrate significant differences in feed conversion ratios and production efficiency factor between treatment groups, however, the influence of organic acid supplement was marginal. Organic acid blend significantly increased cecal acetic and butyric acids concentrations when compared to unsupplemented groups and resulted in minor alterations of intestinal bacterial communities., Competing Interests: The authors have declared that no competing interests exist.

This study was designed to determine the incidence of Clostridium perfringens and their toxin genes in children with autism spectrum disorder (ASD), and determine the antimicrobial susceptibility of C. perfringens isolates. A hundred and fourteen fecal samples were obtained from children aged 3-12 years old (57 samples from ASD children and 57 from healthy controls). Children were divided into four groups based on their gastrointestinal (GI) symptoms as follows: ASD group with and without GI symptoms, and control group with and without GI symptoms. Selective anaerobic media and VITEK®2 ANC ID card were used for isolation and identification of C. perfringens from fecal samples. Antimicrobial susceptibility of C. perfringnes isolates were performed using (E-Test) strips against clindamycin, penicillin and metronidazole antibiotics. Conventional PCR was used to detect the alpha toxin gene (Cpa) and the beta-2 toxin gene (Cpb2). Genetic Analyzer 3130Xi was used to confirm the sequencing of Cpb2 gene. Our findings indicated that 38.6% of ASD group samples had a significantly (p = 0.003) higher incidence of C. perfringens than that of the control group (14%). The highest incidence of C. perfringens was found in the ASD group with GI symptoms (53.8%; p = 0.001). C. perfringens isolated from the ASD group (54.5%) were significantly (p = 0.047) more resistant to clindamycin than those isolated from the control group (12.5%). The C. perfringens isolates from the ASD and the control group showed 95.5% and 100% susceptibility to penicillin, respectively. All C. perfringens isolates of ASD and control group were susceptible to metronidazole. The Cpa toxin gene was also detected in all the C. perfringens isolates of ASD and control group, both with and without GI symptoms. Cpb2 toxin gene showed 100% incidence in ASD samples with GI symptoms and in the control groups both with or without GI symptoms, while it was present at significantly lower levels (25%) in the ASD samples without GI symptoms (p = 0.001). Our findings suggests that a high incidence of C. perfringens and its toxin gene (Cpb2) are associated with the GI complications in ASD which may affect the severity of the disease., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: This study evaluated the extent of bacterial contamination in water from dental unit waterlines (DUWLs)., Methodology: Water samples were collected (before flushing, 1 min post-flushing, and 3 min post-flushing) from 24 clinics (Group A: no disinfection, Group B: citric acid disinfectant) of a Government Dental College. Bacterial counts, identification, antibiotic sensitivity tests, determination of endotoxin levels, and scanning electron microscopy (to confirm the presence of biofilm) were performed., Results: The most common opportunistic bacteria were P. aeruginosa (95%), S. aureus (58%), S. auricularis (49%), P. fluorescens (44%), and A. baumannii (20%). Approximately 50% of the bacterial isolates were resistant to two or more antibiotics. Flushing for 3 min did not reduce the contamination of water from Group A clinics which exceeded the recommendation of ≤500 CFU/ml. No bacterial growth was seen in Group B samples. Endotoxin levels were >5.00 endotoxin units (EU)/ml in Group A and ranged from 1.33 to 5.00 EU/ml in Group B clinics. Scanning electron microscopy images showed bacterial biofilms on the surfaces of the tubes., Conclusions: DUWL contamination is a serious issue in dentistry, and the microbiological quality of the water must be monitored regularly. Further studies on endotoxin exposure and prevention are therefore necessary., Competing Interests: The authors declared that there is no conflict of interest., (© 2019 The Authors.)

Free of Salmonella infection, a total of 300 broiler chicks (Ross 308) were randomly allotted to six dietary treatments (10 replicates) as follows: Negative control (only the basal diet); positive control (infected only); T1, infection + antibiotic (avilamycin); T2, infection + Bacillus subtilis and T3, Salmonella infection + Bacillus licheniformis. The results revealed that production performance was severely affected in the infected group. Also the supplementation of Bacillus subtilis (T2) significantly (P < 0.01) improved feed intake, body weight and performance efficiency factor as compared to the positive control. In addition, feed conversion ratio was significantly (P < 0.01) improved in T2 and T3 compared to the positive control. The results of intestinal health showed that significantly (P < 0.01) higher villus height and total surface area were found in T2 compared to positive control. The results of blood cholesterol, glucose, globulin and total protein concentration were significantly (P < 0.05) higher in T3 compared to the infected birds (positive control). It was concluded that Bacillus subtilis produced superior results in comparison with Bacillus licheniformis in term of growth and intestinal features in broiler by mitigating the deleterious effects of Salmonella infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Antibiotic growth promoters (AGPs) have been used for many years as supplements in various livestock diets, including those for poultry. However, the use of AGPs in feed was also associated with an increasing number of antibiotic-resistant bacteria in livestock. In this study, the in vitro antibacterial efficacies of eight commercially available non-AGPs suitable for use in poultry were investigated. Assessments included a combination of antibacterial activity assays and estimations of the minimal inhibitory and bactericidal concentrations along with scanning electron microscopy analysis. The results showed that the probiotic, CloStat ® exerted a bacteriostatic effect against all tested bacteria, namely Salmonella Typhimurium, Escherichia coli, Staphylococcus aureus , and Clostridium perfringens , whereas Gallipro Tect ® and Bacillus Blend ® demonstrated bacteriostatic activity towards most of the pathogens tested. Other commercial non-AGPs, Sangrovit ® , Fysal ® , and Mix oil blend ® showed a stronger or equal antibacterial activity compared to the positive control (AGP Maxus ® G100) againsts all bacteria tested, except C. perfringens . Nor-Spice AB ® and Varium™ did not show any significant effect against the tested bacteria. Several of the tested AGP substitutes exhibited good antibacterial efficiency against pathogenic bacteria and thus may be good candidates for second-stage in vivo investigations into reducing pathogen colonization in broilers., Competing Interests: The authors declare no conflict of interest., (2020, Japan Poultry Science Association.)

Silver nanoparticles (AgNPs) are gaining considerable importance due to their attractive physicochemical properties for many applications. In the present study, (Ag NPs) were synthesized by the reduction of aqueous solutions of silver nitrate (AgNO 3 ) with powder and solvent extracts of Padina pavonia (brown algae). The obtained nanoparticles exhibited high stability, rapid formation of the biogenic process (2 min -3 h), small size (49.58-86.37 nm) (the diameter of formed nanoparticles was measured by TEM and DLS) and variable shapes (spherical, triangular, rectangle, polyhedral and hexagonal). Preliminary characterization of nanoparticles was monitored by using UV-visible spectroscopy (UV-vis), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and finally by Fourier Transform Infrared spectroscopy (FTIR). The ratios of converted Ag NPs were recorded as 88.5; 86.2 and 90.5% in case of P. pavonia powder. extract and chloroform extract, respectively.

Seven fungal species were isolated from soil samples collected from the University of Sultan Qaboos, Muscat, Sultanate of Oman. The fungal isolates were identified as Aspergillus athecius , A. terreus var. africans , A. flavus , A. terreus , A. foetidus , Fusarium chlamydosporum and F. nygamai . Phytochemical and chromatographic investigation showed variety of secondary metabolites in all of the fugal extracts (extra and intra cellular). The antimicrobial activity of internal and external extracts of the isolated fungal species were screened against Candida albicans , C. glabrata , C. parapsilosis , C. tropicalis , Pseudomonas aeruginosa , Lactobacillus acidophilus , Streptococcus gordonii , S. mutans . The antimicrobial activity of external secondary metabolites was generally better than the internal metabolites. The highest antimicrobial activity (32 mm, 30 mm and 29 mm) was obtained from external secondary metabolites of Aspergillus flavus against Candida tropicals , Candida parapsilosis and Candida albicans , respectively.

With the increasing production of ethanol for biofuels, a by-product of corn-based ethanol fermentation, dried distillers grains with solubles (DDGS) is finding its way into the feed of agricultural animals including cattle, pigs, poultry, sheep, goats, aquaculture species and horses. Corn DDGS contains very high levels of non-starch polysaccharides and could be considered a good source of fibre. Despite knowledge of the role of the fibre in modulating intestinal microbiota and consequently influencing health, there is currently little information on the interactions between DDGS and intestinal microbiota. We assessed the changes in the cecal microbiota of broilers feed rations supplemented with DDGS (five concentrations: 0, 6, 12, 18 and 24% w/w) with and without presence of digestive enzymes. DDGS concentration was strongly positively correlated (P = 3.7e -17 , r = 0.74) with feed conversion efficiency (FCR), diminishing broiler performance with higher concentrations. Additionally, DDGS concentrations positively correlated with Richness index (P = 1.5e -3 , r = 0.5), increasing the number of detectable species in the cecum. Among the most affected genera, Faecalibacterium (P = 0.032, r = -0.34) and Streptococcus (P = 7.9e -3 , r = -0.39) were negatively correlated with DDGS, while Turicibacter (P = 2.8e -4 , r = 0.52) was positively correlated with the DDGS concentration. Enzymes showed minimal effect on cecal microbiota.