Various investigations of ameliorative role of Ashwagandha seeds (Withania somnifera) against amoxicillin toxicity.

Several studies showed the adverse effects of amoxicillin on various body organs. So, this research has been designed to evaluate the modulatory role of Ashwagandha seed extract (ASE) against amoxicillin (AM) toxicity. Rats treated with AM (90 mg/kg), protected by ASE doses (100, 200 and 300 mg/kg), and treated by ASE at the same three doses. At the end of the experimental period, DNA comet assay, cytogenetic examinations, sperm-shape analysis, evaluation of the malondialdehyde (MDA) percentages, histopathological examinations, and biophysical tests (modulus, relaxation time, permittivity, entropy, and internal energy change of brain) were documented. The results confirmed that AM treatment induced significant elevation of DNA damage, cytogenetic aberrations, and MDA content in brain, liver, and testis tissues and sperm-shape anomalies. ASE treatment significantly minimized the genetic changes, sperm-shape anomalies, and MDA generation. These enhancements were more pronounced by protective ASE and increased by increasing the dose level. In histopathological examinations, AM treatment caused neurotoxicity in brain tissue. ASE treatment, partially, minimized these damages and the positive effects of therapeutic ASE were more noticeable. Biophysical parameters showed that therapeutic ASE was better for relaxation time, permittivity, and free energy change. Protective and therapeutic ASE were able to recover entropy and internal energy changes in variant degrees.

Carbapenem and colistin-resistant hypervirulent Klebsiella pneumoniae: An emerging threat transcending the egyptian food chain.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a great public health problem and is associated with many disease outbreaks and high mortality rates. Alarmingly, K. pneumoniae has been isolated from food in several recent studies. This study aimed to investigate the prevalence and characteristics of CRKP in food samples from Egypt. METHODS: A total of 311 food samples (including 116 minced meat, 92 chicken meat, 75 diced meat, and 28 mutton) were collected from local markets in Egypt and were screened for CRKP with the determination of their antimicrobial resistance profiles. The whole genome sequence was done for 23 CRKP isolates to clarify the relationship between CRKP from food and human cases in Egypt using the SNP core genome. The conjugation probability of the blaNDM-5 harboring plasmid was identified using oriTfinder RESULTS: CRKP was isolated from 11% (35/311) of the samples, with 45.71% (16/35) of them showing resistance to colistin, one of the last-resort options for treating CRKP-mediated infections. In addition to the carbapenem and colistin resistance, the CRKP isolates frequently exhibited resistance to multiple antimicrobials including ß-lactams, fluoroquinolones, aminoglycosides, tetracyclines, and chloramphenicol. In addition, most of the CRKP were potentially hypervirulent K. pneumoniae (HvKP) identified as phylogroup Kp1 and of high-risk groups as detected in STs reported in many human outbreaks globally, such as ST383 and ST147. The core-genome phylogeny showed similarities between the isolates from this study and those previously isolated from clinical human samples in Egypt. In addition, analysis of the plasmid on which blaNDM is encoded revealed that several antimicrobial resistance genes such as blaOXA-9, blaCTX-M-15, aac(6')-Ib, qnrS1, and several virulence genes are encoded on the same plasmid. CONCLUSIONS: This study is significant for food safety and public health and is important to further identify the change in the epidemiology of CRKP infections, especially the consumption of contaminated food products.

Emergence of carbapenem resistant gram-negative pathogens with high rate of colistin resistance in Egypt: A cross sectional study to assess resistance trends during the COVID-19 pandemic.

The current study investigated the temporal phenotypic and genotypic antimicrobial resistance (AMR) trends among multi-drug resistant and carbapenem-resistant Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa recovered from Egyptian clinical settings between 2020 and 2021. Bacterial identification and antimicrobial sensitivity of 111 clinical isolates against a panel of antibiotics were performed. Molecular screening for antibiotic resistance determinants along with integrons and associated gene cassettes was implemented. An alarming rate (98.2%) of these isolates were found to be phenotypically resistant to carbapenem. Although 23.9 % K. pneumoniae isolates were phenotypically resistant to colistin, no mobile colistin resistance (mcr) genes were detected. Among carbapenem-resistant isolates, blaNDM and blaOXA-48-like were the most prevalent genetic determinants and were significantly overrepresented among K. pneumoniae. Furthermore, 84.78% of K. pneumoniae isolates co-produced these two carbapenemase genes. The plasmid-mediated quinolone resistance genes (qnrS and qnrB) were detected among the bacterial species and were significantly more prevalent among K. pneumoniae. Moreover, Class 1 integron was detected in 82% of the bacterial isolates. This study alarmingly reveals elevated resistance to last-resort antibiotics such as carbapenems as well as colistin which impose a considerable burden in the health care settings in Egypt. Our future work will implement high throughput sequencing-based antimicrobial resistance surveillance analysis for characterization of novel AMR determinants. This information could be applied as a step forward to establish a robust antibiotic stewardship program in Egyptian clinical settings, thereby addressing the rising challenges of AMR.

Deciphering the dynamics of methicillin-resistant Staphylococcus aureus biofilm formation: from molecular signaling to nanotherapeutic advances.

Methicillin-resistant Staphylococcus aureus (MRSA) represents a global threat, necessitating the development of effective solutions to combat this emerging superbug. In response to selective pressures within healthcare, community, and livestock settings, MRSA has evolved increased biofilm formation as a multifaceted virulence and defensive mechanism, enabling the bacterium to thrive in harsh conditions. This review discusses the molecular mechanisms contributing to biofilm formation across its developmental stages, hence representing a step forward in developing promising strategies for impeding or eradicating biofilms. During staphylococcal biofilm development, cell wall-anchored proteins attach bacterial cells to biotic or abiotic surfaces; extracellular polymeric substances build scaffolds for biofilm formation; the cidABC operon controls cell lysis within the biofilm, and proteases facilitate dispersal. Beside the three main sequential stages of biofilm formation (attachment, maturation, and dispersal), this review unveils two unique developmental stages in the biofilm formation process for MRSA; multiplication and exodus. We also highlighted the quorum sensing as a cell-to-cell communication process, allowing distant bacterial cells to adapt to the conditions surrounding the bacterial biofilm. In S. aureus, the quorum sensing process is mediated by autoinducing peptides (AIPs) as signaling molecules, with the accessory gene regulator system playing a pivotal role in orchestrating the production of AIPs and various virulence factors. Several quorum inhibitors showed promising anti-virulence and antibiofilm effects that vary in type and function according to the targeted molecule. Disrupting the biofilm architecture and eradicating sessile bacterial cells are crucial steps to prevent colonization on other surfaces or organs. In this context, nanoparticles emerge as efficient carriers for delivering antimicrobial and antibiofilm agents throughout the biofilm architecture. Although metal-based nanoparticles have been previously used in combatting biofilms, its non-degradability and toxicity within the human body presents a real challenge. Therefore, organic nanoparticles in conjunction with quorum inhibitors have been proposed as a promising strategy against biofilms. As nanotherapeutics continue to gain recognition as an antibiofilm strategy, the development of more antibiofilm nanotherapeutics could offer a promising solution to combat biofilm-mediated resistance.

Long-term exposure to food-grade disinfectants causes cross-resistance to antibiotics in Salmonella enterica serovar Typhimurium strains with different antibiograms and sequence types.

BACKGROUND: Disinfectants are important in the food industry to prevent the transmission of pathogens. Excessive use of disinfectants may increase the probability of bacteria experiencing long-term exposure and consequently resistance and cross-resistance to antibiotics. This study aims to investigate the cross-resistance of multidrug-resistant, drug-resistant, and drug-susceptible isolates of Salmonella enterica serovar Typhimurium (S. Typhimurium) with different sequence types (STs) to a group of antibiotics after exposure to different food-grade disinfectants. METHODS: A panel of 27 S. Typhimurium strains with different antibiograms and STs were exposed to increasing concentrations of five food-grade disinfectants, including hydrogen peroxide (H2O2), benzalkonium chloride (BAC), chlorine dioxide (ClO2), sodium hypochlorite (NaClO), and ethanol. Recovered evolved strains were analyzed using genomic tools and phenotypic tests. Genetic mutations were screened using breseq pipeline and changes in resistance to antibiotics and to the same disinfectant were determined. The relative fitness of evolved strains was also determined. RESULTS: Following exposure to disinfectants, 22 out of 135 evolved strains increased their resistance to antibiotics from a group of 14 clinically important antibiotics. The results also showed that 9 out of 135 evolved strains had decreased resistance to some antibiotics. Genetic mutations were found in evolved strains. A total of 77.78% of ST34, 58.33% of ST19, and 66.67% of the other STs strains exhibited changes in antibiotic resistance. BAC was the disinfectant that induced the highest number of strains to cross-resistance to antibiotics. Besides, H2O2 induced the highest number of strains with decreased resistance to antibiotics. CONCLUSIONS: These findings provide a basis for understanding the effect of disinfectants on the antibiotic resistance of S. Typhimurium. This work highlights the link between long-term exposure to disinfectants and the evolution of resistance to antibiotics and provides evidence to promote the regulated use of disinfectants.

Recombination-mediated dissemination of Methicillin-resistant S. aureus clonal complex 1 in the Egyptian health care settings.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a rapidly evolving pathogen that is frequently associated with outbreaks and sustained epidemics. This study investigated the population structure, resistome, virulome, and the correlation between antimicrobial resistance determinants with phenotypic resistance profiles of 36 representative hospital-acquired MRSA isolates recovered from hospital settings in Egypt. RESULTS: The community-acquired MRSA lineage, clonal complex 1 (CC1) was the most frequently detected clone, followed by three other globally disseminated clones, CC121, CC8, and CC22. Most isolates carried SCCmec type V and more than half of isolates demonstrated multi-drug resistant phenotypes. Resistance to linezolid, a last resort antibiotic for treating multidrug resistant MRSA, was observed in 11.11% of the isolates belonging to different genetic backgrounds. Virulome analysis indicated that most isolates harboured a large pool of virulence factors and toxins. Genes encoding aureolysin, gamma hemolysins, and serine proteases were the most frequently detected virulence encoding genes. CC1 was observed to have a high pool of AMR resistance determinants including cfr, qacA, and qacB genes, which are involved in linezolid and quaternary ammonium compounds resistance, as well as high content of virulence-related genes, including both of the PVL toxin genes. Molecular clock analysis revealed that CC1 had the greatest frequency of recombination (compared to mutation) among the four major clones, supporting the role of horizontal gene transfer in modulating AMR and hypervirulence in this clone. CONCLUSIONS: This pilot study provided evidence on the dissemination success of CA-MRSA clone CC1 among Egyptian hospitals. Co-detection of multiple AMR and virulence genes in this lineage pose a broad public health risk, with implications for successful treatment. The results of this study, together with other surveillance studies in Egypt, should be used to develop strategies for controlling MRSA infections in Egyptian health-care settings.

Benzalkonium chloride forces selective evolution of resistance towards antibiotics in Salmonella enterica serovar Typhimurium.

BACKGROUND: Although food-grade disinfectants are extensively used worldwide, it has been reported that the long-term exposure of bacteria to these compounds may represent a selective force inducing evolution including the emergence of antibiotic resistance. However, the mechanism underlying this correlation has not been elucidated. This study aims to investigate the genomic evolution caused by long-term disinfectant exposure in terms of antibiotic resistance in Salmonella enterica Typhimurium. METHODS: S. Typhimurium isolates were exposed to increasing concentrations of benzalkonium chloride (BAC) and variations of their antibiotic susceptibilities were monitored. Strains that survived BAC exposure were analyzed at whole genome perspective using comparative genomics, and Sanger sequencing-confirmed mutations in ramR gene were identified. Next, the efflux activity in ramR-mutated strains shown as bisbenzimide accumulation and expression of genes involved in AcrAB-TolC efflux pump using quantitative reverse transcriptase PCR were determined. RESULTS: Mutation rates of evolved strains varied from 5.82 × 10-9 to 5.56 × 10-8, with fold increase from 18.55 to 1.20 when compared with strains evolved without BAC. Mutations in ramR gene were found in evolved strains. Upregulated expression and increased activity of AcrAB-TolC was observed in evolved strains, which may contribute to their increased resistance to clinically relevant antibiotics. In addition, several indels and point mutations in ramR were identified, including L158P, A37V, G42E, F45L, and R46H which have not yet been linked to antimicrobial resistance. Resistance and mutations were stable after seven consecutive cultivations without BAC exposure. These results suggest that strains with sequence type (ST) ST34 were the most prone to mutations in ramR among the three STs tested (ST34, ST19, ST36). CONCLUSIONS: This work demonstrated that disinfectants, specifically BAC forces S. Typhimurium to enter a specific evolutionary trajectory towards antibiotic resistance illustrating the side effects of long-term exposure to BAC and probably also to other disinfectants. Most significantly, this study provides new insights in understanding the emergence of antibiotic resistance in modern society.

Dyslexia with and without Irlen syndrome: A study of influence on abilities and brain-derived neurotrophic factor level.

The presence of comorbid Irlen syndrome (IS) in children with developmental dyslexia (DD) may have an impact on their reading and cognitive abilities. Furthermore, the brain-derived neurotrophic factor (BDNF) was reported to be expressed in brain areas involved in cognitive and visual processing. The aim of this study was to evaluate some cognitive abilities of a group of dyslexic children with IS and to measure and compare the plasma BDNF level to dyslexic children without IS and neurotypical (NT) children. The participants were 60 children with DD (30 in the DD + IS group; 30 in the DD group) and 30 NT children. The Irlen reading perceptual scale, the Stanford Binet intelligence scale, 4th ed, the dyslexia assessment test, and the Illinois test of psycholinguistic abilities were used. The BDNF level was measured using the enzyme-linked immunosorbent assay. One-minute writing and visual closure deficits were more prevalent, while phonemic segmentation deficits were less prevalent in the DD + IS group compared to the DD group. The BDNF level in the DD groups was lower than that in NT children (p < 0.001). Some reading and non-reading tasks were influenced by the presence of a coexisting IS. The reduced BDNF level could play a role in the deficits noticed in the abilities of children with DD.

Biosynthetic gene cluster signature profiles of pathogenic Gram-negative bacteria isolated from Egyptian clinical settings.

Biosynthetic gene clusters (BGCs) are a subset of consecutive genes present within a variety of organisms to produce specialized metabolites (SMs). These SMs are becoming a cornerstone to produce multiple medications including antibacterial and anticancer agents. Natural products (NPs) also play a pivotal role in enhancing the virulence of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), which represent a global health threat. We aimed to sequence and computationally analyze the BGCs present in 66 strains pertaining to three different ESKAPE pathogenic species: 21 A. baumannii, 28 K. pneumoniae, and 17 P. aeruginosa strains recovered from clinical settings in Egypt. DNA was extracted using QIAamp DNA Mini kit and Illumina NextSeq 550 was used for whole-genome sequencing. The sequences were quality-filtered by fastp and assembled by Unicycler. BGCs were detected by antiSMASH, BAGEL, GECCO, and PRISM, and aligned using Clinker. The highest abundance of BGCs was detected in P. aeruginosa (590), then K. pneumoniae (146) and the least in A. baumannii strains (133). P. aeruginosa isolates shared mostly the non-ribosomal peptide synthase (NRPS) type, K. pneumoniae isolates shared the ribosomally synthesized and post-translationally modified peptide-like (RiPP-like) type, while A. baumannii isolates shared the siderophore type. Most of the isolates harbored non-ribosomal peptide (NRP) BGCs with few K. pneumoniae isolates encoding polyketide BGCs. Sactipeptides and bottromycin BGCs were the most frequently detected RiPP clusters. We hypothesize that each species' BGC signature confers its virulence. Future experiments will link the detected clusters with their species and determine whether the encoded SMs are produced and cause their virulence. IMPORTANCE Our study analyzes the biosynthetic gene clusters (BGCs) present in 66 assemblies from clinical ESKAPE pathogen isolates pertaining to Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa strains. We report their sequencing and assembly followed by the analysis of their BGCs using several bioinformatics tools. We then focused on the most abundant BGC type in each species and we discussed their potential roles in the virulence of each species. This study is pivotal to further build on its experimental work that deciphers the role in virulence, possible antibacterial effects, and characterization of the encoded specialized metabolites (SMs). The study highlights the importance of studying the "harmful" BGCs and understanding the pathogenicity and virulence of those species, as well as possible benefits if the SMs were used as antibacterial agents. This could be the first study of its kind from Egypt and would shed light on BGCs from ESKAPE pathogens from Egypt.

BDNF Val66Met Polymorphism: Suggested Genetic Involvement in Some Children with Learning Disorder.

Brain-derived neurotrophic factor (BDNF) plays an essential role in neuronal survival, especially in areas responsible for memory and learning. The BDNF Val66Met polymorphism has been described as a cognitive modifier in people with neuropsychiatric disorders. BDNF levels have been found to be low in children with learning disorder (LD). However, Val66Met polymorphism has not been studied before in such children. The aim was to investigate the presence of BDNF val66Met polymorphism in a group of children with specific LD and to verify its impact on their cognitive abilities. The participants in this cross-sectional study (N = 111) were divided into two groups: one for children with LD and the other for neurotypical (NT) ones. Children with LD (N = 72) were diagnosed according to the DSM-5 criteria. Their abilities were evaluated using Stanford-Binet Intelligence Scale, dyslexia assessment test, Illinois Test of Psycholinguistic Abilities, and phonological awareness test. Genotyping of BDNF Val66Met polymorphism was performed for all participants. The frequency of the Met allele was 26% among children with LD (6 children had homozygous, 26 had heterozygous genotype). The percentage of participants with deficits in reading, writing, and phonemic segmentation was higher in Met allele carriers when compared to non-Met allele carriers in LD group. The frequency of Met allele among NT children was 3.85% (0 homozygous, 3 children had heterozygous genotype) (p = 0.00001). The high frequency of Val66Met polymorphism among children with LD introduces the BDNF gene as a genetic modifier of learning performance in some children who manifest specific learning disorder (developmental dyslexia).

Genomic insights into zoonotic transmission and antimicrobial resistance in Campylobacter jejuni from farm to fork: a one health perspective.

BACKGROUND: Campylobacteriosis represents a global public health threat with various socio-economic impacts. Among different Campylobacter species, Campylobacter jejuni (C. jejuni) is considered to be the foremost Campylobacter species responsible for most of gastrointestinal-related infections. Although these species are reported to primarily inhabit birds, its high genetic and phenotypic diversity allowed their adaptation to other animal reservoirs and to the environment that may impact on human infection. MAIN BODY: A stringent and consistent surveillance program based on high resolution subtyping is crucial. Recently, different epidemiological investigations have implemented high-throughput sequencing technologies and analytical pipelines for higher resolution subtyping, accurate source attribution, and detection of antimicrobial resistance determinants among these species. In this review, we aim to present a comprehensive overview on the epidemiology, clinical presentation, antibiotic resistance, and transmission dynamics of Campylobacter, with specific focus on C. jejuni. This review also summarizes recent attempts of applying whole-genome sequencing (WGS) coupled with bioinformatic algorithms to identify and provide deeper insights into evolutionary and epidemiological dynamics of C. jejuni precisely along the farm-to-fork continuum. CONCLUSION: WGS is a valuable addition to traditional surveillance methods for Campylobacter. It enables accurate typing of this pathogen and allows tracking of its transmission sources. It is also advantageous for in silico characterization of antibiotic resistance and virulence determinants, and hence implementation of control measures for containment of infection.

Tracking the distribution, genetic diversity and lineage of Brucella melitensis recovered from humans and animals in Egypt based on core-genome SNP analysis and in silico MLVA-16.

Brucellosis is one of the most common neglected zoonotic diseases globally, with a public health significance and a high economic loss in the livestock industry caused by the bacteria of the genus Brucella. In this study, 136 Egyptian Brucella melitensis strains isolated from animals and humans between 2001 and 2020 were analysed by examining the whole-core-genome single-nucleotide polymorphism (cgSNP) in comparison to the in silico multilocus variable number of tandem repeat analysis (MLVA-16). Almost all Egyptian isolates were belonging to the West Mediterranean clade, except two isolates from buffalo and camel were belonging to the American and East Mediterranean clades, respectively. A significant correlation between the human case of brucellosis and the possible source of infection from animals was found. It seems that several outbreak strains already existing for many years have been spread over long distances and between many governorates. The cgSNP analysis, in combination with epidemiological metadata, allows a better differentiation than the MLVA-16 genotyping method and, hence, the source definition and tracking of outbreak strains. The MLVA based on the currently used 16 markers is not suitable for this task. Our results revealed 99 different cgSNP genotypes with many different outbreak strains, both older and widely distributed ones and rather newly introduced ones as well. This indicates several different incidents and sources of infections, probably by imported animals from other countries to Egypt. Comparing our panel of isolates to public databases by cgSNP analysis, the results revealed near relatives from Italy. Moreover, near relatives from the United States, France, Austria and India were found by in silico MLVA.

Cyclodextrin metal-organic framework by ultrasound-assisted rapid synthesis for caffeic acid loading and antibacterial application.

Cyclodextrin metal-organic framework by ultrasound-assisted rapid synthesis for caffeic acid (CA) loading and antibacterial application (U-CD-MOF) was successfully studied and this method shortened the preparation time to a few minutes. It was found that the ultrasonic power, reaction time and temperature would affect the morphology and size of the obtained crystal. Under the optimal conditions, U-CD-MOF had a cubic structure with uniform size of 8.60 ± 1.95 µm. U-CD-MOF was used to load the antibacterial natural product CA to form the composite (CA@U-CD-MOF) and the loading rate of CA@U-CD-MOF to CA could reach 19.63 ± 2.53%, which was more than twice that of γ-CD. Various techniques were applied to characterize the synthesized crystal, including Powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and N2 adsorption. In addition, antibacterial tests were performed on the obtained crystal. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CA@U-CD-MOF for Escherichia coli O157: H7 (E. coli O157: H7) were both 25 mg·mL-1, and the MIC for Staphylococcus aureus (S. aureus). was 25 mg·mL-1. The sustained release behavior of CA@U-CD-MOF to CA in ethanol fitted well to Higuchi model and the loading of CA was supported by molecular docking results. In general, U-CD-MOF was successfully achieved by ultrasound-assisted rapid synthesis and the obtained crystal was further evaluated for potential antibacterial application.

Isolation, Characterization, and Genomic Analysis of Three Novel E. coli Bacteriophages That Effectively Infect E. coli O18.

Escherichia coli (E. coli) is one of the most common pathogenic bacteria worldwide. Avian pathogenic E. coli (APEC) causes severe systemic disease in poultry (Colibacillosis), and accordingly, has an extreme risk to the poultry industry and public health worldwide. Due to the increased rate of multi-drug resistance among these bacteria, it is necessary to find an alternative therapy to antibiotics to treat such infections. Bacteriophages are considered one of the best solutions. This study aimed to isolate, characterize, and evaluate the potential use of isolated bacteriophages to control E. coli infections in poultry. Three novel phages against E. coli O18 were isolated from sewage water and characterized in vitro. The genome size of the three phages was estimated to be 44,776 bp, and the electron microscopic analysis showed that they belonged to the Siphoviridae family, in the order Caudovirales. Phages showed good tolerance to a broad range of pH and temperature. The complete genomes of three phages were sequenced and deposited into the GenBank database. The closely related published genomes of Escherichia phages were identified using BLASTn alignment and phylogenetic trees. The prediction of the open reading frames (ORFs) identified protein-coding genes that are responsible for functions that have been assigned such as cell lysis proteins, DNA packaging proteins, structural proteins, and DNA replication/transcription/repair proteins.

A pilot study revealing host-associated genetic signatures for source attribution of sporadic Campylobacter jejuni infection in Egypt.

Campylobacter jejuni (C. jejuni), is considered among the most common bacterial causes of human bacterial gastroenteritis worldwide. The epidemiology and the transmission dynamics of campylobacteriosis in Egypt remain poorly defined due to the limited use of high-resolution typing methods. In this pilot study, we evaluated the discriminatory power of multiple typing 'gene-by-gene based' techniques to characterize C. jejuni obtained from different sources and estimate the relative contribution of different potential sources of C. jejuni infection in Egypt. Whole genome sequencing (WGS) was performed on 90 C. jejuni isolates recovered from clinical samples, retail chicken, and dairy products in Egypt from 2017 to 2018. Comparative genomic analysis was performed using conventional seven-locus multilocus sequence typing (MLST), ribosomal MLST (rMLST), core genome MLST (cgMLST), allelic variation in 15 host-segregating (HS) markers, and comparative genomic fingerprinting (CGF40). The probabilistic source attribution was performed via STRUCTURE software using MLST, CGF40, cgMLST and allelic variation in HS markers. Comparison of the discriminatory power of the aforementioned genotyping methods revealed cgMLST to be the most discriminative method, followed by HS markers. The source attribution analysis showed the role of retail chicken as a source of infection among clinical cases in Egypt when HS and cgMLST were used (64.2% and 52.3% of clinical isolates were assigned to this source, respectively). Interestingly, the cattle reservoir was also identified as a contributor to C. jejuni infection in Egypt; 35.8% and 47.7% of clinical isolates were assigned to this source by HS and cgMLST, respectively. Here, we provided evidence of the importance of using WGS typing methods to facilitate source tracking of C. jejuni. Our findings suggest the importance of non-poultry sources, together with the previously reported role of retail chicken in human campylobacteriosis in Egypt that can provide insights to inform national control measures.

Tracking the Distribution of Brucella abortus in Egypt Based on Core Genome SNP Analysis and In Silico MLVA-16.

Brucellosis, caused by the bacteria of the genus Brucella, is one of the most neglected common zoonotic diseases globally with a public health significance and a high economic loss among the livestock industry worldwide. Since little is known about the distribution of B. abortus in Egypt, a total of 46 B. abortus isolates recovered between 2012-2020, plus one animal isolate from 2006, were analyzed by examining the whole core genome single nucleotide polymorphism (cgSNP) in comparison to the in silico multilocus variable number of tandem repeat analysis (MLVA). Both cgSNP analysis and MLVA revealed three clusters and one isolate only was distantly related to the others. One cluster identified a rather widely distributed outbreak strain which is repeatedly occurring for at least 16 years with marginal deviations in cgSNP analysis. The other cluster of isolates represents a rather newly introduced outbreak strain. A separate cluster comprised RB51 vaccine related strains, isolated from aborted material. The comparison with MLVA data sets from public databases reveals one near relative from Argentina to the oldest outbreak strain and a related strain from Spain to a newly introduced outbreak strain in Egypt. The distantly related isolate matches with a strain from Portugal in the MLVA profile. Based on cgSNP analysis the oldest outbreak strain clusters with strains from the UK. Compared to the in silico analysis of MLVA, cgSNP analysis using WGS data provides a much higher resolution of genotypes and, when correlated to the associated epidemiological metadata, cgSNP analysis allows the differentiation of outbreaks by defining different outbreak strains. In this respect, MLVA data are error-prone and can lead to incorrect interpretations of outbreak events.

High-throughput sequencing reveals genetic determinants associated with antibiotic resistance in Campylobacter spp. from farm-to-fork.

Campylobacter species are one of the most common causative agents of gastroenteritis worldwide. Resistance against quinolone and macrolide antimicrobials, the most commonly used therapeutic options, poses a serious risk for campylobacteriosis treatment. Owing to whole genome sequencing advancements for rapid detection of antimicrobial resistance mechanisms, phenotypic and genotypic resistance trends along the "farm-to-fork" continuum can be determined. Here, we examined the resistance trends in 111 Campylobacter isolates (90 C. jejuni and 21 C. coli) recovered from clinical samples, commercial broiler carcasses and dairy products in Cairo, Egypt. Multidrug resistance (MDR) was observed in 10% of the isolates, mostly from C. coli. The prevalence of MDR was the highest in isolates collected from broiler carcasses (13.3%), followed by clinical isolates (10.5%), and finally isolates from dairy products (4%). The highest proportion of antimicrobial resistance in both species was against quinolones (ciprofloxacin and/or nalidixic acid) (68.4%), followed by tetracycline (51.3%), then erythromycin (12.6%) and aminoglycosides (streptomycin and/or gentamicin) (5.4%). Similar resistance rates were observed for quinolones, tetracycline, and erythromycin among isolates recovered from broiler carcasses and clinical samples highlighting the contribution of food of animal sources to human illness. Significant associations between phenotypic resistance and putative gene mutations was observed, with a high prevalence of the gyrA T86I substitution among quinolone resistant isolates, tet(O), tet(W), and tet(32) among tetracycline resistant isolates, and 23S rRNA A2075G and A2074T mutations among erythromycin resistant isolates. Emergence of resistance was attributed to the dissemination of resistance genes among various lineages, with the dominance of distinctive clones. For example, sub-lineages of CC828 in C. coli and CC21 in C. jejuni and the genetically related clonal complexes 'CC206 and CC48' and 'CC464, CC353, CC354, CC574', respectively, propagated across different niches sharing semi-homogenous resistance patterns.

In Silico Characterization of Toxin-Antitoxin Systems in Campylobacter Isolates Recovered from Food Sources and Sporadic Human Illness.

Campylobacter spp. represents the most common cause of gastroenteritis worldwide with the potential to cause serious sequelae. The ability of Campylobacter to survive stressful environmental conditions has been directly linked with food-borne illness. Toxin-antitoxin (TA) modules play an important role as defense systems against antimicrobial agents and are considered an invaluable strategy harnessed by bacterial pathogens to survive in stressful environments. Although TA modules have been extensively studied in model organisms such as Escherichia coli K12, the TA landscape in Campylobacter remains largely unexplored. Therefore, in this study, a comprehensive in silico screen of 111 Campylobacter (90 C.jejuni and 21 C.coli) isolates recovered from different food and clinical sources was performed. We identified 10 type II TA systems belonging to four TA families predicted in Campylobacter genomes. Furthermore, there was a significant association between the clonal population structure and distribution of TA modules; more specifically, most (12/13) of the Campylobacter isolates belonging to ST-21 isolates possess HicB-HicA TA modules. Finally, we observed a high degree of shared synteny among isolates bearing certain TA systems or even coexisting pairs of TA systems. Collectively, these findings provide useful insights about the distribution of TA modules in a heterogeneous pool of Campylobacter isolates from different sources, thus developing a better understanding regarding the mechanisms by which these pathogens survive stressful environmental conditions, which will further aid in the future designing of more targeted antimicrobials.