Fine Mapping of Sequences Important For FIV RNA Packaging and Their Mechanism of Function

The mechanism by which retroviruses preferentially encapsidate their unspliced genomic RNA among millions of both spliced viral and cellular mRNAs in the cytoplasm represents a function of great specificity. This selection process requires that the genomic RNA contain packaging determinants unique to its own RNA that can interact specifically with the packaging proteins of the viral particles, the Gag polyproteins. Knowing the exact sequences involved in packaging should provide basic insights into the mechanism of preferentially encapsidating the full length genomic RNA. We have been interested in mapping the packaging determinants of the feline immunodeficiency virus (FIV), a lentivirus that is being considered as a potentially powerful gene delivery system for human gene therapy. Our initial studies have shown that the FIV packaging determinants are located as two discontinuous core regions within the 5\u27 end of the viral genome (Browning et aI., 2003 a & b). The first region extends from the R/U5 in the 5\u27 LTR to the first 120 bp of 5\u27 UTR and the second consists of the first 100 bp of gag, while other regions of the genome may also be involved. Studies undertaken in this thesis carried these observations further to determine whether the region in between the two core determinants was important for packaging or merely acted to maintain the spacing of the two core elements. Additionally, since other regions of the genome, especially the LTR, had been implicated as containing significant packaging determinants in other studies (Kemler et aI., 2002), we dissected the role of the LTR elements away from the untranslated region towards FIV RNA packaging. Towards this end, several series of small FIV transfer vectors were constructed either in the heterologous non-viral or homologous subgenomic context containing various combinations of the LTR, and/or UTR and gag and tested for their packaging potential in our well-established in vivo packaging assay. This was followed by analysis of the amount of transfer vector RNAs packaged directly into the virus particles using a semi-quantitative RT-PCR approach. Test of the various transfer vector RNAs confirmed our earlier observation that the FIV packaging determinants are indeed discontinuous and spread out with the core packaging determinants residing within the first 150 bp UTR and 100 bp of gag (Chapter III). Furthermore, the intervening sequences between these two elements were not required either for vector RNA packaging or propagation (Chapter III). Analysis of the LTR elements revealed the presence of other packaging determinants of lesser strength than the core determinants in the 5\u27 R/U5 and 3\u27 U3/R regions of the viral LTRs (Chapter IV). Folding of the 5\u27 end of the viral RNA using computer analysis software revealed the presence of complex stem loop structures. Correlation of the mutational analysis with the folding algorithms revealed the presence of a conserved stem loop in the 5\u27 UTR that may serve as the principal packaging determinant of FIV. Interestingly, no consistent structural element could be identified within the first 100 bp of gag that could be responsible for the packaging potential of the gag region, suggesting that gag sequences may function at the primary sequence level, perhaps providing the intronic sequences needed to distinguish between genomic and sub genomic mRNAs. Taken together, these data should add to the increasing knowledge of how complex retroviruses package their genomic RNAs and help streamline the design of safer self-inactivating FIV–based vectors for human gene therapy

Elucidating the effect of iron acquisition systems in Klebsiella pneumoniae on susceptibility to the novel siderophore-cephalosporin cefiderocol

BACKGROUND: Cefiderocol (CFDC) is a novel siderophore-cephalosporin, effective against multidrug-resistant Gram-negative bacteria. As it has a siderophore side chain, it can utilize iron acquisition systems for penetration of the bacterial outer membrane. We aimed to elucidate the role of siderophores and iron uptake receptors in defining Klebsiella pneumoniae susceptibility to CFDC. METHODS: Initially, 103 K. pneumoniae strains were characterized for susceptibility to different antibiotics including CFDC. CFDC minimum inhibitory concentrations (MIC) were determined in iron-depleted and iron-enriched conditions. Iron uptake genes including siderophores, their receptors, ferric citrate (fecA) and iron uptake (kfu) receptors were detected by PCR in all the strains. For 10 selected strains, gene expression was tested in iron-depleted media with or without CFDC treatment and compared to expression in iron-enriched conditions. RESULTS: CFDC exhibited 96.1% susceptibility, being superior to all the other antibiotics (MIC50: 0.5 and MIC90: 4 μg/ml). Only three strains (2.9%) were intermediately susceptible and a pandrug resistant strain (0.97%) was resistant to CFDC (MIC: 8 and 256 μg/ml, respectively). The presence of kfu and fecA had a significant impact on CFDC MIC, especially when co-produced, and if coupled with yersiniabactin receptor (fyuA). CFDC MICs were negatively correlated with enterobactin receptor (fepA) expression and positively correlated with expression of kfu and fecA. Thus, fepA was associated with increased susceptibility to CFDC, while kfu and fecA were associated with reduced susceptibility to CFDC. CFDC MICs increased significantly in iron-enriched media, with reduced expression of siderophore receptors, hence, causing less drug uptake. CONCLUSION: Iron acquisition systems have a significant impact on CFDC activity, and their altered expression is a factor leading to reduced susceptibility. Iron concentration is also a major player affecting CFDC susceptibility; therefore, it is essential to explore possible ways to improve the drug activity to facilitate its use to treat infections in iron-rich sites

Genetic support of carbapenemases in double carbapenemase producer Klebsiella pneumoniae isolated in the Arabian Peninsula

Enterobacteriaceae co-producing NDM- and OXA-48-type carbapenemases were encountered in higher frequency in the United Arab Emirates (UAE) than in the neighboring countries in our earlier study. The aim of this investigation was to characterize the seven double carbapenemase producer Klebsiella pneumoniae found in the region to assess factors contributing to their emergence. Three K. pneumoniae ST14 isolated in the UAE harboring blaNDM-1 on IncHI1b and blaOXA-232 on IncColE plasmids were clonally related. Furthermore, two K. pneumoniae from the UAE, ABC106 and ABC137 belonged to ST307 and ST1318, respectively. ABC106 carried blaNDM-1 on IncHI1b, and blaOXA-162 on IncL/M plasmids, whereas ABC137 possessed blaNDM-1 on IncX3 and blaOXA-48 on IncL/M plasmids. The double carbapenemase-producing K. pneumoniae from Oman (OMABC109) and Saudi Arabia (SA54) belonged to ST11 and ST152, respectively. OMABC109 harbored blaNDM-1 on an IncHI1b plasmid highly similar to the NDM-plasmid of ABC106 and carried a chromosomally coded blaOXA-181 located on Tn2013. SA54 possessed a blaNDM-1 on an IncFIb/FII plasmid and a blaOXA-48 on an IncL/M plasmid. Based on these findings, clonal spread and horizontal transfer of carbapenemase genes located on transposons or self-transmissible plasmids contributed equally to the emergence of double carbapenemase-producing Enterobacteriaceae in the region

Genomic profiling of extended-spectrum β-lactamase-producing Escherichia coli from Pets in the United Arab Emirates: Unveiling colistin resistance mediated by mcr-1.1 and its probable transmission from chicken meat – A One Health perspective

Background The United Arab Emirates (UAE) has witnessed rapid urbanization and a surge in pet ownership, sparking concerns about the possible transfer of antimicrobial resistance (AMR) from pets to humans and the environment. This study delves into the whole-genome sequencing analysis of ESBL-producing E. coli strains from healthy cats and dogs in the UAE, which exhibit multidrug resistance (MDR). Additionally, it provides a genomic exploration of the mobile colistin resistance gene mcr-1.1, marking the first instance of its detection in Middle Eastern pets. Methods We investigate 17 ESBL-producing E. coli strains from healthy UAE pets using WGS and bioinformatics analysis to identify genes encoding virulence factors, assign diverse typing schemes to the isolates, and scrutinize the presence of AMR genes. Furthermore, we characterized plasmid contigs housing the mcr-1.1 gene and conducted phylogenomic analysis to evaluate their relatedness to previously identified UAE isolates. Results Our study unveiled a variety of virulence factor-encoding genes within the isolates, with fimH emerging as the most prevalent. Regarding β-lactamase resistance genes, the blaCTX group 1 gene family predominated, with CTX-M-15 found in 52.9% (9/17) of the isolates, followed by CTX-M-55 in 29.4% (5/17). These isolates were categorized into multiple sequence types (STs), with the epidemic ST131 being the most frequent. The presence of the mcr-1.1 gene, linked to colistin resistance, was confirmed in two isolates. These isolates belonged to ST1011 and displayed distinct profiles of β-lactamase resistance genes. Phylogenomic analysis revealed close connections between the isolates and those from chicken meat in the UAE. Conclusion Our study underscores the presence of MDR ESBL-producing E. coli in UAE pets. The identification of mcr-1.1-carrying isolates warrants the urgency of comprehensive AMR surveillance and highlights the role of companion animals in AMR epidemiology. These findings underscore the significance of adopting a One Health approach to mitigate AMR transmission risks effectively

First Report of Colistin-Resistant <i>Escherichia coli</i> Carrying <i>mcr-1</i> IncI2(delta) and IncX4 Plasmids from Camels (<i>Camelus dromedarius</i>) in the Gulf Region.

Addressing the emergence of antimicrobial resistance (AMR) poses a significant challenge in veterinary and public health. In this study, we focused on determining the presence, phenotypic background, and genetic epidemiology of plasmid-mediated colistin resistance (mcr) in Escherichia coli bacteria isolated from camels farmed in the United Arab Emirates (UAE). Fecal samples were collected from 50 camels at a Dubai-based farm in the UAE and colistin-resistant Gram-negative bacilli were isolated using selective culture. Subsequently, a multiplex PCR targeting a range of mcr-genes, plasmid profiling, and whole-genome sequencing (WGS) were conducted. Eleven of fifty camel fecal samples (22%) yielded colonies positive for E. coli isolates carrying the mcr-1 gene on mobile genetic elements. No other mcr-gene variants and no chromosomally located colistin resistance genes were detected. Following plasmid profiling and WGS, nine E. coli isolates from eight camels were selected for in-depth analysis. E. coli sequence types (STs) identified included ST7, ST21, ST24, ST399, ST649, ST999, and STdaa2. Seven IncI2(delta) and two IncX4 plasmids were found to be associated with mcr-1 carriage in these isolates. These findings represent the first identification of mcr-1-carrying plasmids associated with camels in the Gulf region. The presence of mcr-1 in camels from this region was previously unreported and serves as a novel finding in the field of AMR surveillance

First Report of Colistin-Resistant <i>Escherichia coli</i> Carrying <i>mcr-1</i> IncI2(delta) and IncX4 Plasmids from Camels (<i>Camelus dromedarius</i>) in the Gulf Region.

Addressing the emergence of antimicrobial resistance (AMR) poses a significant challenge in veterinary and public health. In this study, we focused on determining the presence, phenotypic background, and genetic epidemiology of plasmid-mediated colistin resistance (mcr) in Escherichia coli bacteria isolated from camels farmed in the United Arab Emirates (UAE). Fecal samples were collected from 50 camels at a Dubai-based farm in the UAE and colistin-resistant Gram-negative bacilli were isolated using selective culture. Subsequently, a multiplex PCR targeting a range of mcr-genes, plasmid profiling, and whole-genome sequencing (WGS) were conducted. Eleven of fifty camel fecal samples (22%) yielded colonies positive for E. coli isolates carrying the mcr-1 gene on mobile genetic elements. No other mcr-gene variants and no chromosomally located colistin resistance genes were detected. Following plasmid profiling and WGS, nine E. coli isolates from eight camels were selected for in-depth analysis. E. coli sequence types (STs) identified included ST7, ST21, ST24, ST399, ST649, ST999, and STdaa2. Seven IncI2(delta) and two IncX4 plasmids were found to be associated with mcr-1 carriage in these isolates. These findings represent the first identification of mcr-1-carrying plasmids associated with camels in the Gulf region. The presence of mcr-1 in camels from this region was previously unreported and serves as a novel finding in the field of AMR surveillance

Staphylococcus spp. in Salad Vegetables: Biodiversity, Antimicrobial Resistance, and First Identification of Methicillin-Resistant Strains in the United Arab Emirates Food Supply

Contamination of leafy greens with Staphylococcus spp. can occur at various supply chain stages, from farm to table. This study comprehensively analyzes the species diversity, antimicrobial resistance, and virulence factors of Staphylococci in salad vegetables from markets in the United Arab Emirates (UAE). A total of 343 salad items were sampled from three major cities in the UAE from May 2022 to February 2023 and tested for the presence of Staphylococcus spp. using standard culture-based methods. Species-level identification was achieved using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Antimicrobial susceptibility testing was conducted using the VITEK-2 system with AST-P592 cards. Additionally, whole genome sequencing (WGS) of ten selected isolates was performed to characterize antimicrobial resistance determinants and toxin-related virulence factors. Nine Staphylococcus species were identified in 37.6% (129/343) of the tested salad items, with coagulase-negative staphylococci (CoNS) dominating (87.6% [113/129]) and S. xylosus being the most prevalent (89.4% [101/113]). S. aureus was found in 4.6% (14/343) of the salad samples, averaging 1.7 log10 CFU/g. One isolate was confirmed as methicillin-resistant S. aureus, harboring the mecA gene. It belonged to multi-locus sequence type ST-672 and spa type t384 and was isolated from imported fresh dill. Among the characterized S. xylosus (n = 45), 13.3% tested positive in the cefoxitin screen test, and 6.6% were non-susceptible to oxacillin. WGS analysis revealed that the cytolysin gene (cylR2) was the only toxin-associated factor found in S. xylosus, while a methicillin-sensitive S. aureus isolate harbored the Panton-Valentine Leukocidin (LukSF/PVL) gene. This research is the first to document the presence of methicillin-resistant S. aureus in the UAE food chain. Furthermore, S. xylosus (a coagulase-negative staphylococcus not commonly screened in food) has demonstrated phenotypic resistance to clinically relevant antimicrobials. This underscores the need for vigilant monitoring of antimicrobial resistance in bacterial contaminants, whether pathogenic or commensal, at the human-food interface

Assessing the Prevalence and Potential Risks of Salmonella Infection Associated with Fresh Salad Vegetable Consumption in the United Arab Emirates

This study aimed to investigate the occurrence and characteristics of Salmonella isolates in salad vegetables in the United Arab Emirates (UAE). Out of 400 samples tested from retail, only 1.25% (95% confidence interval, 0.41–2.89) were found to be positive for Salmonella, all of which were from conventional local produce, presented at ambient temperature, and featured as loose items. The five Salmonella-positive samples were arugula (n = 3), dill (n = 1), and spinach (n = 1). The Salmonella isolates from the five samples were found to be pan-susceptible to a panel of 12 antimicrobials tested using a disc diffusion assay. Based on whole-genome sequencing (WGS) analysis, only two antimicrobial resistance genes were detected—one conferring resistance to aminoglycosides (aac(6′)-Iaa) and the other to fosfomycin (fosA7). WGS enabled the analysis of virulence determinants of the recovered Salmonella isolates from salad vegetables, revealing a range from 152 to 165 genes, collectively grouped under five categories, including secretion system, fimbrial adherence determinants, macrophage-inducible genes, magnesium uptake, and non-fimbrial adherence determinants. All isolates were found to possess genes associated with the type III secretion system (TTSS), encoded by Salmonella pathogenicity island-1 (SPI-1), but various genes associated with the second type III secretion system (TTSS-2), encoded by SPI-2, were absent in all isolates. Combining the mean prevalence of Salmonella with information regarding consumption in the UAE, an exposure of 0.0131 salmonellae consumed per person per day through transmission via salad vegetables was calculated. This exposure was used as an input in a beta-Poisson dose–response model, which estimated that there would be 10,584 cases of the Salmonella infection annually for the entire UAE population. In conclusion, salad vegetables sold in the UAE are generally safe for consumption regarding Salmonella occurrence, but occasional contamination is possible. The results of this study may be used for the future development of risk-based food safety surveillance systems in the UAE and to elaborate on the importance for producers, retailers, and consumers to follow good hygiene practices, particularly for raw food items such as leafy salad greens