Unveiling the microevolution of antimicrobial resistance in selected Pseudomonas aeruginosa isolates from Egyptian healthcare settings: A genomic approach.

The incidence of Pseudomonas aeruginosa infections in healthcare environments, particularly in low-and middle-income countries, is on the rise. The purpose of this study was to provide comprehensive genomic insights into thirteen P. aeruginosa isolates obtained from Egyptian healthcare settings. Phenotypic analysis of the antimicrobial resistance profile and biofilm formation were performed using minimum inhibitory concentration and microtiter plate assay, respectively. Whole genome sequencing was employed to identify sequence typing, resistome, virulome, and mobile genetic elements. Our findings indicate that 92.3% of the isolates were classified as extensively drug-resistant, with 53.85% of these demonstrating strong biofilm production capabilities. The predominant clone observed in the study was ST773, followed by ST235, both of which were associated with the O11 serotype. Core genome multi-locus sequence typing comparison of these clones with global isolates suggested their potential global expansion and adaptation. A significant portion of the isolates harbored Col plasmids and various MGEs, all of which were linked to antimicrobial resistance genes. Single nucleotide polymorphisms in different genes were associated with the development of antimicrobial resistance in these isolates. In conclusion, this pilot study underscores the prevalence of extensively drug-resistant P. aeruginosa isolates and emphasizes the role of horizontal gene transfer facilitated by a diverse array of mobile genetic elements within various clones. Furthermore, specific insertion sequences and mutations were found to be associated with antibiotic resistance.

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.

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.

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.

Cracking the Code: Unveiling the Diversity of Carbapenem-Resistant Klebsiella pneumoniae Clones in the Arabian Peninsula through Genomic Surveillance.

The rise of antimicrobial resistance is a global challenge that requires a coordinated effort to address. In this study, we examined the genetic similarity of carbapenem-resistant Klebsiella pneumoniae (CRKP) in countries belonging to the Gulf Cooperation Council (GCC) to gain a better understanding of how these bacteria are spreading and evolving in the region. We used in silico genomic tools to investigate the occurrence and prevalence of different types of carbapenemases and their relationship to specific sequence types (STs) of CRKP commonly found in the region. We analyzed 720 publicly available genomes of multi-drug resistant K. pneumoniae isolates collected from six GCC countries between 2011 and 2020. Our findings showed that ST-14 and ST-231 were the most common STs, and 51.7% of the isolates carried blaOXA-48-like genes. Additionally, we identified rare carbapenemase genes in a small number of isolates. We observed a clonal outbreak of ST-231 in Oman, and four Saudi isolates were found to have colistin resistance genes. Our study offers a comprehensive overview of the genetic diversity and resistance mechanisms of CRKP isolates in the GCC region that could aid in developing targeted interventions to combat this pressing global issue.

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.

Diversity of carbapenem-resistant Klebsiella pneumoniae ST14 and emergence of a subgroup with KL64 capsular locus in the Arabian Peninsula.

To understand the reasons of successful spread of carbapenem-resistant Klebsiella pneumoniae ST14 (CRKP-ST14) in countries of the Arabian Peninsula, the resistome, capsular locus, carbapenemase carrying plasmid types, and core genome of isolates from the region were compared to global isolates. Thirty-nine CRKP-ST14 strains isolated from 13 hospitals in the United Arab Emirates, Bahrain, and Saudi Arabia were selected for whole genome sequencing on Illumina MiSeq platform based on the variety of carbapenemase genes carried and plasmids bearing these genes. Their resistome, capsular locus, and core genome MLST were compared to 173 CRKP-ST14 genomes available in public databases. The selected 39 CRKP-ST14 produced either NDM-1, OXA-48, OXA-162, OXA-232, KPC-2, or co-produced NDM-1 and an OXA-48-like carbapenemase. cgMLST revealed three clusters: 16 isolates from five UAE cities (C1), 11 isolates from three UAE cities and Bahrain (C2), and 5 isolates from Saudi Arabia (C3), respectively, and seven singletons. Resistance gene profile, carbapenemase genes, and their plasmid types were variable in both C1 and C2 clusters. The majority of CRKP-ST14 had KL2, but members of the C2 cluster and two further singletons possessed KL64 capsular locus. Based on cgMLST comparison of regional and global isolates, CRKP-ST14 with KL64 from four continents formed a distinct cluster, suggesting a recent emergence and spread of this variant. Our findings confirmed clonal transmission coupled with likely horizontal gene transfer in carbapenem-resistant Klebsiella pneumoniae ST14. Dissemination of this genetically flexible, highly resistant clone warrants further monitoring.

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.

Stress resistance associated with multi-host transmission and enhanced biofilm formation at 42 °C among hyper-aerotolerant generalist Campylobacter jejuni.

One of the emerging conundrums of Campylobacter food-borne illness is the bacterial ability to survive stressful environmental conditions. We evaluated the heterogeneity among 90 C. jejuni and 21 C. coli isolates from different sources in Egypt with respect to biofilm formation capabilities (under microaerobic and aerobic atmosphere) and resistance to a range of stressors encountered along the food chain (aerobic stress, refrigeration, freeze-thaw, heat, peracetic acid, and osmotic stress). High prevalence (63%) of hyper-aerotolerant (HAT) isolates was observed, exhibiting also a significantly high tolerance to heat, osmotic stress, refrigeration, and freeze-thaw stress, coupled with high biofilm formation ability which was clearly enhanced under aerobic conditions, suggesting a potential link between stress adaptation and biofilm formation. Most HAT multi-stress resistant and strong biofilm producing C. jejuni isolates belonged to host generalist clonal complexes (ST-21, ST-45, ST-48 and ST-206). These findings highlight the potential role of oxidative stress response systems in providing cross-protection (resistance to other multiple stress conditions) and enhancing biofilm formation in Campylobacter and suggest that selective pressures encountered in hostile environments have shaped the epidemiology of C. jejuni in Egypt by selecting the transmission of highly adapted isolates, thus promoting the colonization of multiple host species by important disease-causing lineages.

Epidemic IncX3 plasmids spreading carbapenemase genes in the United Arab Emirates and worldwide.

PURPOSE: Plasmids of the incompatibility group X type 3 (IncX3) were described carrying various carbapenemase genes in carbapenemase-producing Enterobacteriaceae (CPE) worldwide and in the United Arab Emirates (UAE), as well. To understand the driving force behind the emergence of such plasmids in the UAE, the relationship between IncX3 plasmids encountered locally and globally was investigated. METHODS: CPE strains isolated in the UAE during 2009-2014 were screened by X3 PCR-based replicon typing. The clonal relationship of CPE carrying IncX3 plasmids was determined by multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Complete sequence of selected IncX3 plasmids was determined. Phylogenetic relationship between the carbapenemase carrying IncX3 plasmids from the UAE and of those reported worldwide was established by comparing the plasmid backbones. RESULTS: 10.2% of the 295 CPE tested were identified to carry IncX3 plasmids: 13 Escherichia coli, 13 Klebsiella pneumoniae, two Enterobacter cloacae, one Citrobacter freundii and one Morganella morganii isolate, respectively. Most of them were non-clonal; with small clusters of triplets and pairs of E. coli and K. pneumoniae, and a cluster of five K. pneumoniae ST11 exhibiting >90% similar PFGE patterns, respectively. The 30 isolates harbored either bla NDM-1, bla NDM-4, bla NDM-5, bla NDM-7, bla OXA-181 or bla KPC-2 carbapenemase genes on IncX3 plasmids. Phylogenetic analysis of the backbone region of IncX3 plasmids carrying various beta-lactamase genes from the UAE (n=23) and that of North-America, Europe, Asia and Australia (n=35) revealed three clusters based on the carbapenemase genes carried: plasmids harboring bla OXA-181 and bla NDM-5 formed two distinct groups, whereas backbones of plasmids with bla NDM-1, bla NDM-4 and bla NDM-7 clustered together. Each cluster contained plasmids of diverse geographical origin. CONCLUSION: The findings suggest that different carbapenemase gene carrying IncX3 plasmids encountered in the UAE do not evolve locally, rather are subtypes of this epidemic plasmid emerging in this country due to international transfer.

Clonal emergence of Klebsiella pneumoniae ST14 co-producing OXA-48-type and NDM carbapenemases with high rate of colistin resistance in Dubai, United Arab Emirates.

Few studies have addressed the molecular epidemiology of carbapenem-resistant Enterobacteriaceae (CRE) isolates in the Arabian Peninsula, and such investigations have been missing from Dubai, a major economical, tourism and medical centre of the region. The antibiotic susceptibility, the carbapenemase type produced, and the clonality of 89 CRE strains isolated in five major Dubai hospitals in June 2015 to June 2016 were determined. Thirty-three percent of the collection of 70 Klebsiella pneumoniae, 13 Escherichia coli and 6 other Enterobacteriaceae were extremely drug resistant, 27% were resistant to colistin, and 4.5% (4 K. pneumoniae isolates) were resistant to all antibiotics tested. The colistin resistance rate in K. pneumoniae was 31.4%. None of the isolates carried mobile colistin resistance genes. Seventy-seven isolates produced carbapenemase: 53.3% OXA-48-like, 24.7% NDM and 22.1% both OXA-48-like and NDM, respectively. Pulsed-field gel electrophoresis clustered 50% of K. pneumoniae into a 35-membered group, which showed significant association with double carbapenemase production, with extreme drug resistance, and with being isolated from Emirati patients. Members of the cluster belonged to sequence type ST14. The rate of colistin resistance in K. pneumoniae ST14 was 37.1% vs. 27.1% of K. pneumoniae isolates outside of the cluster. Two of the panresistant K. pneumoniae isolates also belonged to ST14, whereas the other two were ST15 and ST231, respectively. In conclusion, beyond the overall high colistin resistance rate in CRE, the emergence of a highly resistant clone of K. pneumoniae ST14 in all Dubai hospitals investigated is a serious problem requiring immediate attention.