Browsing by Author "Al-Agamy, Mohamed H"

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Correlation of CRISPR/Cas and Antimicrobial Resistance in Klebsiella pneumoniae Clinical Isolates Recovered from Patients in Egypt Compared to Global Strains
(MDPI AG, 2023-08) Alkompoz, Amany K; Hamed, Samira M; Abu Zaid, Ahmed S; Almangour, Thamer A; Al-Agamy, Mohamed H; Aboshanab, Khaled M
The CRISPR/Cas system has been long known to interfere with the acquisition of foreign genetic elements and was recommended as a tool for fighting antimicrobial resistance. The current study aimed to explore the prevalence of the CRISPR/Cas system in Klebsiella pneumoniae isolates recovered from patients in Egypt in comparison to global strains and correlate the CRISPR/Cas to susceptibility to antimicrobial agents. A total of 181 clinical isolates were PCR-screened for cas and selected antimicrobial resistance genes (ARGs). In parallel, 888 complete genome sequences were retrieved from the NCBI database for in silico analysis. CRISPR/Cas was found in 46 (25.4%) isolates, comprising 18.8% type I-E and 6.6% type I-E*. Multidrug resistance (MDR) and extensive drug resistance (XDR) were found in 73.5% and 25.4% of the isolates, respectively. More than 95% of the CRISPR/Cas-bearing isolates were MDR (65.2%) or XDR (32.6%). No significant difference was found in the susceptibility to the tested antimicrobial agents among the CRISPR/Cas-positive and -negative isolates. The same finding was obtained for the majority of the screened ARGs. Among the published genomes, 23.2% carried CRISPR/Cas, with a higher share of I-E* (12.8%). They were confined to specific sequence types (STs), most commonly ST147, ST23, ST15, and ST14. More plasmids and ARGs were carried by the CRISPR/Cas-negative group than others, but their distribution in the two groups was not significantly different. The prevalence of some ARGs, such as blaKPC, blaTEM, and rmtB, was significantly higher among the genomes of the CRISPR/Cas- negative strains. A weak, nonsignificant positive correlation was found between the number of spacers and the number of resistance plasmids and ARGs. In conclusion, the correlation between CRISPR/Cas and susceptibility to antimicrobial agents or bearing resistance plasmids and ARGs was found to be nonsignificant. Plasmid-targeting spacers might not be naturally captured by CRISPR/Cas. Spacer match analysis is recommended to provide a clearer image of the exact behavior of CRISPR/Cas towards resistance plasmids.
Genetic Configuration of Genomic Resistance Islands in Acinetobacter baumannii Clinical Isolates From Egypt
(Frontiers Media S.A., 2022-07) Hamed, Samira M; Hussein, Amira F. A; Al-Agamy, Mohamed H; Radwan, Hesham H; Zafer, Mai M
In Acinetobacter baumannii (A. baumannii), a wide repertoire of resistance genes is often carried within genomic resistance islands (RIs), particularly in high-risk global clones (GCs). As the first in Egypt, the current study aimed at exploring the diversity and genetic configuration of RIs in the clinical isolates of A. baumannii. For this purpose, draft genomes of 18 isolates were generated by Illumina sequencing. Disk diffusion susceptibility profiling revealed multidrug resistance (MDR) and extensive drug resistance (XDR) phenotypes in 27.7 and 72.2%, respectively. The highest susceptibility was noted for tigecycline (100.0%) followed by colistin (94.4%), for which an MIC50 of 0.25µg/ml was recorded by the broth microdilution assay. Sequence typing (ST) showed that the majority of the isolates belonged to high-risk global clones (GC1, GC2, and GC9). A novel Oxford sequence type (ST2329) that also formed a novel clonal complex was submitted to the PubMLST database. A novel blaADC variant (blaADC−258) was also identified in strain M18 (ST85Pas/1089Oxf). In addition to a wide array of resistance determinants, whole-genome sequencing (WGS) disclosed at least nine configurations of genomic RIs distributed over 16/18 isolates. GC2 isolates accumulated the largest number of RIs (three RIs/isolate) followed by those that belong to GC1 (two RIs/isolate). In addition to Tn6022 (44.4%), the comM gene was interrupted by AbaR4 (5.5%) and three variants of A. baumannii genomic resistance island 1(AbGRI)-type RIs (44.4%), including AbaR4b (16.6%) and two novel configurations of AbGRI1-like RIs (22.2%). Three of which (AbaR4, AbaR4b, and AbGRI1-like-2) carried blaOXA−23 within Tn2006. With less abundance (38.8%), IS26-bound RIs were detected exclusively in GC2 isolates. These included a short version of AbGRI2 (AbGRI2-15) carrying the genes blaTEM−1 and aphA1 and two variants of AbGRI3 RIs carrying up to seven resistance genes [mphE-msrE-armA-sul1-aadA1-catB8-aacA4]. Confined to GC1 (22.2%), sulfonamide resistance was acquired by an ISAba1 bracketed GIsul2 RI. An additional RI (RI-PER-7) was also identified on a plasmid carried by strain M03. Among others, RI-PER-7 carried the resistance genes armA and blaPER−7. Here, we provided a closer view of the diversity and genetic organization of RIs carried by a previously unexplored population of A. baumannii.
Genomic Characterization of Extensively Drug-Resistant NDM- Producing Acinetobacter baumannii Clinical Isolates With the Emergence of Novel bla ADC-257
(Frontiers, 22/11/2021) Zafer, Mai M; Hussein, Amira F. A; Al-Agamy, Mohamed H; Radwan, Hesham H; Hamed, Samira M
Acinetobacter baumannii has become a major challenge to clinicians worldwide due to its high epidemic potential and acquisition of antimicrobial resistance. This work aimed at investigating antimicrobial resistance determinants and their context in four extensively drug-resistant (XDR) NDM-producing A. baumannii clinical isolates collected between July and October 2020 from Kasr Al-Ainy Hospital, Cairo, Egypt. A total of 20 A. baumannii were collected and screened for acquired carbapenemases (blaNDM, blaVIM and blaIMP) using PCR. Four NDM producer A. baumannii isolates were identified and selected for whole- genome sequencing, in silico multilocus sequence typing, and resistome analysis. Antimicrobial susceptibility profiles were determined using disk diffusion and broth microdilution tests. All blaNDM-positive A. baumannii isolates were XDR. Three isolates belonged to high-risk international clones (IC), namely, IC2 corresponding to ST570Pas/1701Oxf (M20) and IC9 corresponding to ST85Pas/ST1089Oxf (M02 and M11). For the first time, we report blaNDM-1 gene on the chromosome of an A. baumannii strain that belongs to sequence type ST164Pas/ST1418Oxf. Together with AphA6, blaNDM-1 was bracketed by two copies of ISAba14 in ST85Pas isolates possibly facilitating co-transfer of amikacin and carbapenem resistance. A novel blaADC allele (blaADC-257) with an upstream ISAba1 element was identified in M19 (ST/CC164Pas and ST1418Oxf/CC234Oxf). blaADC genes harbored by M02 and M11 were uniquely interrupted by IS1008. Tn2006-associated blaOXA-23 was carried by M20. blaOXA-94 genes were preceded by ISAba1 element in M02 and M11. AbGRI3 was carried by M20 hosting the resistance genes aph(3`)-Ia, aac(6`)-Ib`, catB8, ant(3``)-Ia, sul1, armA, msr(E), and mph(E). Nonsynonymous mutations were identified in the quinolone resistance determining regions (gyrA and parC) of all isolates. Resistance to colistin in M19 was accompanied by missense mutations in lpxACD and pmrABC genes. The current study provided an insight into the genomic background of XDR phenotype in A. baumannii recovered from patients in Egypt. WGS revealed strong association between resistance genes and diverse mobile genetic elements with novel insertion sites and genetic organizations.
Retained colistin susceptibility in clinical Acinetobacter baumannii isolates with multiple mutations in pmrCAB and lpxACD operons
(Frontiers Media S.A., 2023-08) Zafer, Mai M; Hussein, Amira F. A; Al-Agamy, Mohamed H; Radwan, Hesham H; Hamed, Samira M
The progressive increase in the resistance rates to first- and second-line antibiotics has forced the reuse of colistin as last-line treatment for Acinetobacter baumannii infections, but the emergence of colistin-resistant strains is not uncommon. This has been long linked to acquired chromosomal mutations in the operons pmrCAB and lpxACD. Hence, such mutations are routinely screened in colistin-resistant strains by most studies. The current study was designed to explore the possible existence of pmrCAB and lpxACD mutations in colistin-susceptible isolates. For this purpose, the whole genome sequences of eighteen multi-/extensively drug resistant A. baumannii were generated by Illumina sequencing and screened for missense mutations of the operons pmrCAB and lpxACD. Most of the isolates belonged to global clones (GCs) including GC1 (n=2), GC2 (n=7), GC7 (n=2), GC9 (n=3), and GC11 (n=1). The minimum inhibitory concentrations (MICs) of colistin were determined by the broth microdilution assay. Seventeen isolates were fully susceptible to colistin with MICs ranging from (≤0.125 to 0.5 µg/ml). Interestingly, all colistin- susceptible isolates carried missense mutations in pmrCAB and lpxACD operons with reference to A. baumannii ATCC 19606. Overall, 34 mutations were found. Most substitutions were detected in pmrC (n=20) while no mutations were found in pmrA or lpxA. Notably, the mutation pattern of the two operons was almost conserved among the isolates that belonged to the same sequence type (ST) or GC. This was also confirmed by expanding the analysis to include A. baumannii genomes deposited in public databases. Here, we demonstrated the possible existence of missense mutations in pmrCAB and lpxACD operons in colistin- susceptible isolates, shedding light on the importance of interpreting mutations with reference to colistin-susceptible isolates of the same ST/GC to avoid the misleading impact of the ST/GC-related polymorphism. In turn, this may lead to misinterpretation of mutations and, hence, overlooking the real players in colistin resistance that are yet to be identified.
Tn7382, a novel transposon harboring blaNDM-1 and aphA6 in Acinetobacter baumannii
(Elsevier BV, 2022-08) Hamed, Samira M; Hussein, Amira F.A; Al-Agamy, Mohamed H; Radwan, Hesham H; Zafer, Mai M
Objectives: Co-transfer of carbapenem and amikacin resistance might contribute to the evolution of extensively drug resistant (XDR) Acinetobacter baumannii. The current study is aimed at in silico investigating the potential mobility of a novel composite transposon co- harboring blaNDM-1 and aphA6 using bioinformatic tools. Methods: The transposon, named here Tn7382, was recently described in the chromosomes of two XDR A. baumannii isolates (M02 and M11) from Egypt. The draft genomes of M02 and M11 were generated by Illumina sequencing. Nucleotide homology of Tn7382 and flanking regions was analyzed using the BLASTN tool. Results: Tn7382 is derived from Tn125 and encompasses seven orfs [aphA6, ISAba125 transposase-coding gene, blaNDM-1, ble, iso, tat, cutA] enclosed by two direct copies of ISAba14. While described for the first time, Tn7382 was found in the chromosomes of five A. baumannii strains deposited in the NCBI database. Using Artemis Comparison Tool, the potential mobility of Tn7382 was demonstrated in silico by comparative genomic analysis of two A. baumannii strains (TP1 and TP2) retrieved from the NCBI database. The transposon was acquired by TP2 at the same location as an ISAba14 element in the ancestral variant TP1 isolated from the same patient in USA 11 days earlier. Conclusions: Here, we present the characteristics of Tn7382, a composite transposon flanked by ISAba14 and harboring the aphA6 and blaNDM-1 resistance genes. In silico analysis inferred the potential mobility of Tn7382 but experimental validation is still required.