الفهرس 
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Abstract
The CRISPR/Cas system (Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated genes (Cas)) is a component of the prokaryotes genomes that provides acquired immunity, has been long known to interfere with the acquisition of foreign genetic elements, and was recommended as a tool for fighting antimicrobial resistance. It was later found in approximately all archaeal genomes and in about half of the bacterial genomes, but it has not been found in any eukaryote so far.
Multiple CRISPR loci may exist in bacterial genomes; each CRISPR array is made up of short direct repeats (22–56 nucleotides) interspaced by unique DNA fragments called “spacers,” whose origins are mobile genetic elements (MGEs), such as bacteriophages, plasmids, or transposons. Such spacers serve as sequence-specific memories of preceding infections and mediate the visibility of the invaders during subsequent infections. CRISPR arrays are transcribed into CRISPR RNA (crRNA) that guides Cas proteins, encoded by Cas genes, to identify and destroy the invader’s foreign genetic elements via three mechanistic stages: adaptation, expression, and interference.
The CRISPR/Cas system is grouped into two classes that were further categorized into six types and over forty subtypes. A multi-Cas effector protein complex is used by Class 1 members for interference, while Class 2 members use a single effector protein. Type I-E and a subset of the type I-E CRISPR/Cas carried in the chromosome of K. pneumoniae are designated I-E*.
K. pneumoniae is an important gram-negative opportunistic pathogen. The widespread dissemination of MDR, XDR, and hypervirulent (hv) strains of K. pneumoniae is associated with the acquisition of plasmids loaded with antibiotic resistance or hypervirulence genes. The frequent outbreak of K. pneumoniae nosocomial infections is due to plasmids encoding ESBLs and carbapenemase enzymes, which are facilitated by high genetic transfer (HGT) to other species.
The current study aimed to investigate the prevalence of CRISPR/Cas in K. pneumoniae clinical isolates obtained from patients admitted to Nasser Institute Hospital in Cairo, Egypt, and to study its association with antimicrobial resistance in comparison to global strains. In parallel, 888 K. pneumoniae published genomes were retrieved for comparison and further analysis. Additionally, the correlation between CRISPR/Cas and the number of resistance plasmids, ARGs, and STs distribution was also assessed in the published genomes.
A total of 181 clinical K. pneumoniae isolates were recovered from various clinical specimens from the microbiology lab at Nasser Institute Hospital, Cairo, Egypt, for 6 months (April to September 2021). The majority of isolates were from blood (39%), followed by urine (27%), wound swap (18%), CSF (7%), sputum (6%), and pus (3%). The collected isolates were identified using conventional microbiological techniques.
All the recovered isolates were tested for their susceptibility to a panel of antimicrobial agents by disc diffusion and broth microdilution tests. Multi-drug resistance (MDR) and XDR were found in 73.5% and 25.4% of the collected isolates, respectively. The highest activity was shown by colistin (R = 2%), tigecycline (R = 15%), tetracycline (R = 55%), and chloramphenicol (R = 59%). Almost all isolates (99%) were non susceptible to amoxicillin/clavulanate. The resistance to cephalosporins ranged from 85 to 100%, with cefepime and cefoxitin showing the lowest and highest activity, respectively. More than 90% of the isolates were resistant to aztreonam (93%), ciprofloxacin (93%), and nitrofurantoin (92%). High resistance levels were also found for trimethoprim-sulfamethoxazole (87%), the aminoglycoside gentamicin (72%), and amikacin (70%). Carbapenem resistance was phenotypically evident in 80% of the isolates.
The observed resistance to most of the tested antimicrobial agents was less prevalent in the cas-positive isolates compared to others. Exceptions included gentamicin, tetracycline, chloramphenicol, tigecycline, and colistin. No significant difference was found in the susceptibility to the tested antimicrobial agents among the CRISPR/Cas-positive and negative isolates. The isolates harbouring type I-E* CRISPR/Cas systems were more susceptible to all tested antimicrobial agents among the 17 antibiotic susceptibilities tested. Imipenem (58.3%), gentamicin (58.3%), amikacin (50%), tetracycline (41.7%), chloramphenicol (41.7%), and tigecycline (16.7%) exhibited the highest activity compared to those that carried I-E, with the exception for colistin. Only gentamicin and aztreonam showed statistical significance (Fisher’s exact test, p-values = 0.013 and 0.020, respectively). Lower activity against ß-lactams, ciprofloxacin, and nitrofurantoin antibiotics was detected in both types.
PCR was used for screening the CRISPR/Cas signature genes, which were found in 25.4% of the tested isolates. According to the sequence of cas1/cas3 genes, 34 (18.7%) isolates carried type I-E, while 12 (6.6%) were positive for type I-E*. The cas genes are more prevalent in blood and urine cultures compared to others; no significant difference was found in their distribution in the isolates that belong to different specimen types. More than 95% of the CRISPR/Cas-bearing isolates were MDR (65.2%) or XDR (32.6%).
Of all the tested ARGs, blaSHV and qnrS were found to have the highest prevalence. With respect to CRISPR/Cas, a higher prevalence was found for most of the ARGs in the cas-positive group compared to the cas-negative group. An exception included the blaNDM and armA genes, which were more prevalent in the cas-negative isolates. No significant difference was found in the distribution of the ARGs across the two groups. Upon examination of the number of ARGs carried by the isolates in each group, we found that the cas-positive isolates carried more combinations of ARGs compared to the cas-negative group, but this was not statistically significant, according to the Mann–Whitney U test (p-value = 0.070). A higher prevalence was found for all tested genes in the IE-positive isolates compared to the I-E*-positive ones; this was statistically significant only for qnrS.
Analysis of the draft genomes of two representative isolates for I-E and I-E* showed more plasmid replicons and resistance genes in the I-E*-positive isolate. The CRISPR array of isolate K57 contained 50 spacers. Of them, only four (8.0%) spacers matched plasmid sequences, and seven (14.0%) spacers showed no similarity in the NCBI non-redundant nucleotide (nr/nt) database. Likewise, only one spacer was harboured by one of the CRISPR arrays of isolate 117 matched plasmid sequences. Only one plasmid replicon was identified in isolate K57, six replicons were found in isolate 117, and more ARGs were carried by the I-E* positive isolates 117. Unlike the strong biofilm-forming K. pneumoniae hvKp NTUH K2044, none of the sequenced strains had the plasmid-mediated virulence genes iucABCD/iutA. More prophages were found in isolate 117 than in the K57 isolate. This finding correlates with the number of spacers in each isolate.
In silico screening of the CRISPR/Cas system among 888 published K. pneumoniae genomes showed that only 206/888 (23.2%) harboured complete CRISPR/Cas systems. While at least one CRISPR array was found in 26.0% of the genomes, only those that also carried cas gene clusters were considered to have a functional CRISPR/Cas system and were selected for further analysis. In silico PCR analysis of the genomes using cas-specific primers showed that type I-E* (114/888, 12.8%) was more prevalent than type I-E (92/888, 10.4%).
CRISPR/Cas systems were confined to 46 (24.0%) out of 191 STs assigned to the studied genomes. Most frequently, CRISPR/Cas was found in ST147 (16.5%), ST23 (15.0%), ST15 (12.1%), and ST14 (10.2%). Type I-E CRISPR/Cas was found in 28 STs, most commonly in ST147 (37.0%), ST45 (10.9%), and ST1565 (5.4%), while I-E* was distributed over 18 STs and prevailed in ST23 (27.9%), ST15 (21.6%), and ST14 (18.9%).
More plasmids and resistance genes were carried by the CRISPR/Cas-negative group than others; at least one resistance plasmid was found in 75.8% of the CRISPR/Cas-negative strains versus 69.4% of those carrying CRISPR/Cas. With respect to CRISPR/Cas type, at least one resistance plasmid was carried by 73.9% of the I-E-positive strains versus 66.7% of those carrying I-E*, but their distribution in the two groups was not significantly different.
Furthermore, CRISPR arrays were also found on the plasmids of 33 strains. Of these, 17/33 (51.5%) also carried full CRISPR/Cas systems on their chromosomes. The majority (13/17; 76.5%) had the replicon type [IncHI1B, IncFIB], while other replicon types found included [IncFIA, IncR, IncFIB, IncHI1B], [IncHI1B, IncFIB, IncN], [IncFIB (K), IncFII (K)], and [IncHI1B], each of which was found in one plasmid associated with higher resistance prevalence and more resistance genes.
The prevalence of some resistance genes, such as blaKPC, blaTEM, and rmtB, was significantly higher among the genomes of the CRISPR/Cas-negative strains. Unexpectedly, a weak positive correlation was found between the total number of spacers and the total number of resistance plasmids carried by the CRISPR/Cas-positive isolates (Spearman’s Rho correlation coefficient = 0.033, p-value = 0.633). The same finding was obtained for the correlation between the number of spacers and the total number of ARGs (Spearman’s Rho correlation coefficient = 0.004, p-value = 0.949).
In conclusion, no significant correlation could be established between CRISPR/Cas and susceptibility to antimicrobial agents or bearing resistance plasmids and genes. Plasmid-targeting spacers might not be naturally captured by the CRISPR/Cas system.
There are a lot of reasons why the existence of the CRSPR-Cas systems on the bacterial genome does not always impede the dissemination of ARGs, starting from the adaptation stage. Point mutations and insertion sequence-mediated mutations in the adaptation genes (cas1 and cas2) correlated with the spread of MDR strains. In addition, strong selective pressure for antibiotic resistance may result in CRISPR repression, and many CRISPR-harboring strains may be immunologically inactive owing to the existence of self-targeting spacers, which would be anticipated to induce an autoimmune response via erroneous incorporation of protospacers from the host genome into a CRISPR cassette, resulting in damage to the host chromosome and consequently host cell death via cytotoxic effect.
Furthermore, the CRISPR/Cas systems may be inactivated by phages expressing anti-CRISPR proteins (Acrs), resulting in the dissemination of ARGs. Since matched proto-spacer sequences are required for CRISPR scanning, point mutations in PAM or mismatches between spacer and invader DNA abolish CRISPR interference and significantly reduce the affinity of the cascade-crRNA complex to target DNA, and cleavage does not occur, even though CRISPR exist. In addition to DNA-binding proteins, H-NS proteins bind to the promoter of the cas operon, resulting in a decrease in cas3 expression and, consequently, a loss of CRISPR/Cas immunity. Taken together, these reasons raise the possibility of mobile genetic elements evading CRISPR immunity. Analysis of CRISPR spacers and identification of spacer protospacer matches might provide a clear image of the exact behavior of CRISPR/Cas towards resistance plasmids