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Association of CRISPR/Cas System with the Drug Resistance in Klebsiella pneumoniae

Background Klebsiella pneumoniae is a common opportunistic pathogen and its production of extended-spectrum β-lactamases (ESBL) and carbapenemases leads to drug resistance. Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated genes (Cas) are widespread in the genome of many bacteria and are a defense mechanism against foreign invaders such as plasmids and viruses. Purpose To investigate the prevalence of the CRISPR/Cas system in wild type strains of K. pneumoniae in the hospital and its association with drug resistance. Materials and Methods A total of 136 strains were collected and characterized their susceptibility to antimicrobial agents. The prevalence of CRISPR/Cas system was detected by PCR and DNA sequencing was analyzed by CRISPRFinder. The statistical analysis of the results was performed by SPSS. Results We found that 50/136 (37%) isolates produced ESBL and 30/136 (22%) isolates were resistant to carbapenems. These isolates were liable to be multidrug resistant against β-lactams, quinolones, and aminoglycosides. Among the carbapenem-resistant isolates, blaKPC was the main drug resistance-associated gene and different types of ESBL and AmpC genes were present. Resistance to β-lactams, quinolones, aminoglycosides, tetracyclines, and β-lactams/enzyme inhibitor were higher in absence of the CRISPR/Cas system. Eighteen spacers within the CRISPR arrays matched with the genomes of plasmids or phages, some of which carried drug resistance genes. Conclusion ESBL-producing and carbapenem-resistant K. pneumoniae are more likely to develop multidrug resistance and show an inverse correlation between drug resistance and CRISPR/Cas system. Absence of CRISPR/Cas modules allow for the acquisition of external drug resistance genes.


Introduction
The evolutionary ability of bacteria to adapt to new environments has been favored by the acquisition of genes through horizontal gene transfer (HGT) mechanisms such as plasmid insertion. 1 The clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated proteins (Cas) modules are encoded by most archaea and many bacteria and are the adaptive defence systems against invading genetic elements such as viruses and plasmids. 2 CRISPR arrays are composed of small direct repeats of 21 to 48 base pairs (bp), which are separated by similarly-sized spacer sequences ranging from 26 to 72 bp. 3 Accompanying CRISPR sequences, there are 4-10 Cas genes which are highly conserved and encode the Cas proteins. The CRISPR/Cas system integrates a small piece of foreign DNA from invaders such as plasmids and viruses into their direct repeat sequences and will recognize and degrade the same external DNA elements during future invasions. 4 Overall, the CRISPR/ Cas system mediates immunity to invading genetic elements through a three-step process: adaptation, expression, and interference. During the adaptation stage, short pieces of DNA homologous to virus or plasmid sequences are recognized by a several nucleotides long protospacer adjacent motifs (PAMs) of the CRISPR/Cas system and are integrated into the CRISPR loci with assistance of Cas proteins. The second stage is expression, during which the long primary transcript of a CRISPR locus (pre-crRNA) is generated and processed into short crRNAs. At the third step, the foreign DNA or RNA is targeted and cleaved within the protospacer sequence under guidance of crRNAs in association with Cas protein complexes. 5 For many bacteria, antibiotic resistance is mediated by acquisition of new DNA that is frequently encoded on mobile gene elements from plasmids and transposons. Studies have shown a highly significant inverse correlation between the presence of CRISPR/Cas system and acquired antibiotic resistance in Enterococcus faecalis. 6 Some studies proposed the potential of CRISPR/Cas system to regulate bacterial virulence. In Pseudomonas aeruginosa, CRISPR/Cas system enable modulation of biofilm production, which is an important virulence factor for various pathogenic microorganisms. 7 In Streptococcus pyogenes, the presence of CRISPR/Cas modulates prophage contents and hence its virulence. 8 However, a study on Escherichia coli demonstrated that CRISPR/Cas system seems to ineffectively block the spread of plasmids and antibiotic resistance. 9 K. pneumoniae has emerged as a dominant opportunistic pathogen in hospital environments due to the high rate of antibiotic resistance and high degree of dissemination. 10 The multidrug resistance of this bacterium has been associated with the presence of high molecular weight plasmids. 11 The aim of this study was to detect the prevalence of CRISPR/ Cas in wild type strains of K. pneumoiae and analyze the correlation with the drug resistance patterns.

Statistical Analysis
Chi-squared test and Fisher's exact test were used for the statistical analysis and P < 0.05 was considered as statistical significance. All data were analyzed using SPSS version 25.0. We categorized all isolates into CRISPR/Cas-positive or CRISPR/Cas-negative and compared the distribution of ESBL, AmpC, and carbapenemase genes as well as antimicrobial susceptibility.

Prevalence of CRISPR/Cas System in K. pneumoniae
Using PCR for detecting Cas1, Cas3, CRISPR1, CRISPR2, and CRISPR3, we found that all of the Cas1positive isolates were also positive for Cas3 and had at least one CRISPR array (Figures 1-3). In total, CRISPR/ Cas was detected in 29/136 isolates, of which 14 isolates were CRISPR2-and CRISPR3-positive, 13 isolates were CRISPR2-positive, and 2 isolates were CRISPR3-positive. After the DNA sequencing and analysis by CRISPR Finder, the CRISPR arrays had conserved direct repeats of 28 bp: 5ʹ-GAAACACCCCCACGCATGTGGGGA AGAC-3ʹ for CRISPR2 and 5ʹ-GTCTTCCCCACG CACGTGGGGGTGTTTC-3ʹ for CRISPR3. They were separated by different spacers ranging from 32 to 40 bp. The number of spacers for CRISPR2 arrays varied from 5 to 9 and CRISPR3 arrays contained more spacers from 9 to 14. Among those spacers, 18 unique spacers were revealed to match with the genomes of plasmids or phages ( Table 2).

Discussion
A recent bioinformatic study demonstrated that only 6/52 K. pneumoniae strains with accessible complete or draft genomes were equipped with a complete CRISPR/Cas system, which indicated CRISPR/Cas is not widely distributed in Klebsiella pneumoniae. 3 We found CRISPR/ Cas in 29/136 (21.3%) K. pneumoniae isolates from patient material in the hospital and the prevalence of CRISPR/Cas system was reported to be different varying from 30.7% (54/176) to 12.4% (27/217). 16,17 The number, sequence, and length of CRISPR arrays and the number and sequence of the Cas proteins are highly variable between bacterial species. 2 Direct repeats can be both added and deleted so that CRISPR can evolve while maintaining an identical number of repeats. 18,19 The PCR results confirmed that the CRISPR arrays were at different lengths among different isolates due to the integration of spacers and the altering number of repeat-spacer units. According to a new CRISPR/Cas classification, CRISPR/ Cas systems are divided into two classes and six types (class 1, including type I, III, IV and class 2, including types II, V and VI). 2 The classical type I CRISPR/Cas is prevalent in bacteria, which can be divided into several subtypes. Several studies demonstrated that CRISPR/Cas systems in K. pneumoniae are classified as type I-E and I-E*. 3,20 In addition to highly conserved Cas1 and Cas2, Cas3 functions as an indispensable part of type I CRISPR/ Cas and has separate helicase and DNase activities. 21 Both type I-E and I-E* share eight common Cas genes comprising of Cas1, Cas2, Cas3, Cas5, Cas6, Cas7, Cse1, and Cse2. Type I-E Cas3 gene location is downstream of Cas7 -Cas5, which moves upstream of Cas7 -Cas5 in type I-E*. 22 In our study, we detected that K. pneumoniae isolates possess the CRISPR/Cas system based on the PCR data where Cas1 and Cas3 are present in conjunction with CRISPR arrays. Among our 136 isolates, Cas1 and Cas3 were co-expressed in 29 isolates, of which 27 isolates were CRISPR2-positive and 16 isolates were CRISPR3-positive. As described previously, the 29 isolates were classified into subtype I-E* CRISPR/Cas system. 15 We compared the resistance rate of K. pneumoniae to different antimicrobial drugs based on the presence of CRISPR/Cas. Our data showed that isolates carrying this system had a lower resistance to β-lactams, quinolones, aminoglycosides, tetracyclines, and β-lactams/enzyme inhibitors. This suggests an inverse correlation between the presence of CRISPR/Cas and antibiotic resistance in   wild type strains of K. pneumoniae. All the carbapenemresistant isolates harbored blaKPC gene and AmpC or ESBL genes, which suggests that these were prone to be multidrug resistant on a genetic level. Based on the detection of seven drug resistance-associated genes, we found no statistical difference of the distribution of ESBL genes and the CRISPR/Cas system. However, blaKPC, blaFOX, and blaDHA were not detected in CRISPR/Cas-positive isolates, whether CRISPR/Cas was present significantly affected the distribution of AmpC and carbapenem genes. A previous study revealed that deletion of CRISPR/Cas cassette in K. pneumoniae increased the transformation success of blaKPC plasmids, indicating that CRISPR/Cas system mediated resistance to blaKPC plasmid invasion. 15 A study revealed that decreased numbers of plasmids, phages and acquired drug resistance genes in the presence of CRISPR/Cas within the genomes of 97 K. pneumoniae. The spacers hit plasmid and phage were identified as matching with the genes of phage-related proteins. 17 In our study, most of spacers matched the K. pneumoniae chromosomes and 18 unique spacers were identified matching the identity of plasmids or phages, which indicated the immunity of CRISPR/Cas system against mobile genetic elements. In addition, some of those matched foreign DNA elements that carry drug resistance genes such as blaKPC. However, it was similar to the reported finding that no plasmid-or phage-targeted spacers were identified as matching any drug resistance genes. Moreover, the two spacers (5ʹ-CCGCCGTTTAATC GCGGTGATGATATCCGGCA-3ʹ; 5ʹ-TGCCGGATATC ATCACCGCGATTAAACGGCGG-3ʹ) that exist within the 25 CRISPR/Cas-positive strains matched different plasmids, which carry the genes mediating resistance to carbapenems and cephalosporins. These results demonstrated that the CRISPR/Cas system may interfere with the acquired drug resistance of K. pneumoniae to carbapenems and cephalosporins, especially for multidrug-resistant strains, which have more mobile elements linked to drug resistance genes like integrons. 23 Further studies are needed to explore the interaction of CRISPR/Cas with mobile genetic elements related to drug resistance. Above all, the CRISPR/Cas system may have a very specific function to interfere with the horizontal gene transfer of drug resistance genes in K. pneumoniae.

Conclusion
The CRISPR/Cas is an adaptive defense system against foreign genetic elements and is not widely distributed in K. pneumoniae. The presence of subtype I-E* CRISPR/Cas system is associated with lower drug resistance. The spacers is a evidence that foreign genetic elements are targeted by the CRISPR/Cas system, which implies that the CRISPR arrays to some extent may prevent the acquisition of drug resistance genes in K. pneumoniae.

Ethical Approval
There is no ethical concern in this study, which was approved by The Medical Ethics Committee of the First Affiliated Hospital of Anhui Medical University. The written informed consent was obtained from patients in accordance with the Declaration of Helsinki.