1 | Introduction
Klebsiella variicola ( K. variicola), a Gram-negative and facultative anaerobic bacillus, belongs to theKlebsiella genus of the Enterobacteriaceae family [1]. Such bacteria have been found in human, animals, insects, plants, and environments [2-4]. K. variicola infections can cause a series of human diseases, such as bloodstream infections, respiratory tract infections, and neonatal sepsis [5-7]. Moreover, bloodstream infections due to K. variicola harbored a higher 30-day mortality rate than its notorious relative,Klebsiella pneumoniae (K. pneumoniae ) [8]. Notably, most cases caused by K. variicola infections were often misidentified as being infected withK. pneumoniae because of their high similarities in biochemical and phenotypic features, which leads to the underestimation of the real prevalence of K. variicola infections in clinical practice [9]. Additionally, K. variicola could cause bovine mastitis and affect the milk production and quality [10]. With the misuse and abuse of antibiotics in clinical and agriculture environments, the last few years has witnessed the rapid increase of multidrug-resistant (MDR) K. variicola strains [11-13]. Accumulating evidence has reported that MDR K. variicola strains were detected in several countries, such as USA, Australia, Tanzania, Germany and China [3, 14]. Horizontal gene transfer (HGT) is deemed as the main mechanism for the transmission of antibiotic resistance genes (ARGs), in which plasmid serves a vector role. It has been documented that K. variicola harbored an open genome and shared similar plasmid types with other members of theKlebsiella genus, especially K. pneumoniae, thereby indicating frequent plasmid-mediated HGT among these species [1, 15]. In particular, ARGs-related IncFIBk, IncFIIk and IncFII replicons were often shared betweenK. variicola and K. pneumoniae [16, 17].
The clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR-associated genes constitute an RNA-guided adaptive immunity system (CRISPR/Cas system), which protects bacteria against HGT of mobile genetic elements (MGEs), such as plasmids and phages [18]. CRISPR/Cas system is composed of three main parts: i) CRISPR loci that consist of several non-contiguous direct repeats (DRs) separated by stretches of variable sequences called as spacers; ii) a group of cas genes essential for adaptive immunity; iii) the leader sequence that acts as the promoter [19]. Generally, the CRISPR/Cas system operates in three stages: adaptation, expression, and interference [20]. During adaptation, a fragment of foreign DNA is captured and integrated into CRISPR array in an ordinal manner, thus forming a new R-S unit. Subsequently, the R-S unit is transcribed into precursor CRISPR RNA (pre-crRNA), which is then processed to produce a mature CRISPR RNA (crRNA). Upon subsequent infection, the crRNA targets and recognizes complementary DNA sequence to degrade exogenous genes with the guide of Cas protein. According to the content of cas genes, two classes, six types, and over 45 subtypes of CRISPR/Cas systems have been identified [21]. Class I CRISPR/Cas systems (type I, III, and IV) rely on heteromeric multi-protein effector complexes, whereas class 2 systems (type II, V, and VI) depend on single multi-domain effector proteins, such as Cas9, Cpf1, and C2c2 [22]. In addition to the adaptive immunity, CRISPR/Cas system performs several other biological roles, including regulating gene expression, and participating in DNA repair and genomic evolution [23-25]. Several studies have explored the diversity of CRISPR/Cas systems in multiple species, including Bifidobacterium[26], Escherichia coli [27], and even the relatives toK. variicola, K. pneumoniae [28]. It has been demonstrated that K. pneumoniae mainly carried type I-E, I-E* and IV-A CRISPR/Cas systems, which played different roles in the dissemination of antibiotic resistance in K. pneumoniae [29]. AlthoughK. variicola is becoming another species of concern afterK. pneumoniae in Klebsiella genus due to its ability to acquire and spread ARGs, little is known about the role of CRISPR/Cas system in this species.
Here, we employed a genome mining approach to analyze the characterization of the CRISPR/Cas system in K. variicolastrains, explore the association between CRISPR evolution and MLST (multiple locus sequence typing), and discussed the potential role of CRISPR/Cas system in ARGs spread.