In silico analysis reveals the co-existence of CRISPR-Cas type I-F1 and type I-F2 systems and its association with restricted phage invasion in Acinetobacter baumannii.

Introduction: Acinetobacter baumannii, an opportunistic pathogen, rapidly acquires antibiotic resistance, thus compelling researchers to develop alternative treatments at utmost priority. Phage-based therapies are of appreciable benefit; however, CRISPR-Cas systems are a major constraint in this approach. Hence for effective implementation and a promising future of phage-based therapies, a multifaceted understanding of the CRISPR-Cas systems is necessary. Methods: This study investigated 4,977 RefSeq genomes of A. baumannii from the NCBI database to comprehend the distribution and association of CRISPR-Cas systems with genomic determinants. Results: Approximately 13.84% (n = 689/4,977) isolates were found to carry the CRSIPR-Cas system, and a small fraction of isolates, 1.49% (n = 74/4,977), exhibited degenerated CRISPR-Cas systems. Of these CRISPR-Cas positive (+) isolates, 67.48% (465/689) isolates harbored type I-F1, 28.59% (197/689) had type I-F2, and 3.7% (26/689) had co-existence of both type I-F1 and type I-F2 systems. Co-existing type I-F1 and type I-F2 systems are located distantly (∼1.733 Mb). We found a strong association of CRISPR-Cas systems within STs for type I-F1 and type I-F2, whereas the type I-F1 + F2 was not confined to any particular ST. Isolates with type I-F1 + F2 exhibited a significantly high number of mean spacers (n = 164.58 ± 46.41) per isolate as compared to isolates with type I-F2 (n = 82.87 ± 36.14) and type I-F1 (n = 54.51 ± 26.27) with majority targeting the phages. Isolates with type I-F1 (p < 0.0001) and type I-F2 (p < 0.0115) displayed significantly larger genome sizes than type I-F1 + F2. A significantly reduced number of integrated phages in isolates with co-existence of type I-F1 + F2 compared with other counterparts was observed (p = 0.0041). In addition, the isolates carrying type I-F1 + F2 did not exhibit reduced resistance and virulence genes compared to CRISPR-Cas(-) and CRISPR-Cas (+) type I-F1 and type I-F2, except for bap, abaI, and abaR. Conclusion: Our observation suggests that the co-existence of type I-F1 and F2 is more effective in constraining the horizontal gene transfer and phage invasion in A. baumannii than the isolates exhibiting only type I-F1 and only type I-F2 systems.

Colistin Resistance Among Multiple Sequence Types of Klebsiella pneumoniae Is Associated With Diverse Resistance Mechanisms: A Report From India.

Background: The resistance to colistin and carbapenems in Klebsiella pneumoniae infections have been associated with increased morbidity and mortality worldwide. A retrospective observational study was conducted to determine the prevalence and molecular events contributing to colistin resistance. Methods: Clinical samples were screened for colistin resistance and underlying mechanisms were studied by PCR-based amplification and sequence analysis of genes of two-component regulatory system (phoPQ and pmrAB), regulatory transmembrane protein-coding mgrB, and mobilized colistin resistance genes (mcr-1-8). Gene expression of pmrC and pmrK was analyzed by qRT-PCR, and the genetic relationship was assessed by MLST. The putative effect of amino-acid substitutions was predicted by a combination of bioinformatics tools. Results: Of 335 Klebsiella spp. screened, 11 (3.2%) were identified as colistin-resistant (MIC range, 8 to >128 µg/ml). K. pneumoniae isolates belonged to clonal complex-11 (CC11) with sequence types (STs): 14, 16, 43, 54, 147 and 395, whereby four isolates conferred three novel STs (3986, 3987 and 3988) profiles. Sequence analysis revealed non-synonymous potentially deleterious mutations in phoP (T151A), phoQ (del87-90, del263-264, L30Q, and A351D), pmrA (G53S), pmrB (D150V, T157P, L237R, G250C, A252G, R315P, and Q331H), and mgrB (C28G) genes. The mgrB gene in three strains was disrupted by insertion sequences encoding IS1-like and IS5/IS1182 family-like transposase genes. All 11 isolates showed an elevation in the transcription level of pmrC gene. Mobilized colistin-resistance (mcr) genes were not detected. All but one of the colistin-resistant isolates was also resistant to carbapenems; ß-lactamase genes blaNDM-1-like , blaOXA-48-like , and blaCTX-M-like were detected in eight, five, and nine isolates, respectively. Conclusion: All the studied colistin- and carbapenem-resistant K. pneumoniae isolates were genetically distinct, and various mechanisms of colistin resistance were detected, indicating its spontaneous emergence in this bacterial species.