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Raza A, Fatima P, Yasmeen B, Rana ZA, Ellakwa DES. From resistance to remedy: the role of clustered regularly interspaced short palindromic repeats system in combating antimicrobial resistance-a review. NAUNYN-SCHMIEDEBERG'S ARCHIVES OF PHARMACOLOGY 2025; 398:2259-2273. [PMID: 39404843 DOI: 10.1007/s00210-024-03509-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/30/2024] [Accepted: 10/01/2024] [Indexed: 03/19/2025]
Abstract
The growing challenge of antimicrobial resistance (AMR) poses a significant and increasing risk to public health worldwide, necessitating innovative strategies to restore the efficacy of antibiotics. The precise genome-editing abilities of the CRISPR-Cas system have made it a potent instrument for directly targeting and eliminating antibiotic resistance genes. This review explored the mechanisms and applications of CRISPR-Cas systems in combating AMR. The latest developments in CRISPR technology have broadened its potential use, encompassing programmable antibacterial agents and improved diagnostic methods for antibiotic-resistant infections. Nevertheless, several challenges must be overcome for clinical success, including the survival of resistant bacteria, generation of anti-CRISPR proteins that reduce effectiveness, and genetic modifications that change target sequences. Additionally, the efficacy of CRISPR-Cas systems differs across bacterial species, making their universal application challenging. After overcoming these challenges, CRISPR-Cas has the potential to revolutionize AMR treatment, restore antibiotic efficacy, and reshape infection control.
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Affiliation(s)
- Ali Raza
- Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Turkey.
| | - Pakiza Fatima
- Department of Wildlife & Ecology, Faculty of Fisheries and Wildlife, University of Veterinary and Animal Sciences, Lahore, Pakistan
| | - Bushra Yasmeen
- Department of Wildlife & Ecology, Faculty of Fisheries and Wildlife, University of Veterinary and Animal Sciences, Lahore, Pakistan
| | - Zulqarnain Amjad Rana
- Faculty of Veterinary Science, Khan Bahadar Choudhry Mushtaq Ahmed College of Veterinary and Animal Sciences, Narowal, Pakistan
| | - Doha El-Sayed Ellakwa
- Department of Biochemistry and Molecular Biology, Faculty of Pharmacy for Girls, Al-Azhar University, Cairo, Egypt.
- Department of Biochemistry, Faculty of Pharmacy, Sinai University, Kantra Branch, Ismailia, Egypt.
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Li X, Wang L, Lin J, Gu Y, Liu Z, Hu J. Detection of CRISPR‒Cas and type I R-M systems in Klebsiella pneumoniae of human and animal origins and their relationship to antibiotic resistance and virulence. Microbiol Spectr 2025; 13:e0000924. [PMID: 39699265 PMCID: PMC11792477 DOI: 10.1128/spectrum.00009-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2024] [Accepted: 10/31/2024] [Indexed: 12/20/2024] Open
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)‒CRISPR-associated protein (Cas) and restriction‒modification (R-M) systems are important immune systems in bacteria. Information about the distributions of these two systems in Klebsiella pneumoniae from different hosts and their mutual effect on antibiotic resistance and virulence is still limited. In this study, the whole genomes of 520 strains of K. pneumoniae from GenBank, including 325 from humans and 195 from animals, were collected for CRISPR‒Cas systems and type I R-M systems, virulence genes, antibiotic resistance genes, and multilocus sequence typing detection. The results showed that host origin had no obvious influence on the distributions of the two systems (CRISPR‒Cas systems in 29.8% and 24.1%, type I R-M systems in 9.8% and 11.8% of human-origin and animal-origin strains, respectively) in K. pneumoniae. Identical spacer sequences from different hosts demonstrated there was a risk of human-animal transmission. All virulence genes (yersiniabactin, colibactin, aerobactin, salmochelin, rmpADC, and rmpA2) detection rates were higher when only the CRISPR‒Cas systems were present but were all reduced when coexisting with type I R-M systems. However, a lower prevalence of most antibiotic-resistance genes was found when the CRISPR‒Cas systems were alone, and when type I R-M systems were coexisting, some of the antibiotic resistance gene incidence rates were even lower (quinolones, macrolides, tetracyclines and carbapenems), and some of them were higher instead (aminoglycosides, clindamycins, rifampicin-associated, sulfonamides, methotrexates, beta-lactamases and ultrabroad-spectrum beta-lactamases). The synergistic and opposed effects of the two systems on virulence and antibiotic-resistance genes need further study.IMPORTANCEK. pneumoniae is an important opportunistic pathogen responsible for both human and animal infections, and the emergence of hypervirulent and multidrug-resistant K. pneumoniae has made it difficult to control this pathogen worldwide. Here, we find that CRISPR‒Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, have synergistic and opposed effects on virulence and antibiotic resistance genes in K. pneumoniae. Moreover, this study provides insights into the distributions of the two systems in K. pneumoniae from different hosts, and there is no significant difference in the prevalence of the two systems among K. pneumoniae spp. In addition, this study also characterizes the CRISPR arrays of K. pneumoniae from different hosts, suggesting that the strains sharing the same spacer sequences have the potential to spread between humans and animals.
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Affiliation(s)
- Xue Li
- Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China
| | - Ling Wang
- Department of Nosocomial Infection Administration, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China
| | - Jinghuan Lin
- Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China
| | - Yingjuan Gu
- Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China
| | - Zhihua Liu
- Department of Infectious Disease, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
| | - Jing Hu
- Department of Nosocomial Infection Administration, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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Kadkhoda H, Gholizadeh P, Ghotaslou R, Nabizadeh E, Pirzadeh T, Ahangarzadeh Rezaee M, Feizi H, Samadi Kafil H, Aghazadeh M. Role of CRISPR-cas system on virulence traits and carbapenem resistance in clinical Klebsiella pneumoniae isolates. Microb Pathog 2025; 199:107151. [PMID: 39615707 DOI: 10.1016/j.micpath.2024.107151] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2024] [Revised: 11/13/2024] [Accepted: 11/22/2024] [Indexed: 12/08/2024]
Abstract
BACKGROUND AND OBJECTIVES The bacterial adaptive immune system known as CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated protein) is engaged in defense against various mobile genetic elements (MGEs) such as plasmids and bacteriophages. The purpose of this study was to characterize the CRISPR-Cas systems in carbapenem-resistant Klebsiella pneumoniae isolates and assess any possible correlation between these systems with antibiotic susceptibility, biofilm formation, and bacterial virulence. MATERIALS AND METHODS A total of 156 CRKP isolates were collected from different specimens of the inpatients. Biofilm formation and antibiotic susceptibility testing were evaluated using standard methods. Furthermore, the CRISPR-Cas system subtype genes, 11 carbapenemase genes, and 17 virulence genes were identified using separate standard PCR reactions. The diversity of the isolates was determined by random amplified polymorphic DNA (RAPD)-PCR. RESULTS The development of biofilms and antibiotic susceptibility of several CRKP isolates were significantly correlated with the absence or presence of the CRISPR-Cas system. PCR analysis of carbapenemase genes revealed that the frequency of the blaNDM-1 gene was significantly higher in the isolates with the subtype I-E CRISPR-Cas system. Moreover, the isolates with the subtype I-E CRISPR-Cas system exhibited a propensity to possess more virulence genes such as allS, k2A, wcaG, aerobactin, rmpA, iroN, magA, rmpA2, kfu, iutA, iucB, ybtS, repA, and terW. CONCLUSION CRISPR-Cas systems could affect the antibiotic susceptibility, capacity for biofilm formation, and virulence of Klebsiella pneumoniae. Our findings showed that the isolates containing the CRISPR-Cas system were moderate or strong biofilm producers and had a higher frequency of virulence genes.
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Affiliation(s)
- Hiva Kadkhoda
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Pourya Gholizadeh
- Zoonoses Research Center, Ardabil University of Medical Sciences, Ardabil, Iran
| | - Reza Ghotaslou
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran; Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Edris Nabizadeh
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Tahereh Pirzadeh
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mohammad Ahangarzadeh Rezaee
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran; Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Hadi Feizi
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Medical Microbiology, Aalinasab Hospital, Social Security Organization, Tabriz, Iran
| | - Hossein Samadi Kafil
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran; Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
| | - Mohammad Aghazadeh
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran; Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
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Owaid HA, Al-Ouqaili MT. Molecular characterization and genome sequencing of selected highly resistant clinical isolates of Pseudomonas aeruginosa and its association with the clustered regularly interspaced palindromic repeat/Cas system. Heliyon 2025; 11:e41670. [PMID: 39866497 PMCID: PMC11761341 DOI: 10.1016/j.heliyon.2025.e41670] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2024] [Revised: 12/21/2024] [Accepted: 01/02/2025] [Indexed: 01/28/2025] Open
Abstract
The presence of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in the superbug Pseudomonas (P) aeruginosa presents a unique opportunity to precisely target and edit bacterial genomes to modify their drug resistance. The objective was to detect the prevalence of CRISPR in extensively and pan-drug-resistant Pseudomonas aeruginosa and to determine the utility of whole-genome sequencing (WGS) for the analysis of the entire genome for such strains. The antimicrobial susceptibilities of one hundred isolates were assessed using the antibiotic susceptibility test (AST) card of the VITEK system. The presence of the CRISPR/Cas system was determined via specific primers using conventional polymerase chain reaction (PCR). Further, WGS was conducted using a DNA nanoball sequencing platform via BGI-Tech for the isolates of interest. Out of 54 resistant Pseudomonas aeruginosa isolates, 33 (33.0 %) were metallo-β-lactamase producers. Cas1, Cas3, CRISPR1, and CRISPR2 were positive in 6.0 % of isolates, while incomplete CRISPR1-Cas systems alone were found in 15.0 %. Also, CRISPR2-type was found intact in 26 % of isolates. The prevalence of resistance to antimicrobials in P. aeruginosa isolates was significantly greater in the CRISPR/Cas-negative group compared to the CRISPR/Cas-positive. Significant relationships for variables were examined using Fisher's exact tests using Chi-squared and a P-value of <0.05 as a statistical threshold. Further, on examination of CRs as a collective entity, encompassing both extensive drug resistance (XDR) and pan-drug resistance (PDR), it becomes evident that the vast majority of these strains (n = 29; 87.8 %) lacked CRISPR/Cas systems. In phylogenic analysis, PDR-P. aeruginosa revealed a very close evolutionary relationship with those originating from Kazakhstan, while XDR was globally unique. Further, the entire genome showed the presence of unique virulence and resistant pseudomonal genes. The CRISPR/Cas system and drug resistance are antagonistic to one another. XDR and PDR P. aeruginosa represent a potential threat to public health and contribute to the seriousness of associated illnesses by leading to resistant infections. Further, WGS for the two strains revealed resistance to multiple antibiotics. It is important to examine specific antimicrobial resistance (AMR) pathways, which suggests that a significant number of resistant genes in these isolates indicate a loss of CRISPR genes in the two strains. Furthermore, the WGS approach can lead to a better understanding of the genomic mechanism of pseudomonal resistance to antibiotics.
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Affiliation(s)
- Hekmat A. Owaid
- Department of Biology, College of Science, University of Anbar, Ramadi, Iraq
| | - Mushtak T.S. Al-Ouqaili
- Department of Microbiology, College of Medicine, University of Anbar, Anbar Governorate, Ramadi, Iraq
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Montazeri EA, Saki M, Savari M, Meghdadi H, Akrami S. Association between the presence of CRISPR-Cas system genes and antibiotic resistance in Klebsiella pneumoniae isolated from patients admitted in Ahvaz teaching hospitals. BMC Infect Dis 2024; 24:1117. [PMID: 39375619 PMCID: PMC11460096 DOI: 10.1186/s12879-024-10018-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2024] [Accepted: 09/30/2024] [Indexed: 10/09/2024] Open
Abstract
BACKGROUND This study aims to investigate the frequency of cas1 and cas3 and CRISPR1,2,3 genes in Klebsiella pneumoniae isolates, as well as their connection with antibiotic resistance. MATERIALS AND METHODS 106 K. pneumoniae isolates were identified by biochemical assays and PCR. The susceptibility to antibiotics was determined by Kirby-Bauer disk diffusion method. Screening of ESBLs was undertaken by using double disk diffusion and standard disk diffusion methods. The E-test and mCIM techniques was used to confirm the disc diffusion-based carbapenem resistance profiles. CRISPR-Cas system genes were identified using PCR. RESULTS ESBL production was found in 19% of isolates. Carbapenemase production was found in 46% of the isolates. Furthermore, the bacteria were classified as multidrug (76%), extensively drug-resistant (4%), or pan-drug-resistant (2%). When CRISPR/Cas systems were present, antibiotic resistance was lower; conversely, when they were absent, resistance was higher. CONCLUSIONS If the CRISPR/Cas modules aren't present, the bacteria can still acquire foreign DNA, including antibiotic resistance genes. K. pneumoniae isolates with a CRISPR-Cas system were less likely to carry antibiotic-resistance genes than those lacking this defense system.
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Affiliation(s)
- Effat Abbasi Montazeri
- Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
- Department of Microbiology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Morteza Saki
- Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
- Department of Microbiology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Mohammad Savari
- Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
- Department of Microbiology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Hossein Meghdadi
- Department of Microbiology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Sousan Akrami
- Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
- Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
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Bhalla N, Nanda RK. Pangenome-wide association study reveals the selective absence of CRISPR genes (Rv2816c-19c) in drug-resistant Mycobacterium tuberculosis. Microbiol Spectr 2024; 12:e0052724. [PMID: 38916315 PMCID: PMC11302280 DOI: 10.1128/spectrum.00527-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2024] [Accepted: 05/31/2024] [Indexed: 06/26/2024] Open
Abstract
The presence of intermittently dispersed insertion sequences and transposases in the Mycobacterium tuberculosis (Mtb) genome makes intra-genome recombination events inevitable. Understanding their effect on the gene repertoires (GR), which may contribute to the development of drug-resistant Mtb, is critical. In this study, publicly available WGS data of clinical Mtb isolates (endemic region n = 2,601; non-endemic region n = 1,130) were de novo assembled, filtered, scaffolded into assemblies, and functionally annotated. Out of 2,601 Mtb WGS data sets from endemic regions, 2,184 (drug resistant/sensitive: 1,386/798) qualified as high quality. We identified 3,784 core genes, 123 softcore genes, 224 shell genes, and 762 cloud genes in the pangenome of Mtb clinical isolates from endemic regions. Sets of 33 and 39 genes showed positive and negative associations (P < 0.01) with drug resistance status, respectively. Gene ontology clustering showed compromised immunity to phages and impaired DNA repair in drug-resistant Mtb clinical isolates compared to the sensitive ones. Multidrug efflux pump repressor genes (Rv3830c and Rv3855c) and CRISPR genes (Rv2816c-19c) were absent in the drug-resistant Mtb. A separate WGS data analysis of drug-resistant Mtb clinical isolates from the Netherlands (n = 1130) also showed the absence of CRISPR genes (Rv2816c-17c). This study highlights the role of CRISPR genes in drug resistance development in Mtb clinical isolates and helps in understanding its evolutionary trajectory and as useful targets for diagnostics development.IMPORTANCEThe results from the present Pan-GWAS study comparing gene sets in drug-resistant and drug-sensitive Mtb clinical isolates revealed intricate presence-absence patterns of genes encoding DNA-binding proteins having gene regulatory as well as DNA modification and DNA repair roles. Apart from the genes with known functions, some uncharacterized and hypothetical genes that seem to have a potential role in drug resistance development in Mtb were identified. We have been able to extrapolate many findings of the present study with the existing literature on the molecular aspects of drug-resistant Mtb, further strengthening the relevance of the results presented in this study.
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Affiliation(s)
- Nikhil Bhalla
- Translational Health Group, International Center of Genetic Engineering and Biotechnology, New Delhi, India
| | - Ranjan Kumar Nanda
- Translational Health Group, International Center of Genetic Engineering and Biotechnology, New Delhi, India
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Kadkhoda H, Gholizadeh P, Samadi Kafil H, Ghotaslou R, Pirzadeh T, Ahangarzadeh Rezaee M, Nabizadeh E, Feizi H, Aghazadeh M. Role of CRISPR-Cas systems and anti-CRISPR proteins in bacterial antibiotic resistance. Heliyon 2024; 10:e34692. [PMID: 39149034 PMCID: PMC11325803 DOI: 10.1016/j.heliyon.2024.e34692] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2024] [Revised: 07/08/2024] [Accepted: 07/15/2024] [Indexed: 08/17/2024] Open
Abstract
The emergence and development of antibiotic resistance in bacteria is a serious threat to global public health. Antibiotic resistance genes (ARGs) are often located on mobile genetic elements (MGEs). They can be transferred among bacteria by horizontal gene transfer (HGT), leading to the spread of drug-resistant strains and antibiotic treatment failure. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated genes) is one of the many strategies bacteria have developed under long-term selection pressure to restrict the HGT. CRISPR-Cas systems exist in about half of bacterial genomes and play a significant role in limiting the spread of antibiotic resistance. On the other hand, bacteriophages and other MGEs encode a wide range of anti-CRISPR proteins (Acrs) to counteract the immunity of the CRISPR-Cas system. The Acrs could decrease the CRISPR-Cas system's activity against phages and facilitate the acquisition of ARGs and virulence traits for bacteria. This review aimed to assess the relationship between the CRISPR-Cas systems and Acrs with bacterial antibiotic resistance. We also highlighted the CRISPR technology and Acrs to control and prevent antibacterial resistance. The CRISPR-Cas system can target nucleic acid sequences with high accuracy and reliability; therefore, it has become a novel gene editing and gene therapy tool to prevent the spread of antibiotic resistance. CRISPR-based approaches may pave the way for developing smart antibiotics, which could eliminate multidrug-resistant (MDR) bacteria and distinguish between pathogenic and beneficial microorganisms. Additionally, the engineered anti-CRISPR gene-containing phages in combination with antibiotics could be used as a cutting-edge treatment approach to reduce antibiotic resistance.
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Affiliation(s)
- Hiva Kadkhoda
- Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Pourya Gholizadeh
- Digestive Disease Research Center, Ardabil University of Medical Sciences, Ardabil, Iran
- Zoonoses Research Center, Ardabil University of Medical Sciences, Ardabil, Iran
| | - Hossein Samadi Kafil
- Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
- Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Reza Ghotaslou
- Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
- Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Tahereh Pirzadeh
- Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
- Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mohammad Ahangarzadeh Rezaee
- Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
- Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Edris Nabizadeh
- Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Hadi Feizi
- Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
- Department of Medical Microbiology, Aalinasab Hospital, Social Security Organization, Tabriz, Iran
| | - Mohammad Aghazadeh
- Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
- Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
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Owaid HA, Al-Ouqaili MT. Molecular and bacteriological investigations for the co-existence CRISPR/Cas system and β-lactamases of types extended-spectrum and carbapenemases in Multidrug, extensive drug and Pandrug-Resistant Klebsiella pneumoniae. Saudi J Biol Sci 2024; 31:104022. [PMID: 38817398 PMCID: PMC11137337 DOI: 10.1016/j.sjbs.2024.104022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2024] [Revised: 05/05/2024] [Accepted: 05/10/2024] [Indexed: 06/01/2024] Open
Abstract
The recent approach towards combating the antimicrobial resistance has led to the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated sequence to overcome the challenges of antimicrobial resistance. Thus, this study aimed to detect the underlying resistance mechanisms such as ESBLs and carbapenemases and whether there is a correlation between multidrug, extensive drug and pan drug resistance and the occurrence of CRISPR loci. A total of one hundred study isolates were subjected to antimicrobial susceptibility testing using the AST card of the Vitek technique to detect resistance patterns involving ESBLs and carbapenemase (CRE). An investigation of the genes encoding CRISPR/Cas systems using PCR was achieved. Out of 81 (81.0%) resistant Klebsiella pneumoniae isolates, 71 (71%) and 21 (21.0%) produced ESBLs and carbapenemases, respectively. Also, 53 (53.0%), 19 (19.0%) and 9 (9.0%) were MDR, XDR, and PDR respectively. It was noted that Cas1, Cas3, CRISPR1, CRISPR2 and CRISPR3 were positive in 38 (38.0%) of the isolates, while CRISPR1 for incomplete CRISPR1-Cas systems alone was detected in 78 (78.0%). Further, the number of intact CRISPR1, intact CRISPR2 and intact CRISPR3 types were 7 (27.0%), 34 (34%) and 18 (18.0%) respectively. It is concluded that antibiotic resistance levels were inversely correlated with the existence of CRISPR/Cas systems. The absence of the CRISPR/Cas system increases the prevalence of MDR, XDR and PDR in ESBL and carbapenem-producing Klebsiella pneumoniae. With the increase in the degree of antibiotic resistance (MDR, XDR to PDR), the occurrence ratio of the (CRISPR)/CRISPR-associated sequence decreased.
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Affiliation(s)
- Hekmat A. Owaid
- Department of Biology, College of Science, University of Anbar, Ramadi, Iraq
| | - Mushtak T.S. Al-Ouqaili
- Department of Microbiology, College of Medicine, University of Anbar, Al-Anbar Governorate, Ramadi, Iraq
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Kadkhoda H, Gholizadeh P, Ghotaslou R, Pirzadeh T, Ahangarzadeh Rezaee M, Nabizadeh E, Feizi H, Samadi Kafil H, Aghazadeh M. Prevalence of the CRISPR-cas system and its association with antibiotic resistance in clinical Klebsiella pneumoniae isolates. BMC Infect Dis 2024; 24:554. [PMID: 38831286 PMCID: PMC11149351 DOI: 10.1186/s12879-024-09451-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2024] [Accepted: 05/30/2024] [Indexed: 06/05/2024] Open
Abstract
BACKGROUND AND OBJECTIVE(S) CRISPR-Cas is a prokaryotic adaptive immune system that protects bacteria and archaea against mobile genetic elements (MGEs) such as bacteriophages plasmids, and transposons. In this study, we aimed to assess the prevalence of the CRISPR-Cas systems and their association with antibiotic resistance in one of the most challenging bacterial pathogens, Klebsiella pneumoniae. MATERIALS AND METHODS A total of 105 K. pneumoniae isolates were collected from various clinical infections. Extended-spectrum β-lactamases (ESBLs) phenotypically were detected and the presence of ESBL, aminoglycoside-modifying enzymes (AME), and CRISPR-Cas system subtype genes were identified using PCR. Moreover, the diversity of the isolates was determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR. RESULTS Phenotypically, 41.9% (44/105) of the isolates were found to be ESBL producers. A significant inverse correlation existed between the subtype I-E CRISPR-Cas system's presence and ESBL production in K. pneumoniae isolates. Additionally, the frequency of the ESBL genes blaCTX-M1 (3%), blaCTX-M9 (12.1%), blaSHV (51.5%), and blaTEM (33.3%), as well as some AME genes such as aac(3)-Iva (21.2%) and ant(2'')-Ia (3%) was significantly lower in the isolates with the subtype I-E CRISPR-Cas system in comparison to CRISPR-negative isolates. There was a significant inverse correlation between the presence of ESBL and some AME genes with subtype I-E CRISPR-Cas system. CONCLUSION The presence of the subtype I-E CRISPR-Cas system was correlated with the antibiotic-resistant gene (ARGs). The isolates with subtype I-E CRISPR-Cas system had a lower frequency of ESBL genes and some AME genes than CRISPR-negative isolates.
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Affiliation(s)
- Hiva Kadkhoda
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
- Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Pourya Gholizadeh
- Digestive Disease Research Center, Ardabil University of Medical Sciences, Ardabil, Iran
- Zoonoses Research Center, Ardabil University of Medical Sciences, Ardabil, Iran
| | - Reza Ghotaslou
- Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
- Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Tahereh Pirzadeh
- Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
- Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | | | - Edris Nabizadeh
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
- Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Hadi Feizi
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
- Department of Medical Microbiology, Aalinasab Hospital, Social Security Organization, Tabriz, Iran
| | - Hossein Samadi Kafil
- Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
| | - Mohammad Aghazadeh
- Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
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Shabbir MAB, Ul-Rahman A, Iftikhar MR, Rasheed M, Maan MK, Sattar A, Ahmad M, Khan FA, Ahmad W, Riaz MI, Aslam HB. Exploring the Interplay of the CRISPR-CAS System with Antibiotic Resistance in Staphylococcus aureus: A Poultry Meat Study from Lahore, Pakistan. MEDICINA (KAUNAS, LITHUANIA) 2024; 60:130. [PMID: 38256391 PMCID: PMC10818619 DOI: 10.3390/medicina60010130] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/19/2023] [Revised: 01/02/2024] [Accepted: 01/08/2024] [Indexed: 01/24/2024]
Abstract
Staphylococcus aureus is one of the major pathogens responsible for causing food poisoning worldwide. The emergence of antibiotic resistance in this bacterium is influenced by various factors. Among them, bacterial acquired defense systems described as clustered regularly interspaced short palindromic repeats (CRISPR)-cas system might be involved in antibiotic resistance development in bacteria. The current study was designed to assess the prevalence of S. aureus and its antibiotic resistance profile and identify the relationship of the CRISPR-cas system with antimicrobial resistance, followed by phylogenetic analysis. Total samples (n = 188) of poultry meat were collected from the poultry bird market of Lahore, Punjab, Pakistan. We used both phenotypic (antibiotic disc diffusion) and genotypic methods (PCR) to identify multi-drug resistant (MDR) strains of S. aureus. Additionally, the role of the CRISPR-Cas system in the isolated MDR S. aureus was also assessed. In addition, real-time quantitative PCR (qRT-PCR) was used to evaluate the association of the CRISPR-cas system with antimicrobial resistance. All of the S. aureus isolates showed 100% resistance against erythromycin, 97.5% were resistant to tetracycline, and 75% were resistant to methicillin. Eleven isolates were MDR in the current study. The CRISPR system was found in all MDR isolates, and fifteen spacers were identified within the CRISPR locus. Furthermore, MDR S. aureus isolates and the standard strain showed higher expression levels of CRISPR-associated genes. The correlation of said system with MDR isolates points to foreign gene acquisition by horizontal transfer. Current knowledge could be utilized to tackle antibiotic-resistant bacteria, mainly S. aureus.
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Affiliation(s)
- Muhammad Abu Bakr Shabbir
- Institute of Microbiology, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan (F.A.K.)
| | - Aziz Ul-Rahman
- Department of Pathobiology and Biomedical Sciences, Faculty of Veterinary and Animal Sciences, Muhammad Nawaz Shareef (MNS) University of Agriculture, Multan 66000, Pakistan;
| | - Muhammad Rizwan Iftikhar
- Institute of Microbiology, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan (F.A.K.)
| | - Majeeda Rasheed
- Department of life Sciences, Khwaja Fareed University of Engineering and Information Technology, Rahim Yar Khan 64200, Pakistan;
| | - Muhammad Kashif Maan
- Department of Veterinary Surgery, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan
| | - Adeel Sattar
- Department of Pharmacology and Toxicology, Faculty of Biosciences, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan
| | - Mehmood Ahmad
- Department of Pharmacology and Toxicology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan;
| | - Farid Ahmed Khan
- Institute of Microbiology, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan (F.A.K.)
| | - Waqas Ahmad
- Department of Pathology, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan;
| | - Muhammad Ilyas Riaz
- Institute of Microbiology, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan (F.A.K.)
| | - Hassaan Bin Aslam
- Institute of Microbiology, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan (F.A.K.)
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Alkompoz AK, Hamed SM, Zaid ASA, Almangour TA, Al-Agamy MH, Aboshanab KM. Correlation of CRISPR/Cas and Antimicrobial Resistance in Klebsiella pneumoniae Clinical Isolates Recovered from Patients in Egypt Compared to Global Strains. Microorganisms 2023; 11:1948. [PMID: 37630508 PMCID: PMC10459600 DOI: 10.3390/microorganisms11081948] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2023] [Revised: 07/27/2023] [Accepted: 07/28/2023] [Indexed: 08/27/2023] Open
Abstract
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.
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Affiliation(s)
| | - Samira M. Hamed
- Department of Microbiology and Immunology, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), 6th of October, Giza 12451, Egypt;
| | - Ahmed S. Abu Zaid
- Department of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University, Cairo 11566, Egypt;
| | - Thamer A. Almangour
- Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia;
| | - Mohamed H. Al-Agamy
- Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia;
- Department of Microbiology and Immunology, Faculty of Pharmacy, Al-Azhar University, Cairo 11651, Egypt
| | - Khaled M. Aboshanab
- Department of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University, Cairo 11566, Egypt;
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Kannadasan AB, Sumantran VN, Vaidyanathan R. A Global Comprehensive Study of the Distribution of Type I-E and Type I-E* CRISPR-Cas Systems in Klebsiella pneumoniae. Indian J Community Med 2023; 48:567-572. [PMID: 37662134 PMCID: PMC10470566 DOI: 10.4103/ijcm.ijcm_486_22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2022] [Accepted: 05/03/2023] [Indexed: 09/05/2023] Open
Abstract
Background The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems are the short DNA sequences and RNA-dependent nuclease involved in the adaptive immunity in bacteria and archaea. The type of CRISPR-Cas system influences antibiotic susceptibility in Klebsiella pneumoniae. Here, our objective was to study the diversity of CRISPR-Cas system in the genome of K. pneumoniae from the available whole genome sequencing (WGS) data. Material and Methods We identified the CRISPR-Cas systems of K. pneumoniae using the CRISPR-CasFinder database. The complete genome sequence and its submission details were obtained from the National Center for Biotechnology Information (NCBI) database. Results A total of 1607 K. pneumoniae whole genome sequences were analyzed. The major contributors of WGS data of K. pneumoniae were China (26.6%), United States (21.5%), Australia (10%), South Korea (8%), India (5.5%), and United Kingdom (4.9%). Out of 1607 genomes analyzed, almost one-fourth were CRISPR-Cas positive (403/1607) and three-fourth were CRISPR-Cas negative (1204/1607). Among CRISPR-Cas positive strains, 220 belonged to type I-E* and 183 were type I-E. Furthermore, type I-E* CRISPR-Cas systems were significantly higher in Asia (P < 0.001), whereas type I-E were significantly higher in Europe (P < 0.01). Among countries, typically, type I-E* strains were found to be higher in China (P < 0.01) and India (P < 0.01), whereas type I-E strains were higher in Germany (P < 0.01). Conclusion Hence, it is important to know the type of CRISPR-Cas systems in K. pneumoniae strains across the countries and it can help to understand the diversity of CRISPR-Cas systems worldwide.
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Affiliation(s)
- Anand Babu Kannadasan
- Dr. A.P.J. Abdul Kalam Center of Excellence in Innovation and Entrepreneurship, Dr. MGR Educational and Research Institute, Adayalampattu, Chennai, Tamil Nadu, India
| | - Venil Naranan Sumantran
- Dr. A.P.J. Abdul Kalam Center of Excellence in Innovation and Entrepreneurship, Dr. MGR Educational and Research Institute, Adayalampattu, Chennai, Tamil Nadu, India
| | - Rama Vaidyanathan
- Dr. A.P.J. Abdul Kalam Center of Excellence in Innovation and Entrepreneurship, Dr. MGR Educational and Research Institute, Adayalampattu, Chennai, Tamil Nadu, India
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Jwair NA, Al-Ouqaili MTS, Al-Marzooq F. Inverse Association between the Existence of CRISPR/Cas Systems with Antibiotic Resistance, Extended Spectrum β-Lactamase and Carbapenemase Production in Multidrug, Extensive Drug and Pandrug-Resistant Klebsiella pneumoniae. Antibiotics (Basel) 2023; 12:980. [PMID: 37370299 DOI: 10.3390/antibiotics12060980] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2023] [Revised: 05/19/2023] [Accepted: 05/24/2023] [Indexed: 06/29/2023] Open
Abstract
Antimicrobial resistance, with the production of extended-spectrum β-lactamases (ESBL) and carbapenemases, is common in the opportunistic pathogen, Klebsiella pneumoniae. This organism has a genome that can contain clustered regularly interspaced short palindromic repeats (CRISPRs), which operate as a defense mechanism against external invaders such as plasmids and viruses. This study aims to determine the association of the CRISPR/Cas systems with antibiotic resistance in K. pneumoniae isolates from Iraqi patients. A total of 100 K. pneumoniae isolates were collected and characterized according to their susceptibility to different antimicrobial agents. The CRISPR/Cas systems were detected via PCR. The phenotypic detection of ESBLs and carbapenemases was performed. The production of ESBL was detected in 71% of the isolates. Carbapenem-resistance was detected in 15% of the isolates, while only 14% were susceptible to all antimicrobial agents. Furthermore, the bacteria were classified into multidrug (77%), extensively drug-resistant (11.0%) and pandrug-resistant (4.0%). There was an inverse association between the presence of the CRISPR/Cas systems and antibiotic resistance, as resistance was higher in the absence of the CRISPR/Cas system. Multidrug resistance in ESBL-producing and carbapenem-resistant K. pneumoniae occurred more frequently in strains negative for the CRISPR/Cas system. Thus, we conclude that genes for exogenous antibiotic resistance can be acquired in the absence of the CRISPR/Cas modules that can protect the bacteria against acquiring foreign DNA.
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Affiliation(s)
- Noor A Jwair
- Department of Microbiology, College of Medicine, University of Anbar, Ramadi P.O. Box 55431, Iraq
| | - Mushtak T S Al-Ouqaili
- Department of Microbiology, College of Medicine, University of Anbar, Ramadi P.O. Box 55431, Iraq
| | - Farah Al-Marzooq
- Department of Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain P.O. Box 15551, United Arab Emirates
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14
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Hu Y, Jiang J, Wang D, Guo Q, Wang M. Coexistence of blaKPC-IncFII plasmids and type I-E* CRISPR-Cas systems in ST15 Klebsiella pneumoniae. Front Microbiol 2023; 14:1125531. [PMID: 36970694 PMCID: PMC10030501 DOI: 10.3389/fmicb.2023.1125531] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2022] [Accepted: 02/17/2023] [Indexed: 03/11/2023] Open
Abstract
The CRISPR-Cas system in Klebsiella pneumoniae can prevent the entry of blaKPC-IncF plasmids. However, some clinical isolates bear the KPC-2 plasmids despite carrying the CRISPR-Cas system. The purpose of this study was to characterize the molecular features of these isolates. A total of 697 clinical K. pneumoniae isolates were collected from 11 hospitals in China, and tested for the presence of CRISPR-Cas systems using polymerase chain reaction. Overall, 164 (23.5%) of 697 K. pneumoniae isolates had type I-E* (15.9%) or type I-E (7.7%) CRISPR-Cas systems. The most prevalent sequence type among isolates carrying type I-E* CRISPR was ST23 (45.9%), followed by ST15 (18.9%). Isolates with CRISPR-Cas system were more susceptible to ten antimicrobials tested, including carbapenems, compared with the CRISPR-negative isolates. However, there were still 21 CRISPR-Cas-carrying isolates that showed resistance to carbapenems, and these isolates were subjected to whole-genome sequencing. Thirteen of these 21 isolates carried blaKPC-2-bearing plasmids, of which nine had a new plasmid type, IncFIIK34, and two had IncFII(PHN7A8) plasmids. In addition, 12 of these 13 isolates belonged to ST15, while only eight (5.6%, 8/143) isolates belonged to ST15 in carbapenem-susceptible K. pneumoniae carrying CRISPR-Cas systems. In conclusion, we found that blaKPC-2-bearing IncFII plasmids could co-exist with the type I-E* CRISPR-Cas systems in ST15 K. pneumoniae.
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Affiliation(s)
- Yiyi Hu
- Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Clinical Pharmacology of Antibiotics, National Health Commission of People’s Republic of China, Shanghai, China
| | - Jianping Jiang
- Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Clinical Pharmacology of Antibiotics, National Health Commission of People’s Republic of China, Shanghai, China
| | - Dongliang Wang
- The First Department of Critical Care Medicine, Gansu Provincial Hospital, Gansu, China
| | - Qinglan Guo
- Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Clinical Pharmacology of Antibiotics, National Health Commission of People’s Republic of China, Shanghai, China
| | - Minggui Wang
- Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Clinical Pharmacology of Antibiotics, National Health Commission of People’s Republic of China, Shanghai, China
- *Correspondence: Minggui Wang,
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15
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CRISPR in Modulating Antibiotic Resistance of ESKAPE Pathogens. Mol Biotechnol 2023; 65:1-16. [PMID: 35939207 DOI: 10.1007/s12033-022-00543-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2021] [Accepted: 07/13/2022] [Indexed: 01/11/2023]
Abstract
The ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) isolates both from the clinical settings and food products are demonstrated to gain resistance to multiple antimicrobials. Therefore, the ESKAPE pathogens pose a serious threat to public health, which warrants specific attention to developing alternative novel therapeutics. The clustered regularly interspaced short palindromic repeats associated (CRISPR-Cas) system is one of the novel methods for managing antibiotic-resistant strains. Specific Cas nucleases can be programmed against bacterial genomic sequences to decrease bacterial resistance to antibiotics. Moreover, a few CRISPR-Cas nucleases have the ability to the sequence-specific killing of bacterial strains. However, some pathogens acquire antibiotic resistance due to the presence of the CRISPR-Cas system. In brief, there is a wide range of functional diversity of CRISPR-Cas systems in bacterial pathogens. Hence, to be an effective and safe infection treatment strategy, a comprehensive understanding of the role of CRISPR-Cas systems in modulating antibiotic resistance in ESKAPE pathogens is essential. The present review summarizes all the mechanisms by which CRISPR confers and prevents antibiotic resistance in ESKAPE. The review also emphasizes the relationship between CRISPR-Cas systems, biofilm formation, and antibiotic resistance in ESKAPE.
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16
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Tao S, Chen H, Li N, Fang Y, Xu Y, Liang W. Association of CRISPR-Cas System with the Antibiotic Resistance and Virulence Genes in Nosocomial Isolates of Enterococcus. Infect Drug Resist 2022; 15:6939-6949. [PMID: 36474907 PMCID: PMC9719680 DOI: 10.2147/idr.s388354] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2022] [Accepted: 10/26/2022] [Indexed: 04/07/2024] Open
Abstract
Purpose This study aimed to investigate the prevalence of the CRISPR-Cas system in nosocomial isolates of Enterococcus and their possible association with antibiotic resistance and virulence genes. Materials and Methods Identification and antimicrobial susceptibility of the microorganism were performed by the automatized VITEK 2 Compact system (bioMerieux, France). A total of 100 Enterococcus isolates were collected and identified by VITEK 2 Compact automatic microbial identification drug susceptibility analyzer. The prevalence of various CRISPR-Cas systems, antibiotic resistance genes and virulence genes were investigated by polymerase chain reaction (PCR). The prevalence of CRISPR-Cas systems associated with antibiotic resistance and virulence genes was performed by appropriate statistical tests. Results A total of 100 isolates of Enterococcus were identified and there were 62/100(62.0%) Enterococcus faecalis isolates and 38/100(38.0%) Enterococcus faecalis isolates. In total, 46 (46.0%) of 100 isolates had at least one CRISPR-Cas locus. CRISPR elements were more prevalent in Enterococcus faecalis isolates. The results of PCR demonstrated that CRISPR1-Cas, orphan CRISPR2, and CRISPR3-Cas were present in 23 (23.0%), 42 (42.0%) and 5 (5.0%) Enterococcus isolates, respectively. Compared with CRISPR-Casnegative isolates, the CRISPR-Cas positive isolates showed significant lower resistance rates against ampicillin, erythromycin, levofloxacin, tetracycline, vancomycin, gentamicin, streptomycin, and rifampicin. Presumably consistent with drug susceptibility, fewer CRISPR loci were identified in vanA, tetM, ermB, aac6'-aph(2"), aadE, and ant(6) positive isolates. There was a significant negative correlation between the CRISPR-Cas locus and the enterococcal virulence factors enterococcal surface protein (esp) gene. Conclusion In conclusion, the results indicated that the absence of the CRISPR-Cas system was negatively associated with some antibiotic resistance in clinical isolates of Enterococcus faecalis and Enterococcus faecium. Also, there was a negative correlation with the carriage of antibiotic resistance genes. Furthermore, CRISPR-Cas may prevent some isolates from acquiring certain virulence factors.
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Affiliation(s)
- Shuan Tao
- School of Medicine, Jiangsu University, Zhenjiang, People’s Republic of China
- Department of Clinical Laboratory, Ningbo First Hospital, Ningbo, People’s Republic of China
| | - Huimin Chen
- School of Medicine, Jiangsu University, Zhenjiang, People’s Republic of China
| | - Na Li
- Bengbu Medical College, Bengbu, People’s Republic of China
| | - Yewei Fang
- Department of Clinical Laboratory, Ningbo First Hospital, Ningbo, People’s Republic of China
| | - Yao Xu
- School of Medicine, Ningbo University, Ningbo, People’s Republic of China
| | - Wei Liang
- Department of Clinical Laboratory, Ningbo First Hospital, Ningbo, People’s Republic of China
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17
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Iordache D, Baci GM, Căpriță O, Farkas A, Lup A, Butiuc-Keul A. Correlation between CRISPR Loci Diversity in Three Enterobacterial Taxa. Int J Mol Sci 2022; 23:12766. [PMID: 36361556 PMCID: PMC9658729 DOI: 10.3390/ijms232112766] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2022] [Revised: 10/20/2022] [Accepted: 10/20/2022] [Indexed: 11/05/2022] Open
Abstract
CRISPR-Cas is an adaptive immunity system of prokaryotes, composed of CRISPR arrays and the associated proteins. The successive addition of spacer sequences in the CRISPR array has made the system a valuable molecular marker, with multiple applications. Due to the high degree of polymorphism of the CRISPR loci, their comparison in bacteria from various sources may provide insights into the evolution and spread of the CRISPR-Cas systems. The aim of this study was to establish a correlation between the enterobacterial CRISPR loci, the sequence of direct repeats (DR), and the number of spacer units, along with the geographical origin and collection source. For this purpose, 3474 genomes containing CRISPR loci from the CRISPRCasdb of Salmonella enterica, Escherichia coli, and Klebsiella pneumoniae were analyzed, and the information regarding the isolates was recorded from the NCBI database. The most prevalent was the I-E CRISPR-Cas system in all three studied taxa. E. coli also presents the I-F type, but in a much lesser percentage. The systems found in K. pneumoniae can be classified into I-E and I-E*. The I-E and I-F systems have two CRISPR loci, while I-E* has only one locus upstream of the Cas cluster. PCR primers have been developed in this study for each CRISPR locus. Distinct clustering was not evident, but statistically significant relationships occurred between the different CRISPR loci and the number of spacer units. For each of the queried taxa, the number of spacers was significantly different (p < 0.01) by origin (Africa, Asia, Australia and Oceania, Europe, North America, and South America) but was not linked to the isolation source type (human, animal, plant, food, or laboratory strains).
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Affiliation(s)
- Dumitrana Iordache
- Doctoral School of Integrative Biology, Babeș-Bolyai University, 44 Republicii street, 400015 Cluj-Napoca, Romania
- Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, Babeș-Bolyai University, 1 M. Kogalniceanu Street, 400084 Cluj-Napoca, Romania
- Centre for Systems Biology, Biodiversity and Bioresources, Babeș-Bolyai University, 5–7 Clinicilor Street, 400006 Cluj-Napoca, Romania
| | - Gabriela-Maria Baci
- Faculty of Animal Science and Biotechnology, University of Agricultural Sciences and Veterinary Medicine, 400372 Cluj-Napoca, Romania
| | - Oana Căpriță
- Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, Babeș-Bolyai University, 1 M. Kogalniceanu Street, 400084 Cluj-Napoca, Romania
| | - Anca Farkas
- Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, Babeș-Bolyai University, 1 M. Kogalniceanu Street, 400084 Cluj-Napoca, Romania
- Centre for Systems Biology, Biodiversity and Bioresources, Babeș-Bolyai University, 5–7 Clinicilor Street, 400006 Cluj-Napoca, Romania
| | - Andreea Lup
- Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, Babeș-Bolyai University, 1 M. Kogalniceanu Street, 400084 Cluj-Napoca, Romania
| | - Anca Butiuc-Keul
- Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, Babeș-Bolyai University, 1 M. Kogalniceanu Street, 400084 Cluj-Napoca, Romania
- Centre for Systems Biology, Biodiversity and Bioresources, Babeș-Bolyai University, 5–7 Clinicilor Street, 400006 Cluj-Napoca, Romania
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CRISPR-Cas in Acinetobacter baumannii Contributes to Antibiotic Susceptibility by Targeting Endogenous AbaI. Microbiol Spectr 2022; 10:e0082922. [PMID: 35938813 PMCID: PMC9430643 DOI: 10.1128/spectrum.00829-22] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Acinetobacter baumannii is a well-known human opportunistic pathogen in nosocomial infections, and the emergence of multidrug-resistant Acinetobacter baumannii has become a complex problem for clinical anti-infective treatments. The ways this organism obtains multidrug resistance phenotype include horizontal gene transfer and other mechanisms, such as altered targets, decreased permeability, increased enzyme production, overexpression of efflux pumps, metabolic changes, and biofilm formation. A CRISPR-Cas system generally consists of a CRISPR array and one or more operons of cas genes, which can restrict horizontal gene transfer in bacteria. Nevertheless, it is unclear how CRISPR-Cas systems regulate antibiotic resistance in Acinetobacter baumannii. Thus, we sought to assess how CRISPR-Cas affects biofilm formation, membrane permeability, efflux pump, reactive oxygen species, and quorum sensing to clarify further the mechanism of CRISPR-Cas regulation of Acinetobacter baumannii antibiotic resistance. In the clinical isolate AB43, which has a complete I-Fb CRISPR-Cas system, we discovered that the Cas3 nuclease of this type I-F CRISPR-Cas system regulates Acinetobacter baumannii quorum sensing and has a unique function in changing drug resistance. As a result of quorum sensing, synthase abaI is reduced, allowing efflux pumps to decrease, biofilm formation to become weaker, reactive oxygen species to generate, and drug resistance to decrease in response to CRISPR-Cas activity. These observations suggest that the CRISPR-Cas system targeting endogenous abaI may boost bacterial antibiotic sensitivity. IMPORTANCE CRISPR-Cas systems are vital for genome editing, bacterial virulence, and antibiotic resistance. How CRISPR-Cas systems regulate antibiotic resistance in Acinetobacter baumannii is almost wholly unknown. In this study, we reveal that the quorum sensing regulator abaI mRNA was a primary target of the I-Fb CRISPR-Cas system and the cleavage activity of Cas3 was the most critical factor in regulating abaI mRNA degradation. These results advance our understanding of how CRISPR-Cas systems inhibit drug resistance. However, the mechanism of endogenous targeting of abaI by CRISPR-Cas needs to be further explored.
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Tao S, Chen H, Li N, Liang W. The Application of the CRISPR-Cas System in Antibiotic Resistance. Infect Drug Resist 2022; 15:4155-4168. [PMID: 35942309 PMCID: PMC9356603 DOI: 10.2147/idr.s370869] [Citation(s) in RCA: 44] [Impact Index Per Article: 14.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2022] [Accepted: 06/17/2022] [Indexed: 11/28/2022] Open
Abstract
The emergence and global epidemic of antimicrobial resistance (AMR) poses a serious threat to global public health in recent years. AMR genes are shared between bacterial pathogens mainly via horizontal gene transfer (HGT) on mobile genetic elements (MGEs), thereby accelerating the spread of antimicrobial resistance (AMR) and increasing the burden of drug resistance. There is an urgent need to develop new strategies to control bacterial infections and the spread of antimicrobial resistance. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are an RNA-guided adaptive immune system in prokaryotes that recognizes and defends against invasive genetic elements such as phages and plasmids. Because of its specifically target and cleave DNA sequences encoding antibiotic resistance genes, CRISPR/Cas system has been developed into a new gene-editing tool for the prevention and control of bacterial drug resistance. CRISPR-Cas plays a potentially important role in controlling horizontal gene transfer and limiting the spread of antibiotic resistance. In this review, we will introduce the structure and working mechanism of CRISPR-Cas systems, followed by delivery strategies, and then focus on the relationship between antimicrobial resistance and CRISPR-Cas. Moreover, the challenges and prospects of this research field are discussed, thereby providing a reference for the prevention and control of the spread of antibiotic resistance.
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Affiliation(s)
- Shuan Tao
- School of Medical, Jiangsu University, Zhenjiang, Jiangsu Province, 212013, People’s Republic of China
- Lianyungang Clinical College of Jiangsu University, Lianyungang, Jiangsu Province, 222023, People’s Republic of China
| | - Huimin Chen
- School of Medical, Jiangsu University, Zhenjiang, Jiangsu Province, 212013, People’s Republic of China
| | - Na Li
- Bengbu Medical College, Bengbu, Anhui Province, 233030, People’s Republic of China
| | - Wei Liang
- Lianyungang Clinical College of Jiangsu University, Lianyungang, Jiangsu Province, 222023, People’s Republic of China
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20
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Huang YY, Zhang XY, Zhu P, Ji L. Development of clustered regularly interspaced short palindromic repeats/CRISPR-associated technology for potential clinical applications. World J Clin Cases 2022; 10:5934-5945. [PMID: 35949837 PMCID: PMC9254185 DOI: 10.12998/wjcc.v10.i18.5934] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/03/2021] [Revised: 01/10/2022] [Accepted: 04/24/2022] [Indexed: 02/06/2023] Open
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) proteins constitute the innate adaptive immune system in several bacteria and archaea. This immune system helps them in resisting the invasion of phages and foreign DNA by providing sequence-specific acquired immunity. Owing to the numerous advantages such as ease of use, low cost, high efficiency, good accuracy, and a diverse range of applications, the CRISPR-Cas system has become the most widely used genome editing technology. Hence, the advent of the CRISPR/Cas technology highlights a tremendous potential in clinical diagnosis and could become a powerful asset for modern medicine. This study reviews the recently reported application platforms for screening, diagnosis, and treatment of different diseases based on CRISPR/Cas systems. The limitations, current challenges, and future prospectus are summarized; this article would be a valuable reference for future genome-editing practices.
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Affiliation(s)
- Yue-Ying Huang
- School of Medical Laboratory, Weifang Medical University, Weifang 261053, Shandong Province, China
| | - Xiao-Yu Zhang
- School of Medical Laboratory, Weifang Medical University, Weifang 261053, Shandong Province, China
| | - Ping Zhu
- School of Medical Laboratory, Weifang Medical University, Weifang 261053, Shandong Province, China
| | - Ling Ji
- Department of Laboratory Medicine, Peking University Shenzhen Hospital, Shenzhen 518035, Guangdong Province, China
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Wang Y, Ge H, Wei X, Zhao X. Research progress on antibiotic resistance of Salmonella. FOOD QUALITY AND SAFETY 2022. [DOI: 10.1093/fqsafe/fyac035] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/14/2022]
Abstract
Abstract
Antibiotic abuse results in various antibiotic resistance among a number of foodborne bacteria, posing a severe threat to food safety. Antibiotic resistance genes are commonly detected in foodborne pathogens, which has sparked much interest in finding solutions to these issues. Various strategies against these drug-resistant pathogens have been studied, including new antibiotics and phages. Recently, a powerful tool has been introduced in the fight against drug-resistant pathogens, namely, clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) system aggregated by a prokaryotic defense mechanism. This review summarized the mechanism of antibiotic resistance in Salmonella and resistance to common antibiotics, analysed the relationship between Salmonella CRISPR-Cas and antibiotic resistance, discussed the changes in antibiotic resistance on the structure and function of CRISPR-Cas, and finally predicted the mechanism of CRISPR-Cas intervention in Salmonella antibiotic resistance. In the future, CRISPR-Cas is expected to become an important tool to reduce the threat of antibiotic-resistant pathogens in food safety.
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Affiliation(s)
- Yizhe Wang
- Research Center for Environmental Ecology and Engineering, Key Laboratory for Green Chemical Process of Ministry of Education, Hubei Key Laboratory of Novel Reactor and Green Chemical Technology, School of Environmental Ecology and Biological Engineering, Wuhan Institute of Technology, Wuhan 430205, China
| | - Hengwei Ge
- Research Center for Environmental Ecology and Engineering, Key Laboratory for Green Chemical Process of Ministry of Education, Hubei Key Laboratory of Novel Reactor and Green Chemical Technology, School of Environmental Ecology and Biological Engineering, Wuhan Institute of Technology, Wuhan 430205, China
| | - Xinyue Wei
- Research Center for Environmental Ecology and Engineering, Key Laboratory for Green Chemical Process of Ministry of Education, Hubei Key Laboratory of Novel Reactor and Green Chemical Technology, School of Environmental Ecology and Biological Engineering, Wuhan Institute of Technology, Wuhan 430205, China
| | - Xihong Zhao
- Research Center for Environmental Ecology and Engineering, Key Laboratory for Green Chemical Process of Ministry of Education, Hubei Key Laboratory of Novel Reactor and Green Chemical Technology, School of Environmental Ecology and Biological Engineering, Wuhan Institute of Technology, Wuhan 430205, China
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CRISPR-Cas System: An Adaptive Immune System’s Association with Antibiotic Resistance in Salmonella enterica Serovar Enteritidis. BIOMED RESEARCH INTERNATIONAL 2022; 2022:9080396. [PMID: 35386307 PMCID: PMC8979702 DOI: 10.1155/2022/9080396] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/11/2022] [Accepted: 03/15/2022] [Indexed: 12/16/2022]
Abstract
Several factors are involved in the emergence of antibiotic-resistant bacteria and pose a serious threat to public health safety. Among them, clustered regularly interspaced short palindromic repeat- (CRISPR-) Cas system, an adaptive immune system, is thought to be involved in the development of antibiotic resistance in bacteria. The current study was aimed at determining not only the presence of antibiotic resistance and CRISPR-Cas system but also their association with each other in Salmonella enteritidis isolated from the commercial poultry. A total of 139 samples were collected from poultry birds sold at the live bird markets of Lahore City, and both phenotypic and genotypic methods were used to determine antimicrobial resistance. The presence of the CRISPR-Cas system was determined by PCR, followed by sequencing. All isolates of S. enteritidis (100%) were resistant to nalidixic acid, whereas 95% of isolates were resistant to ampicillin. Five multidrug-resistant isolates (MDR) such as S. enteritidis isolate (S. E1, S. E2, S. E4, S. E5, and S. E8) were found in the present study. The CRISPR-Cas system was detected in all of these MDR isolates, and eight spacers were detected within the CRISPR array. In addition, an increased expression of CRISPR-related genes was observed in the standard strain and MDR S. enteritidis isolates. The association of the CRISPSR-Cas system with multiple drug resistance highlights the exogenous acquisition of genes by horizontal transfer. The information could be used further to combat antibiotic resistance in pathogens like Salmonella.
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Alduhaidhawi AHM, AlHuchaimi SN, Al- Mayah TA, Al-Ouqaili MTS, Alkafaas SS, Muthupandian S, Saki M. Prevalence of CRISPR-Cas Systems and Their Possible Association with Antibiotic Resistance in Enterococcus faecalis and Enterococcus faecium Collected from Hospital Wastewater. Infect Drug Resist 2022; 15:1143-1154. [PMID: 35340673 PMCID: PMC8942119 DOI: 10.2147/idr.s358248] [Citation(s) in RCA: 40] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2022] [Accepted: 03/12/2022] [Indexed: 12/25/2022] Open
Abstract
PURPOSE This study aimed to evaluate the presence of CRISPR-Cas system genes and their possible association with antibiotic resistance patterns of Enterococcus faecalis and Enterococcus faecium species isolated from hospital wastewater (HWW) samples of several hospitals. METHODS HWW samples (200 mL) were collected from wastewater discharged from different hospitals from October 2020 to March 2021. The isolation and identification of enterococci species were performed by standard bacteriology tests and polymerase chain reaction (PCR). Antibiotic resistance was determined using the disc diffusion. The presence of various CRISPR-Cas systems was investigated by PCR. The association of the occurrence of CRISPR-Cas systems with antibiotic resistance was analyzed with appropriate statistical tests. RESULTS In total, 85 different enterococci species were isolated and identified using phenotypic methods. The results of PCR confirmed the prevalence of 50 (58.8%) E. faecalis and 35 (41.2%) E. faecium, respectively. In total, 54 (63.5%) of 85 isolates showed the presence of CRISPR-Cas loci. The incidence of CRISPR-Cas was more common in E. faecalis. CRISPR1, CRISPR2, and CRISPR3 were present in 35 (41.2%), 47 (55.3%), and 30 (35.3%) enterococci isolates, respectively. The CRISPR-Cas positive isolates showed significant lower resistance rates against vancomycin, ampicillin, chloramphenicol, erythromycin, rifampin, teicoplanin, tetracycline, imipenem, tigecycline, and trimethoprim-sulfamethoxazole in comparison with CRISPR-Cas negative isolates. The results showed that the presence of CRISPR-Cas genes was lower in multidrug-resistant (MDR) isolates (53.1%, n = 26/49) compared to the non-MDR enterococci isolates (77.8%, n = 28/36) (P = 0.023). CONCLUSION This study revealed the higher prevalence of E. faecalis than E. faecium in HWWs. Also, the lack of CRISPR-Cas genes was associated with more antibiotic resistance rates and multidrug resistance in E. faecalis and E. faecium isolates with HWW origin.
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Affiliation(s)
| | | | | | - Mushtak T S Al-Ouqaili
- Department of Microbiology, College of Medicine, University of Anbar, Ramadi, Al-Anbar Governorate, Iraq
| | - Samar Sami Alkafaas
- Department of Chemistry, Division of Biochemistry, Faculty of Science, Tanta University, Tanta, Egypt
| | - Saravanan Muthupandian
- Department of Microbiology and Immunology, Division of Biomedical Sciences, School of Medicine, College of Health Sciences, Mekelle University, Mekelle, 1871, Ethiopia
- Department of Pharmacology, AMR and Nanomedicine Laboratory, Center for Transdisciplinary Research, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences (SIMATS), Chennai, 60007, India
| | - Morteza Saki
- Department of Microbiology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
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Guo T, Sun X, Li M, Wang Y, Jiao H, Li G. The Involvement of the csy1 Gene in the Antimicrobial Resistance of Acinetobacter baumannii. Front Med (Lausanne) 2022; 9:797104. [PMID: 35155494 PMCID: PMC8825777 DOI: 10.3389/fmed.2022.797104] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2021] [Accepted: 01/03/2022] [Indexed: 12/26/2022] Open
Abstract
Acinetobacter baumannii is an important, opportunistic nosocomial pathogen that causes a variety of nosocomial infections, and whose drug resistance rate has increased in recent years. The CRISPR-Cas system exists in several bacteria, providing adaptive immunity to foreign nucleic acid invasion. This study explores whether CRISPR-Cas is related to drug resistance. Antibiotics were used to treat strains ATCC19606 and AB43, and the expression of CRISPR-related genes was found to be changed. The Csy proteins (Csy1–4) were previously detected to promote target recognition; however, the potential function of csy1 gene is still unknown. Thus, the RecAb homologous recombination system was utilized to knock out the csy1 gene from A. baumannii AB43, which carries the Type I-Fb CRISPR-Cas system, and to observe the drug resistance changes in wild-type and csy1-deleted strains. The AB43Δcsy1 mutant strain was found to become resistant to antibiotics, while the wild-type strain was sensitive to antibiotics. Moreover, transcriptome analysis revealed that the csy1 gene regulates genes encoding CRISPR-Cas-related proteins, drug-resistant efflux pumps, membrane proteins, and oxidative phosphorylation-related proteins, inhibiting antimicrobial resistance in A. baumannii. The in vitro resistance development assay revealed that the complete CRISPR-Cas system could inhibit the development of bacterial resistance. Our findings expand our understanding of the role of CRISPR-Cas csy1 gene in A. baumannii and link the CRISPR-Cas system to the biogenesis of bacterial drug-resistant structures.
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Affiliation(s)
- Tingting Guo
- Department of Microbiology, School of Medicine, Yangzhou University, Yangzhou, China.,Jiangsu Key Laboratory of Zoonosis/Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China.,Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, Yangzhou, China
| | - Xiaoli Sun
- Department of Microbiology, School of Medicine, Yangzhou University, Yangzhou, China
| | - Mengying Li
- Department of Microbiology, School of Medicine, Yangzhou University, Yangzhou, China.,Department of Pharmacy, Suzhou Hospital of Integrated Traditional Chinese and Western Medicine, Suzhou, China
| | - Yuhang Wang
- Department of Microbiology, School of Medicine, Yangzhou University, Yangzhou, China
| | - Hongmei Jiao
- Department of Microbiology, School of Medicine, Yangzhou University, Yangzhou, China
| | - Guocai Li
- Department of Microbiology, School of Medicine, Yangzhou University, Yangzhou, China.,Jiangsu Key Laboratory of Zoonosis/Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China.,Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, Yangzhou, China
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Wang G, Song G, Xu Y. A Rapid Antimicrobial Susceptibility Test for Klebsiella pneumoniae Using a Broth Micro-Dilution Combined with MALDI TOF MS. Infect Drug Resist 2021; 14:1823-1831. [PMID: 34025124 PMCID: PMC8132464 DOI: 10.2147/idr.s305280] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2021] [Accepted: 04/22/2021] [Indexed: 12/27/2022] Open
Abstract
Background Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a novel method that can be used to identify pathogens and has potential applications in the detection of drug-resistant bacteria. Purpose To evaluate the ability of a MALDI-TOF MS-based broth micro-dilution method in detecting the minimum inhibitory concentration (MIC) values of Klebsiella pneumoniae to ceftriaxone and imipenem. Materials and Methods Sixty strains of K. pneumoniae with different levels of resistance to carbapenems and cephalosporins were randomly collected. The 0.5 McFarland (Mc) concentration of the bacterial suspension was inoculated in cation-adjusted Mueller-Hinton broth (CAMHB) with a final cell turbidity of 5×105 CFU/mL. The broth was incubated with serial concentrations of antibiotics. After centrifuging the bacterial suspensions, the lysed cells were analyzed by MALDI-TOF MS to identify the growth-promoting or inhibitory effects on K. pneumoniae. The molecular mechanisms of resistance were investigated by PCR and DNA sequencing analysis. Results The expression of known resistance genes (blaKPC, blaFOX, blaDHA, blaCTX-M and blaTEM) was detected in the 30 carbapenems-resistant strains. The agreement between the MIC values derived from the MALDI-TOF MS analysis and from the broth micro-dilution method was 61.7% for ceftriaxone and 71.7% for imipenem. According to the Clinical and Laboratory Standards Institute (CLSI) breakpoint of resistance to ceftriaxone and imipenem, the 60 isolates were accurately classified as resistant or susceptible isolates with 100% sensitivity and 100% specificity. Conclusion The transmission and infection of multidrug-resistant bacteria could be better managed and treated with the rapid identification of strains and antimicrobial susceptibility. A MALDI-TOF MS-based susceptibility test could be used to identify resistance of K. pneumoniae within a short time-frame. This approach could potentially be used as a supplementary antimicrobial susceptibility test that could be investigated on more bacterial species combined with different antibiotics.
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Affiliation(s)
- Gang Wang
- Department of Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui Province, People's Republic of China
| | - Guobin Song
- Department of Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui Province, People's Republic of China.,Department of Clinical Laboratory, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China
| | - Yuanhong Xu
- Department of Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui Province, People's Republic of China
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Abstract
CRISPR-Cas systems, widespread in bacteria and archaea, are mainly responsible for adaptive cellular immunity against exogenous DNA (plasmid and phage). However, the latest research shows their involvement in other functions, such as gene expression regulation, DNA repair and virulence. In recent years, they have undergone intensive research as convenient tools for genomic editing, with Cas9 being the most commonly used nuclease. Gene editing may be of interest in biotechnology, medicine (treatment of inherited disorders, cancer, etc.), and in the development of model systems for various genetic diseases. The dCas9 system, based on a modified Cas9 devoid of nuclease activity, called CRISPRi, is widely used to control gene expression in bacteria for new drug biotargets validation and is also promising for therapy of genetic diseases. In addition to direct use for genomic editing in medicine, CRISPR-Cas can also be used in diagnostics, for microorganisms’ genotyping, controlling the spread of drug resistance, or even directly as “smart” antibiotics. This review focuses on the main applications of CRISPR-Cas in medicine, and challenges and perspectives of these approaches.
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