Dataset Information

Integrating CRISPR-Enabled Trackable Genome Engineering and Transcriptomic Analysis of Global Regulators for Antibiotic Resistance Selection and Identification in Escherichia coli.

ABSTRACT: It is important to expedite our understanding of antibiotic resistance to address the increasing numbers of fatalities and environmental pollution due to the emergence of antibiotic resistance and multidrug-resistant strains. Here, we combined the CRISPR-enabled trackable genome engineering (CREATE) technology and transcriptomic analysis to investigate antibiotic tolerance in Escherichia coli We developed rationally designed site saturation mutagenesis libraries targeting 23 global regulators to identify fitness-conferring mutations in response to diverse antibiotic stresses. We identified seven novel mutations that confer resistance to the ribosome-targeting antibiotics doxycycline, thiamphenicol, and gentamicin in E. coli To the best of our knowledge, these mutations that we identified have not been reported previously during treatment with the indicated antibiotics. Transcriptome sequencing-based transcriptome analysis was further employed to evaluate the genome-wide changes in gene expression in E. coli for SoxR G121P and cAMP receptor protein (CRP) V140W reconstructions, and improved fitness in response to doxycycline and gentamicin was seen. In the case of doxycycline, we speculated that SoxR G121P significantly increased the expression of genes involved in carbohydrate metabolism and energy metabolism to promote cell growth for improved adaptation. In the CRP V140W mutant with improved gentamicin tolerance, the expression of several amino acid biosynthesis genes and fatty acid degradation genes was significantly changed, and these changes probably altered the cellular energy state to improve adaptation. These findings have important significance for understanding such nonspecific mechanisms of antibiotic resistance and developing new antibacterial drugs.IMPORTANCE The growing threat of antimicrobial resistance poses a serious threat to public health care and motivates efforts to understand the means by which resistance acquisition occurs and how this can be combatted. To address these challenges, we expedited the identification of novel mutations that enable complex phenotypic changes that result in improved tolerance to antibiotics by integrating CREATE and transcriptomic analysis of global regulators. The results give us a better understanding of the mechanisms of resistance to tetracycline antibiotics and aminoglycoside antibiotics and also indicate that the method may be used for quickly identifying resistance-related mutations.

SUBMITTER: Chen C

PROVIDER: S-EPMC7174635 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Integrating CRISPR-Enabled Trackable Genome Engineering and Transcriptomic Analysis of Global Regulators for Antibiotic Resistance Selection and Identification in Escherichia coli.

Chen Cong C Choudhury Alaksh A Zhang Shuanghong S Garst Andrew D AD Song Xin X Liu Xunli X Chen Tao T Gill Ryan T RT Wang Zhiwen Z

mSystems 20200421 2

It is important to expedite our understanding of antibiotic resistance to address the increasing numbers of fatalities and environmental pollution due to the emergence of antibiotic resistance and multidrug-resistant strains. Here, we combined the CRISPR-enabled trackable genome engineering (CREATE) technology and transcriptomic analysis to investigate antibiotic tolerance in <i>Escherichia coli</i> We developed rationally designed site saturation mutagenesis libraries targeting 23 global regula ...[more]

PMID: 32317390

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Project description:BackgroundThe majority of studies that link antibiotic usage and resistance focus on simple associations between the resistance against a specific antibiotic and the use of that specific antibiotic. However, the relationship between antibiotic use and resistance is more complex. Here we evaluate selection and co-selection by assessing which antibiotics, including those mainly prescribed for respiratory tract infections, are associated with increased resistance to various antibiotics among Escherichia coli isolated from urinary samples.MethodsMonthly primary care prescribing data were obtained from National Health Service (NHS) Digital. Positive E. coli records from urine samples in English primary care (n = 888,207) between April 2014 and January 2016 were obtained from the Second Generation Surveillance System. Elastic net regularization was used to evaluate associations between prescribing of different antibiotic groups and resistance against amoxicillin, cephalexin, ciprofloxacin, co-amoxiclav and nitrofurantoin at the clinical commissioning group (CCG) level. England is divided into 209 CCGs, with each NHS practice prolonging to one CCG.ResultsAmoxicillin prescribing (measured in DDD/ 1000 inhabitants / day) was positively associated with amoxicillin (RR 1.03, 95% CI 1.01-1.04) and ciprofloxacin (RR 1.09, 95% CI 1.04-1.17) resistance. In contrast, nitrofurantoin prescribing was associated with lower levels of resistance to amoxicillin (RR 0.92, 95% CI 0.84-0.97). CCGs with higher levels of trimethoprim prescribing also had higher levels of ciprofloxacin resistance (RR 1.34, 95% CI 1.10-1.59).ConclusionAmoxicillin, which is mainly (and often unnecessarily) prescribed for respiratory tract infections is associated with increased resistance against various antibiotics among E. coli causing urinary tract infections. Our findings suggest that when predicting the potential impact of interventions on antibiotic resistances it is important to account for use of other antibiotics, including those typically used for other indications.

Project description:Pathogenic Escherichia coli and Salmonella enterica pose serious public health threats due to their ability to cause severe gastroenteritis and life-threatening sequela, particularly in young children. Moreover, the emergence and dissemination of antibiotic resistance in these bacteria have complicated control of infections. Alternative strategies that effectively target these enteric pathogens and negate or reduce the need of antibiotics are urgently needed. Such an alternative is the CRISPR-Cas9 system because it can generate sequence-specific lethal double stranded DNA breaks. In this study, two self-transmissible broad host range conjugative plasmids, pRK24 and pBP136, were engineered to deliver multiplexed CRSIPR-Cas9 systems that specifically target Enterohemorrhagic and Enteropathogenic strains of E. coli (EHEC and EPEC), S. enterica, and blaCMY-2 antibiotic resistance plasmids. Using in vitro mating assays, we show that the conjugative delivery of pRK24-CRISPR-Cas9 carrying guide RNAs to the EPEC/EHEC eae (intimin) gene can selectively kill enterohemorrhagic E. coli O157 eae+ cells (3 log kill at 6 h) but does not kill the isogenic Δeae mutant (P<0.001). Similar results were also obtained with a pBP136 derivative, pTF16, carrying multiplexed guide RNAs targeting E. coli eae and the S. enterica ssaN gene coding for the type III secretion ATPase. Another pBP136 derivative, TF18, carries guide RNAs targeting S. enterica ssaN and the antibiotic resistance gene, blaCMY-2, carried on the multi-drug resistant pAR06302. Introduction of pTF18 into bacteria harboring pAR06302 showed plasmids were cured at an efficiency of 53% (P<0.05). Using a murine neonate EPEC infection model, pTF16 was delivered by a murine derived E. coli strain to EPEC infected mice and showed significant reductions of intestinal EPEC (P<0.05). These results suggest that establishing conjugative CRISPR-Cas9 antimicrobials in the intestinal microbiome may provide protection from enteric pathogens and reduce antibiotic resistance without disrupting the normal microbiota.

Project description:BackgroundManipulations in Saccharomyces cerevisiae classically depend on use of auxotrophy selection markers. There are several disadvantages to this in a microbial cell factory setting: (1) auxotrophies must first be engineered in prototrophic strains, and many industrial strains are polyploid/aneuploid prototrophs (2) available strain auxotrophies must be paired with available repair plasmids (3) remaining auxotrophies must be repaired prior to development of industrial bioprocesses. Use of dominant antibiotic resistance markers can circumvent these problems. However, there are relatively few yeast antibiotic resistance marker vectors available; furthermore, available vectors contain only one expression cassette, and it is often desirable to introduce more than one gene at a time.ResultsTo overcome these problems, eight new shuttle vectors have been developed. The plasmids are maintained in yeast under a 2 μm ori and in E. coli by a pUC ori. They contain two yeast expression cassettes driven by either (1) the constitutive TEF1 and PGK1 promoters, or (2) the constitutive TEF1 promoter and the inducible GAL10 or HXT7 promoters. Expression strength of these promoters over a typical production time frame in glucose/galactose medium was examined, and identified the TEF1 and HXT7 promoters as preferred promoters over long term fermentations. Selection is provided by either aphA1 (conferring resistance to G418 in yeast and kanamycin/neomycin in E. coli) or ble (conferring resistance to phleomycin in both yeast and E. coli). Selection conditions for these plasmids/antibiotics in defined media were examined, and selection considerations are reviewed. In particular, medium pH has a strong effect on both G418 and phleomycin selection.ConclusionsThese vectors allow manipulations in prototrophic yeast strains with expression of two gene cassettes per plasmid, and will be particularly useful for metabolic engineering applications. The vector set expands the (currently limited) selection of antibiotic marker plasmids available for use in yeast, and in addition makes available dual gene expression cassettes on individual plasmids using antibiotic selection. The resistance gene cassettes are flanked by loxP recognition sites to allow CreA-mediated marker removal and recycling, providing the potential for genomic integration of multiple genes. Guidelines for selection using G418 and phleomycin are provided.

Dataset Information

Integrating CRISPR-Enabled Trackable Genome Engineering and Transcriptomic Analysis of Global Regulators for Antibiotic Resistance Selection and Identification in Escherichia coli.

Publications

Integrating CRISPR-Enabled Trackable Genome Engineering and Transcriptomic Analysis of Global Regulators for Antibiotic Resistance Selection and Identification in Escherichia coli.

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