Award Date

5-1-2020

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry and Biochemistry

First Committee Member

Ernesto Abel-Santos

Second Committee Member

Gary Kleiger

Third Committee Member

Ronald Gary

Fourth Committee Member

Brian Hedlund

Fifth Committee Member

Qingxi Shen

Number of Pages

110

Abstract

Clostridioides difficile infection (CDI) is one of the most common nosocomial infections worldwide. A major risk factor for CDI is antibiotic therapy, due to C. difficile’s resistance to a myriad of antibiotics—one of which are cephalosporins. Cephalosporins are ß-lactam antibiotics that function by binding to the active site of penicillin-binding proteins (PBPs), thus inhibiting peptidoglycan synthesis and leading to cell lysis. Gram-positive bacteria can counteract ß-lactams by (i) producing ß-lactamases, (ii) expressing modified PBPs, or (iii) by expressing efflux pumps.

Genomic analysis of C. difficile strain 630 revealed the presence of at least 31 putative ß-lactam resistance genes that encode putative ß-lactamases, ß-lactamase-like proteins and PBPs. We hypothesized that upon cephalosporin exposure, C. difficile would differentially express one or more of these genes. Many of these genes were differentially expressed when C. difficile was exposed to cefoxitin, but most of them were not upregulated more than 3.5-fold according to RT-qPCR analysis. Strikingly, putative ß-lactamase, blaCDD, was upregulated nearly 600-fold upon cefoxitin treatment. Deletion of blaCDD caused little to no reduction in cephalosporin resistance.

RT-qPCR analysis of the blaCDD-null mutant when treated with cefoxitin did not reveal any drastic changes in expression of the remaining genes that could explain transcription compensation for the deleted ß-lactamase. Deletion of the second-most upregulated gene which encodes a putative PBP, vanY, minimally affected cephalosporin resistance. To elucidate additional putative resistance genes that may be differentially regulated upon cephalosporin exposure, we analyzed the entire C. difficile transcriptome using RNA-Seq. We found that upon cephradine, ceftazidime, and cefepime treatment, expression was similar to

untreated cells. Cefoxitin-treated cells, however, had a higher number of differentially regulated genes. This might be attributed to the activation of starvation responses due to longer treatment time for cefoxitin-treated cells. Nevertheless, all cephalosporin treatments triggered the upregulation of a putative eterodimeric ABC transporter directly downstream of blaCDD. Further functional analysis of this ABC transporter will be necessary to elucidate its possible role in cephalosporin resistance. Future studies will be geared towards using transposon mutagenesis to elucidate genes that are required for C. difficile’s survival upon cephalosporin treatment but that are constitutively expressed regardless of antibiotic exposure.

Keywords

ABC transporter; antibiotic resistance; Clostridioides difficile; Clostridium difficile; ß-lactam; ß-lactamase

Disciplines

Biochemistry

File Format

pdf

File Size

2.9 MB

Degree Grantor

University of Nevada, Las Vegas

Language

English

Rights

IN COPYRIGHT. For more information about this rights statement, please visit http://rightsstatements.org/vocab/InC/1.0/

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Biochemistry Commons

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