The vanRCd Mutation 343A>G, Resulting in a Thr115Ala Substitution, Is Associated with an Elevated Minimum Inhibitory Concentration (MIC) of Vancomycin in Clostridioides difficile Clinical Isolates from Florida

ABSTRACT Clostridioides difficile, the primary cause of nosocomial antibiotic-associated diarrhea, has a complex relationship with antibiotics. While the use of broad-spectrum antibiotics disrupts the gut microbiota and increases the risk of C. difficile infection (CDI), antibiotics are also the primary treatment for CDI. However, only a few antibiotics, including vancomycin, fidaxomicin, and rifaximin, are effective against CDI, and resistance to these antibiotics has emerged recently. In this study, we report the identification of two RT027 C. difficile clinical isolates (TGH35 and TGH64) obtained from symptomatic CDI-diagnosed patients in Tampa, Florida in 2016. These two strains showed an elevated minimum inhibitory concentration (MIC) of vancomycin (MIC = 4 μg/mL, compared to the EUCAST breakpoint of 2 μg/mL) and contained a vanRCd 343A>G mutation resulting in a Thr115Ala substitution in the VanRCd response regulator. This mutation was absent in the vancomycin-sensitive control epidemic strain RT027/R20291. TGH64 was also resistant to rifaximin (MIC ≥ 128 μg/mL) and carried the previously reported Arg505Lys and Ile548Met mutations in RpoB. Furthermore, we report on the antimicrobial resistance (AMR) and genomic characterization of additional C. difficile isolates, including RT106/TGH120, RT017/TGH33, and RT017/TGH51, obtained from the same patient sample cohort representing the highly prevalent and regionally distributed C. difficile ribotypes worldwide. Considering that the VanRCd Thr115Ala mutation was also independently reported in seven C. difficile clinical isolates from Texas and Israel in 2019, we recommend epidemiological surveillance to better understand the impact of this mutation on vancomycin resistance. IMPORTANCE The perpetually evolving antimicrobial resistance (AMR) of C. difficile is an important contributor to its epidemiology and is a grave concern to global public health. This exacerbates the challenge of treating the infections caused by this multidrug-resistant causative organism of potentially life-threatening diarrhea. Further, the novel resistance-determining factors can be transferred between different strains and species of bacteria and cause the spread of AMR in clinical, environmental, and community settings. In this study, we have identified a mutation (vanRCd 343A>G) that causes a Thr115Ala substitution and is linked to an increased MIC of vancomycin in clinical isolates of C. difficile obtained from Florida in 2016. Understanding the mechanisms of AMR, especially those of newly evolving strains, is essential to effectively guide antibiotic stewardship policies to combat antibiotic resistance as well as to discover novel therapeutic targets.

IMPORTANCE The perpetually evolving antimicrobial resistance (AMR) of C. difficile is an important contributor to its epidemiology and is a grave concern to global public health. This exacerbates the challenge of treating the infections caused by this multidrug-resistant causative organism of potentially life-threatening diarrhea. Further, the novel resistance-determining factors can be transferred between different strains and species of bacteria and cause the spread of AMR in clinical, environmental, and community settings. In this study, we have identified a mutation (vanR Cd 343A.G) that causes a Thr115Ala substitution and is linked to an increased MIC of vancomycin in clinical isolates of C. difficile obtained from Florida in 2016. Understanding the mechanisms of AMR, especially those of newly evolving strains, is essential to effectively guide antibiotic stewardship policies to combat antibiotic resistance as well as to discover novel therapeutic targets. C lostridioides difficile, which is a cause of mild to life-threatening diarrhea that was responsible for 12,800 deaths in the USA in 2017, has been declared by the CDC as one of the top five urgent antibiotic-resistant threats (1). The emergence of new strains, which are often more virulent and antibiotic-resistant, has been associated with the recent increase in the prevalence and severity of CDI (2).
As a life-threatening disease with a high incidence of recurrence and limited effective therapeutic options, the resistance of C. difficile to vancomycin, which is the first line of therapy that is recommended for CDI treatment, is a grave public health concern (2, 3). Vancomycin inhibits bacterial cell wall synthesis by binding with high affinity to the D-Ala-D-Ala C terminus of peptidoglycan precursors, which thereby prevents the addition of late precursors to the nascent peptidoglycan chain (4). A gene cluster called the "van operon" has been described to mediate vancomycin resistance in enterococci (4). The two-component regulatory system of van operons encompasses a sensor histidine kinase (VanS) and a response regulator (VanR). The vanG-type of operon contains the resistance genes VanG (D-Ala-D-Ser ligase), VanXY (a bifunctional D,D-dipeptidase/D,D-carboxypeptidase), and VanT (serine racemase). When vancomycin is sensed by the membrane-bound VanS, it undergoes the ATP-dependent autophosphorylation of a histidine residue. This phosphoryl group is then transferred to the cytoplasmic VanR, which, in turn, transcriptionally activates the expression of downstream resistance genes. VanT converts L-Ser to D-Ser and VanG ligates D-Ala and D-Ser, forming low-affinity precursors and modifying the vancomycin binding target. VanXY hydrolyzes peptidoglycan precursors that end with D-Ala residues, thereby eliminating the high-affinity binding targets of vancomycin (4). However, although a functional vanG operon-like gene cluster called "vanG Cd " has been found in about 85% of C. difficile clinical isolates, it was not associated with vancomycin resistance in C. difficile (5).
We previously reported 139 C. difficile clinical isolates that were obtained from symptomatic patients who were diagnosed with CDI in Tampa, FL, USA (6). Based on broth microdilution-based screening for antimicrobial susceptibility, we selected isolates that showed a reduced susceptibility to multiple antibiotics and conducted capillary PCR ribotyping at the Dutch National Reference Laboratory at the Leiden University Medical Center (LUMC), using a standardized protocol (7). Considering that the epidemiology of C. difficile shows distinct geographical distributions, we selected five isolates to represent the most prevalent C. difficile ribotypes that are found in different geographical locations around the world, namely, RT027 (Europe and North America) (2), RT106 (USA) (8), and RT017 (Asia) (9), for further analysis in the present study.
The antibiotic susceptibility testing (AST) of the isolates to the currently clinically used antibiotics for CDI treatment, namely, vancomycin, metronidazole, fidaxomicin, and rifaximin, was conducted using the reference standard agar dilution method, based on the Clinical and Laboratory Standards Institute (CLSI) guidelines M11-A7 (volume 27, no 2, ISBN:1-56238-626-3), using the epidemic RT027/R20291 C. difficile strain as a control with a minimum of three technical replicates in two independent experiments. While all strains were susceptible to metronidazole and fidaxomicin, high resistance to rifaximin (MIC $ 128 mg/mL) was detected only in TGH64. Rifaximin, which is an adjunct therapeutic for CDI, acts by binding to the b-subunit of RNA polymerase (RpoB), which thereby inhibits bacterial RNA synthesis (20). Since mutations in the rifamycin resistance-determining region (RRID) of RpoB have been previously associated with rifamycin resistance in C. difficile without imposing a fitness cost (20), we performed a EMBL Clustal Omega-based multiple     (20). Two isolates that were ribotyped as RT027, namely, TGH35 and TGH64, showed elevated vancomycin MIC (MIC = 4 mg/mL; compared to the EUCAST breakpoint of 2 mg/mL) ( Table 1) values. We further assessed the vancomycin susceptibility of these strains via two other methods: the BHI broth microdilution, based on the CLSI guidelines, and Etest, which was conducted at LUMC. In agreement with the results of previous studies, both of these methods gave lower MIC values than were obtained via the reference agar dilution method for several strains (22,23). However, with all three methods, the elevated vancomycin MIC in TGH35 and TGH64 persisted compared to the other strains.
CARD-based AMR prediction revealed two genes of the vanG Cd operon, namely, vanR Cd and vanXY Cd , in TGH35, TGH64, and TGH120. While vanXY Cd was identical in all three isolates, the vanR Cd of TGH120 differed from the two RT027 strains (Table 1). Therefore, we performed a MSA of the entire vanG Cd operon in these isolates with the reference strains CD630 and R20291. The vanG Cd operon is absent in the TGH33, TGH51, and M68 strains. Remarkably, only one nucleotide difference was detected in the whole .6 kb vanG Cd operon comparison between the five strains: 343A.G in the vanR Cd gene of TGH35 and TGH64 (Fig. 1C). The resulting sense mutation Thr115Ala in the receptor domain of VanR Cd likely affects the expression of the downstream resistance genes. We reported this mutation at the ASM Microbe Annual Conference in June of 2019 (24). Later, the VanR Cd Thr115Ala mutation was also independently reported in seven C. difficile clinical isolates from the Texas Medical Center, USA as well as in two C. difficile clinical isolates from Israel (25). These isolates showed the constitutive expression of the vanG Cd operon and elevated MICs of vancomycin, which could be reversed in the VanR Cd -mutant isolates by silencing vanG Cd , whereas vanG Cd silencing had no effect on the MIC of the control R20291 strain. Shen et al. also used the structural homology modeling of VanR Cd to propose that the Thr115Ala substitution provides better stability for its interaction with DNA, thereby enhancing the capability for the transcriptional activation of downstream genes. Since single base pair mutations under selection can quickly lead to the development of resistance, the present work highlights the need for epidemiological surveillance to monitor the prevalence of this mutation, especially in vancomycin-treated patients, to better understand its effects on the resistance to an antibiotic that is currently crucial in the treatment of C. difficile infection.

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