Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile.

Severe outbreaks and deaths have been linked to the emergence and global spread of fluoroquinolone-resistant Clostridioides difficile over the past two decades. At the same time, metronidazole, a nitro-containing antibiotic, has shown decreasing clinical efficacy in treating C. difficile infection (CDI). Most metronidazole-resistant C. difficile exhibit an unusual resistance phenotype that can only be detected in susceptibility tests using molecularly intact heme. Here, we describe the mechanism underlying this trait. We find that most metronidazole-resistant C. difficile strains carry a T-to-G mutation (which we term PnimBG) in the promoter of gene nimB, resulting in constitutive transcription. Silencing or deleting nimB eliminates metronidazole resistance. NimB is related to Nim proteins that are known to confer resistance to nitroimidazoles. We show that NimB is a heme-dependent flavin enzyme that degrades nitroimidazoles to amines lacking antimicrobial activity. Furthermore, occurrence of the PnimBG mutation is associated with a Thr82Ile substitution in DNA gyrase that confers fluoroquinolone resistance in epidemic strains. Our findings suggest that the pandemic of fluoroquinolone-resistant C. difficile occurring over the past few decades has also been characterized by widespread resistance to metronidazole.

Mechanisms and impact of antimicrobial resistance in Clostridioides difficile.

The evolution of antimicrobial resistance in Clostridioides difficile has markedly shaped its epidemiology and detrimentally impacted patient care. C. difficile exhibits resistance to multiple classes of antimicrobials, due to accumulation of horizontally acquired resistance genes and de novo mutations to drug targets. Particularly worrying is that declines in clinical success of firstline CDI antimicrobials coincide with the spread of strains that are more resistant to these drugs. Yet, there is still much to learn regarding the prevalence of genetic elements in clinical isolates, their molecular mechanisms, and the extent to which this information can be translated to develop molecular diagnostics that improve antimicrobial prescribing and antimicrobial stewardship approaches for CDI. Thus, this perspective discusses current understanding and knowledge gaps of antimicrobial resistance mechanisms in C. difficile, emphasizing on CDI therapies.

Ebselen Not Only Inhibits Clostridioides difficile Toxins but Displays Redox-Associated Cellular Killing.

Ebselen, a reactive organoselenium compound, was shown to inhibit toxins TcdA and TcdB by covalently binding to their cysteine protease domains. It was suggested that ebselen lacked antimicrobial activity against Clostridioides difficile. However, this perception conflicts with C. difficile having essential cysteine-containing enzymes that could be potential targets and the reported antimicrobial activity of ebselen against other species. Hence, we reevaluated the anti-C. difficile properties of ebselen. Susceptibility testing revealed that its activity was either slightly reduced by pyruvate found in Wilkins-Chalgren agar or obliterated by blood in brucella agar. In brain heart infusion (BHI) agar, ebselen inhibited most C. difficile strains (MICs of 2 to 8 µg/ml), except for ribotype 078 that was intrinsically resistant (MIC = 32 to 128 µg/ml). Against C. difficile R20291, at concentrations below its minimal bactericidal concentration (MBC), 16 µg/ml, ebselen inhibited production of toxins and spores. Transcriptome analysis revealed that ebselen altered redox-associated processes and cysteine metabolism and enhanced expression of Stickland proline metabolism, likely to regenerate NAD+ from NADH. In cellular assays, ebselen induced uptake of cysteine, depleted nonprotein thiols, and disrupted the NAD+/NADH ratio. Taken together, killing of C. difficile cells by ebselen occurs by a multitarget action that includes disrupting intracellular redox, which is consistent with ebselen being a reactive molecule. However, the physiological relevance of these antimicrobial actions in treating acute C. difficile infection (CDI) is likely to be undermined by host factors, such as blood, which protect C. difficile from killing by ebselen. IMPORTANCE We show that ebselen kills pathogenic C. difficile by disrupting its redox homeostasis, changing the normal concentrations of NAD+ and NADH, which are critical for various metabolic functions in cells. However, this antimicrobial action is hampered by host components, namely, blood. Future discovery of ebselen analogues, or mechanistically similar compounds, that remain active in blood could be drug leads for CDI or probes to study C. difficile redox biology in vivo.

Reduced Susceptibility to Metronidazole Is Associated With Initial Clinical Failure in Clostridioides difficile Infection.

BACKGROUND: Clinical studies have demonstrated inferior cure rates when metronidazole (MTZ) is used to treat Clostridioides difficile infection (CDI). We hypothesized that a newly identified, heme-inducible form of reduced MTZ susceptibility in C. difficile leads to higher odds of initial clinical failure in patients with CDI treated with MTZ. METHODS: This multicenter cohort study included adults diagnosed with CDI between 2017 and 2018. C. difficile isolated from stool samples underwent agar dilution MTZ susceptibility testing with incorporation of fresh heme. Blinded investigators reviewed medical records for initial clinical failure and other relevant clinical variables. Classification and regression tree (CART) analysis was used to identify the MTZ minimum inhibitory concentration (MIC) breakpoint that was predictive of initial clinical failure. Results were confirmed using univariate and multivariable logistic regression analyses to account for potential confounders. RESULTS: Of the 356 patients included, 72% received MTZ-based therapy and 27% experienced initial clinical failure. CART analysis identified an MTZ MIC ≥1 µg/mL above which patients had a higher rate of initial clinical failure. MTZ MICs ranged from 0.25 to 8 µg/mL (MIC50/90 = 0.25/2 µg/mL), and approximately 18% of isolates had MTZ MICs ≥1 µg/mL. In multivariable analysis, an MTZ MIC ≥1 µg/mL was an independent predictor of initial clinical failure in patients receiving an MTZ-based treatment regimen (odds ratio, 2.27 [95% confidence interval, 1.18-4.34]). CONCLUSIONS: Using a reproducible method to determine C. difficile MICs to MTZ, a breakpoint of ≥1 µg/mL identified patients at higher risk of initial clinical failure.

The Integrity of Heme Is Essential for Reproducible Detection of Metronidazole-Resistant Clostridioides difficile by Agar Dilution Susceptibility Tests.

Metronidazole resistance in clinical Clostridioides difficile is often described as unstable, since resistant strains reportedly appear susceptible following freezer storage or brief passage. This has presented a conundrum for adopting susceptibility testing to accurately evaluate the connection between metronidazole resistance and decreased clinical efficacy of metronidazole in patients with C. difficile infections (CDIs). We discovered that supplementation of microbiological media with the metalloporphyrin heme is crucial for detection of metronidazole-resistant C. difficile using the agar dilution susceptibility testing method. Known metronidazole-resistant strains appeared susceptible to metronidazole in media lacking heme. Similarly, these resistant strains exhibited increased susceptibility to metronidazole when tested on heme-containing agars that were exposed to room light for more than 1 day, likely due to heme photodecomposition. In parallel experiments, resistance was reproducibly detected when heme-containing agars were either prepared and used on the same day or protected from light and then used on subsequent days. Notably, heme did not influence the susceptibilities of drug-susceptible strains that were of the same ribotype as the resistant strains. These findings firmly show that the consistent detection of metronidazole-resistant C. difficile is dependent upon heme and its protection from light. Studies are warranted to determine the extent to which this heme-associated metronidazole-resistant phenotype affects the clinical efficacy of metronidazole in CDI and the underlying genetic and biochemical mechanisms.

Non-proteolytic activity of 19S proteasome subunit RPT-6 regulates GATA transcription during response to infection.

GATA transcription factors play a crucial role in the regulation of immune functions across metazoans. In Caenorhabditis elegans, the GATA transcription factor ELT-2 is involved in the control of not only infections but also recovery after an infection. We identified RPT-6, part of the 19S proteasome subunit, as an ELT-2 binding partner that is required for the proper expression of genes required for both immunity against bacterial infections and recovery after infection. We found that the intact ATPase domain of RPT-6 is required for the interaction and that inhibition of rpt-6 affected the expression of ELT-2-controlled genes, preventing the appropriate immune response against Pseudomonas aeruginosa and recovery from infection by the pathogen. Further studies indicated that SKN-1, which is an Nrf transcription factor involved in the response to oxidative stress and infection, is activated by inhibition of rpt-6. Our results indicate that RPT-6 interacts with ELT-2 in vivo to control the expression of immune genes in a manner that is likely independent of the proteolytic activity of the proteasome.

Role of GATA transcription factor ELT-2 and p38 MAPK PMK-1 in recovery from acute P. aeruginosa infection in C. elegans.

Infectious diseases caused by bacterial pathogens reduce the fitness of their associated host but are generally limited in duration. In order for the diseased host to regain any lost fitness upon recovery, a variety of molecular, cellular, and physiological processes must be employed. To better understand mechanisms underlying the recovery process, we have modeled an acute Pseudomonas aeruginosa infection in C. elegans using brief exposures to this pathogen and subsequent antibiotic treatment. To identify host genes altered during recovery from P. aeruginosa infection, we performed whole genome expression profiling. The analysis of this dataset indicated that the activity of the host immune system is down-regulated upon recovery and revealed shared and pathogen-specific host responses during recovery. We determined that the GATA transcription factor ELT-2 and the p38 MAP kinase PMK-1 are necessary for animals to successfully recover from an acute P. aeruginosa infection. In addition, we found that ELT-2 plays a more prominent and earlier role than PMK-1 during recovery. Our data sheds further light on the molecular mechanisms and transcriptional programs involved in recovery from an acute bacterial infection, which provides a better understanding of the entire infectious disease process.

Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria.

Polymyxins are polycationic antimicrobial peptides that are currently the last-resort antibiotics for the treatment of multidrug-resistant, Gram-negative bacterial infections. The reintroduction of polymyxins for antimicrobial therapy has been followed by an increase in reports of resistance among Gram-negative bacteria. Some bacteria, such as Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii, develop resistance to polymyxins in a process referred to as acquired resistance, whereas other bacteria, such as Proteus spp., Serratia spp., and Burkholderia spp., are naturally resistant to these drugs. Reports of polymyxin resistance in clinical isolates have recently increased, including acquired and intrinsically resistant pathogens. This increase is considered a serious issue, prompting concern due to the low number of currently available effective antibiotics. This review summarizes current knowledge concerning the different strategies bacteria employ to resist the activities of polymyxins. Gram-negative bacteria employ several strategies to protect themselves from polymyxin antibiotics (polymyxin B and colistin), including a variety of lipopolysaccharide (LPS) modifications, such as modifications of lipid A with phosphoethanolamine and 4-amino-4-deoxy-L-arabinose, in addition to the use of efflux pumps, the formation of capsules and overexpression of the outer membrane protein OprH, which are all effectively regulated at the molecular level. The increased understanding of these mechanisms is extremely vital and timely to facilitate studies of antimicrobial peptides and find new potential drugs targeting clinically relevant Gram-negative bacteria.