Dr Arya Gupta

Abstract

Cisplatin was originally discovered through its antibacterial action, and subsequently has found use as a potent broad spectrum anticancer agent. This study determines the effect of growth media and solvent on the antibacterial activity of cisplatin and its analogue, oxaliplatin. E. coli MG1655 or MG1655 ΔtolC were treated with the platinum compounds under different conditions and susceptibility was determined. Our results showed that DMSO reduced the activity of cisplatin by 4-fold (MIC 12.5 mg/L) compared with 0.9% NaCl-solubilized cisplatin (MIC 3.12 mg/L) when tested in MOPS. Surprisingly, complete loss of activity was observed in Mueller Hinton Broth II (MHB II). By supplementing MOPS with individual components of MHB II such as the sulphur containing amino acids, L-cysteine and L-methionine, individually or in combination reduced activity by ≥8-fold (MIC ≥25 mg/L). Oxaliplatin was less active against E. coli (MIC 100 mg/L) but exhibited similar inactivation in the presence of DMSO, MHBII or MOPS spiked with L-cysteine and L-methionine (MIC ≥400 mg/L). Our data suggest that the antibacterial activity of cisplatin and oxaliplatin is modulated by both choice of solvent and composition of growth media. We demonstrate that this is primarily due to sulphur-containing amino acids cysteine and methionine, an essential component of the recommended media for testing antimicrobial susceptibility, MHBII.

Cisplatin was originally discovered through its antibacterial action and subsequently has found use as a potent broad‐spectrum anticancer agent. This study determines the effect of growth media and solvent on the antibacterial activity of cisplatin and its analogue, oxaliplatin. Escherichia coli MG1655 or MG1655 ΔtolC was treated with the platinum compounds under different conditions and susceptibility was determined. Our results showed that DMSO reduced the activity of cisplatin by fourfold (MIC 12·5 mg l−1) compared with 0·9% NaCl‐solubilized cisplatin (MIC 3·12 mg l−1) when tested in MOPS. Surprisingly, complete loss of activity was observed in Mueller–Hinton Broth II (MHB II). By supplementing MOPS with individual components of MHB II such as the sulphur‐containing amino acids, L‐cysteine and L‐methionine, individually or in combination reduced activity by ≥8‐fold (MIC ≥25 mg l−1). Oxaliplatin was less active against E. coli (MIC 100 mg l−1) but exhibited similar inactivation in the presence of DMSO, MHBII or MOPS spiked with L‐cysteine and L‐methionine (MIC ≥400 mg l−1). Our data suggest that the antibacterial activity of cisplatin and oxaliplatin is modulated by both choice of solvent and composition of growth media. We demonstrate that this is primarily due to sulphur‐containing amino acids cysteine and methionine, an essential component of the recommended media for testing antimicrobial susceptibility, MHBII.

N-Leucinyl benzenesulfonamides have been discovered as a novel class of potent inhibitors of E. coli leucyl-tRNA synthetase. The binding of inhibitors to the enzyme was measured by using isothermal titration calorimetry. This provided information on enthalpy and entropy contributions to binding, which, together with docking studies, were used for structure–activity relationship analysis. Enzymatic assays revealed that N-leucinyl benzenesulfonamides display remarkable selectivity for E. coli leucyl-tRNA synthetase compared to S. aureus and human orthologues. The simplest analogue of the series, N-leucinyl benzenesulfonamide (R = H), showed the highest affinity against E. coli leucyl-tRNA synthetase and also exhibited antibacterial activity against Gram-negative pathogens (the best MIC = 8 μg/mL, E. coli ATCC 25922), which renders it as a promising template for antibacterial drug discovery.

Abstract

Bacteriophage (“phage”) are both predators and evolutionary drivers for bacteria, notably contributing to the spread of antimicrobial resistance (AMR) genes by generalised transduction. Our current understanding of the dual nature of this relationship is limited. We used an interdisciplinary approach to quantify how these interacting dynamics can lead to the evolution of multi-drug resistant bacteria. We co-cultured two strains of Methicillin-resistant Staphylococcus aureus , each harbouring a different antibiotic resistance gene, with 80α generalized transducing phage. After a growth phase of 8h, bacteria and phage surprisingly coexisted at a stable equilibrium in our culture, the level of which was dependent on the starting concentration of phage. We detected double-resistant bacteria as early as 7h, indicating that transduction of AMR genes had occurred. We developed multiple mathematical models of the bacteria and phage relationship, and found that phage-bacteria dynamics were best captured by a model in which the phage burst size decreases as the bacteria population reaches stationary phase, and where phage predation is frequency-dependent. We estimated that one in every 10 8 new phage generated was a transducing phage carrying an AMR gene, and that double-resistant bacteria were always predominantly generated by transduction rather than by growth. Our results suggest a shift in how we understand and model phage-bacteria dynamics. Although rates of generalised transduction could be interpreted as too rare to be significant, they are sufficient to consistently lead to the evolution of multi-drug resistant bacteria. Currently, the potential of phage to contribute to the growing burden of AMR is likely underestimated.

Cancer chemotherapeutics kill rapidly dividing cells, which includes cells of the immune system. The resulting neutropenia predisposes patients to infection, which delays treatment and is a major cause of morbidity and mortality. To tackle this problem, we have isolated several compounds that inhibit bacterial DNA repair, alone they are non-toxic, however in combination with DNA damaging anti-cancer drugs, they prevent bacterial growth. These compounds were identified through screening of an FDA-approved drug library in the presence of the anti-cancer compound cisplatin. Using a series of triage tests, the screen was reduced to a handful of drugs that were tested for specific activity against bacterial nucleotide excision DNA repair (NER). Five compounds emerged, of which three possess promising antimicrobial properties including cell penetrance, and the ability to block replication in a multi-drug resistant clinically relevant E. coli strain. This study suggests that targeting NER could offer a new therapeutic approach tailor-made for infections in cancer patients, by combining cancer chemotherapy with an adjuvant that targets DNA repair.

Abstract

Cancer chemotherapeutics kill rapidly dividing cells, which includes cells of the immune system. The resulting neutropenia predisposes patients to infection, which delays treatment and is a major cause of morbidity and mortality. Here we have exploited the cytotoxicity of the anti-cancer compound cisplatin to screen for FDA-approved drugs that impair bacterial nucleotide excision DNA repair (NER), the primary mechanism bacteria use to repair cisplatin lesions. Five compounds have emerged of which three possess ideal antimicrobial properties including cell penetrance, specific activity for NER, and the ability to kill a multi-drug resistant clinically relevant E. coli strain. Targeting NER offers a new therapeutic approach for infections in cancer patients by combining antimicrobial activity with cancer chemotherapy.

Resistance to the lantibiotic nisin (NIS) arises readily in Staphylococcus aureus as a consequence of mutations in the nsaS gene, which encodes the sensor kinase of the NsaRS two-component regulatory system. Here we present a series of studies to establish how these mutational changes result in reduced NIS susceptibility. Comparative transcriptomic analysis revealed upregulation of the NsaRS regulon in a NIS-resistant mutant of S. aureus versus its otherwise-isogenic progenitor, indicating that NIS resistance mutations prompt gain-of-function in NsaS. Two putative ABC transporters (BraDE and VraDE) encoded within the NsaRS regulon that have been reported to provide a degree of intrinsic protection against NIS were shown to be responsible for acquired NIS resistance; as is the case for intrinsic NIS resistance, NIS detoxification was ultimately mediated by VraDE, with BraDE participating in the signaling cascade underlying VraDE expression. Our study revealed new features of this signal transduction pathway, including that BraDE (but not VraDE) physically interacts with NsaRS. Furthermore, while BraDE has been shown to sense stimuli and signal to NsaS in a process that is contingent upon ATP hydrolysis, we established that this protein complex is also essential for onward transduction of the signal from NsaS through energy-independent means. NIS resistance in S. aureus therefore joins the small number of documented examples in which acquired antimicrobial resistance results from the unmasking of an intrinsic detoxification mechanism through gain-of-function mutation in a regulatory circuit.

Increasing antimicrobial drug resistance represents a global existential threat. Infection is a particular problem in immunocompromised individuals, such as patients undergoing cancer chemotherapy, due to the targeting of rapidly dividing cells by antineoplastic agents. We recently developed a strategy that targets bacterial nucleotide excision DNA repair (NER) to identify compounds that act as antimicrobial sensitizers specific for patients undergoing cancer chemotherapy. Building on this, we performed a virtual drug screening of a ~120,000 compound library against the key NER protein UvrA. From this, numerous target compounds were identified and of those a candidate compound, Bemcentinib (R428), showed a strong affinity toward UvrA. This NER protein possesses four ATPase sites in its dimeric state, and we found that Bemcentinib could inhibit UvrA's ATPase activity by ~90% and also impair its ability to bind DNA. As a result, Bemcentinib strongly diminishes NER's ability to repair DNA in vitro. To provide a measure of in vivo activity we discovered that the growth of Escherichia coli MG1655 was significantly inhibited when Bemcentinib was combined with the DNA damaging agent 4-NQO, which is analogous to UV. Using the clinically relevant DNA-damaging antineoplastic cisplatin in combination with Bemcentinib against the urological sepsis-causing E. coli strain EC958 caused complete growth inhibition. This study offers a novel approach for the potential development of new compounds for use as adjuvants in antineoplastic therapy.

As the resistance of Staphylococcus aureus to antibiotics represents a major threat to global health, anti-infectives with novel mechanisms must be developed. Novel compounds were generated as potential phenylalanine tRNA synthetase (PheRS) inhibitors based on the published homology model of S. aureus PheRS to aid the design process using Molecular Operating Environment (MOE) software. PheRS was selected as it is structurally unique enzyme among the aminoacyl-tRNA syntheases (aaRS), it is considerably different from human cytosolic and human mitochondrial aaRS and it is essential and conserved across bacterial species. The designed compounds were synthesized according to different clear schemes. The compounds were confirmed by 1H NMR, 13C NMR, HRMS and/or microanalysis, and they were microbiologically evaluated.

GSK2251052 is a broad-spectrum antibacterial inhibitor of leucyl tRNA-synthetase (LeuRS) that has been evaluated in phase II clinical trials. Here, we report the identification of a clinical isolate of Staphylococcus aureus that exhibits reduced susceptibility to GSK2251052 without prior exposure to the compound and demonstrate that this phenotype is attributable to a single amino acid polymorphism (P329) within the editing domain of LeuRS.

In bacteria, nucleotide excision repair (NER) plays a major role in repairing DNA damage from a wide variety of sources. Therefore, its inhibition offers potential to develop a new antibacterial in combination with adjuvants, such as UV light. To date, only one known chemical inhibitor of NER is 2-(5-amino-1,3,4-thiadiazol-2-yl)benzo(f)chromen-3-one (ATBC) exists and targets Mycobacterium tuberculosis NER. To enable the design of future drugs, we need to understand its mechanism of action. To determine the mechanism of action, we used in silico structure-based prediction, which identified the ATP-binding pocket of Escherichia coli UvrA as a probable target. Growth studies in E. coli showed it was nontoxic alone, but able to impair growth when combined with DNA-damaging agents, and as we predicted, it reduced by an approximately 70% UvrA’s ATPase rate. Since UvrA’s ATPase activity is necessary for effective DNA binding, we used single-molecule microscopy to directly observe DNA association. We measured an approximately sevenfold reduction in UvrA molecules binding to a single molecule of dsDNA suspended between optically trapped beads. These data provide a clear mechanism of action for ATBC, and show that targeting UvrA’s ATPase pocket is effective and ATBC provides an excellent framework for the derivation of more soluble inhibitors that can be tested for activity.

Bacteriophage (phage) are both predators and evolutionary drivers for bacteria, notably contributing to the spread of antimicrobial resistance (AMR) genes by generalized transduction. Our current understanding of this complex relationship is limited. We used an interdisciplinary approach to quantify how these interacting dynamics can lead to the evolution of multidrug-resistant bacteria. We cocultured two strains of methicillin-resistant Staphylococcus aureus, each harboring a different antibiotic resistance gene, with generalized transducing phage. After a growth phase of 8 h, bacteria and phage surprisingly coexisted at a stable equilibrium in our culture, the level of which was dependent on the starting concentration of phage. We detected double-resistant bacteria as early as 7 h, indicating that transduction of AMR genes had occurred. We developed multiple mathematical models of the bacteria and phage relationship and found that phage-bacteria dynamics were best captured by a model in which phage burst size decreases as the bacteria population reaches stationary phase and where phage predation is frequency-dependent. We estimated that one in every 108 new phage generated was a transducing phage carrying an AMR gene and that double-resistant bacteria were always predominantly generated by transduction rather than by growth. Our results suggest a shift in how we understand and model phage-bacteria dynamics. Although rates of generalized transduction could be interpreted as too rare to be significant, they are sufficient in our system to consistently lead to the evolution of multidrug-resistant bacteria. Currently, the potential of phage to contribute to the growing burden of AMR is likely underestimated. IMPORTANCE Bacteriophage (phage), viruses that can infect and kill bacteria, are being investigated through phage therapy as a potential solution to the threat of antimicrobial resistance (AMR). In reality, however, phage are also natural drivers of bacterial evolution by transduction when they accidentally carry nonphage DNA between bacteria. Using laboratory work and mathematical models, we show that transduction leads to evolution of multidrug-resistant bacteria in less than 8 h and that phage production decreases when bacterial growth decreases, allowing bacteria and phage to coexist at stable equilibria. The joint dynamics of phage predation and transduction lead to complex interactions with bacteria, which must be clarified to prevent phage from contributing to the spread of AMR.

Objectives Methicillin-resistant Staphylococcus aureus (MRSA) infections impose a considerable burden on health systems, yet there is remarkable variation in the global incidence and epidemiology of MRSA. The MACOTRA consortium aimed to identify bacterial markers of epidemic success of MRSA isolates in Europe using a representative MRSA collection originating from France, the Netherlands and the United Kingdom. Methods Operational definitions of success were defined in consortium meetings to compose a balanced strain collection of successful and sporadic MRSA isolates. Isolates were subjected to antimicrobial susceptibility testing and whole-genome sequencing; genes were identified and phylogenetic trees constructed. Markers of epidemiological success were identified using genome-based time-scaled haplotypic density analysis and linear regression. Antimicrobial usage data from ESAC-Net was compared with national MRSA incidence data. Results Heterogeneity of MRSA isolate collections across countries hampered the use of a unified operational definition of success; therefore, country-specific approaches were used to establish the MACOTRA strain collection. Phenotypic antimicrobial resistance varied within related MRSA populations and across countries. In time-scaled haplotypic density analysis, fluoroquinolone, macrolide and mupirocin resistance were associated with MRSA success, whereas gentamicin, rifampicin and trimethoprim resistance were associated with sporadicity. Usage of antimicrobials across 29 European countries varied substantially, and β-lactam, fluoroquinolone, macrolide and aminoglycoside use correlated with MRSA incidence. Discussion Our results are the strongest yet to associate MRSA antibiotic resistance profiles and antibiotic usage with the incidence of infection and successful clonal spread, which varied by country. Harmonized isolate collection, typing, resistance profiling and alignment with antimicrobial usage over time will aid comparisons and further support country-specific interventions to reduce MRSA burden.

Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) are both opportunistic pathogens that are known to cause severe respiratory infections in humans. While these pathogens don't commonly cause infections in healthy individuals, they are often found in patients with chronic lung diseases, such as Cystic Fibrosis (CF). Data from people with CF disease indicates that CF lungs are predominantly infected with SA at a younger age with PA appearing later (<10 years) when it then outcompetes SA. There is, however, significant overlap where both pathogens co-exist in the patient's lungs. While clinical data on prevalence is abundant, little data exists on mechanisms of competition of two pathogens in CF lungs. Artificial sputum medium (ASM) is a culture medium that was designed to mimic sputum from CF patients. It contains components of CF sputum, such as amino acids, mucin and DNA. It was shown that PA growth in the ASM is similar to the growth in the lungs of CF patients. We hypothesised that the ASM model could be adapted to study the interaction between SA and PA in a controlled environment that is relevant to the in vivo environment of CF lungs. SA (ATCC29213) and PA (H174) were grown in the artificial sputum media (ASM). Approximately 3x105 bacteria were added to 15 mL ASM and grown at 37oC with constant shaking. Conditioned media was prepared by growing SA in ASM for 24 hours and then removing SA. SA appeared to adapt quicker to the nutrient-poor environment of ASM than PA and enters logarithmic growth immediately upon incubation, as opposed to PA, which exhibited an 8-hour lag period. When both pathogens are added simultaneously to the ASM, SA outcompeted PA and dominated the culture restricting the growth of PA. To better mimic in vivo conditions, ASM was conditioned by adding SA for 24 hours. This allowed creation of an environment that is similar to that found in the CF lungs with established SA infections. When both pathogens were added into the conditioned media, PA did not exhibit the lag phase and outcompeted SA, suppressing its growth. This effect remained even if SA outnumbered PA 10 000-fold. In an attempt to create media that is most similar to that of CF sputum, ASM was mixed 50:50 with conditioned media, which represents the constant renewal of secreted products and nutrients seen in the lungs. When both pathogens were added to the media, rapid growth was observed in both pathogens for 24 hours. After 24 hours the population of SA decreased from 1x109 CFU/ml to 3x106 CFU/ml, while the PA population remained stable at 1x108 CFU/ml. After 48 hours, the population of both pathogens remained constant. By modifying ASM with conditioned ASM, we have created a stable in vitro co-culture of Staphylococcus aureus and Pseudomonas aeruginosa. Our model can be utilised to examine the competition and/or co-existence between clinical strains and other bacterial species that reside within the lungs and identify factors which suppress pathogen growth.

Objectives To gain a more detailed understanding of endogenous (mutational) and exogenous (horizontally acquired) resistance to silver in Gram-negative pathogens, with an emphasis on clarifying the genetic bases for resistance. Methods A suite of microbiological and molecular genetic techniques was employed to select and characterize endogenous and exogenous silver resistance in several Gram-negative species. Results In Escherichia coli, endogenous resistance arose after 6 days of exposure to silver, a consequence of two point mutations that were both necessary and sufficient for the phenotype. These mutations, in ompR and cusS, respectively conferred loss of the OmpC/F porins and derepression of the CusCFBA efflux transporter, both phenotypic changes previously linked to reduced intracellular accumulation of silver. Exogenous resistance involved derepression of the SilCFBA efflux transporter as a consequence of mutation in silS, but was additionally contingent on expression of the periplasmic silver-sequestration protein SilE. Silver resistance could be selected at high frequency (>10−9) from Enterobacteriaceae lacking OmpC/F porins or harbouring the sil operon and both endogenous and exogenous resistance were associated with modest fitness costs in vitro. Conclusions Both endogenous and exogenous silver resistance are dependent on the derepressed expression of closely related efflux transporters and are therefore mechanistically similar phenotypes. The ease with which silver resistance can become selected in some bacterial pathogens in vitro suggests that there would be benefit in improved surveillance for silver-resistant isolates in the clinic, along with greater control over use of silver-containing products, in order to best preserve the clinical utility of silver.

People with cystic fibrosis-related diabetes (CFRD) suffer from chronic infections with Staphylococcus aureus and/or Pseudomonas aeruginosa. In people with CFRD, the concentration of glucose in the airway surface liquid (ASL) was shown to be elevated from 0.4 to 4 mM. The effect of glucose on bacterial growth/interactions in ASL is not well understood and here we studied the relationship between these lung pathogens in artificial sputum medium (ASM), an environment similar to ASL in vivo. S. aureus exhibited more rapid adaptation to growth in ASM than P. aeruginosa. Supplementation of ASM with glucose significantly increased the growth of S. aureus (p < 0.01, n = 5) and P. aeruginosa (p < 0.001, n = 3). ASM conditioned by the presence of S. aureus promoted growth of P. aeruginosa with less lag time compared with non-conditioned ASM, or conditioned medium that had been heated to 121 °C. Stable co-culture of S. aureus and P. aeruginosa could be established in a 50:50 mix of ASM and S. aureus-conditioned supernatant. These data indicate that glucose, in a nutrient depleted environment, can promote the growth of S. aureus and P. aeruginosa. In addition, heat labile factors present in S. aureus pre-conditioned ASM promoted the growth of P. aeruginosa. We suggest that the use of ASM allows investigation of the effects of nutrients such as glucose on common lung pathogens. ASM could be further used to understand the relationship between S. aureus and P. aeruginosa in a co-culture scenario. Our model of stable co-culture could be extrapolated to include other common lung pathogens and could be used to better understand disease progression in vitro.