Impact of multidrug resistance on the virulence and fitness of Pseudomonas aeruginosa: a microbiological and clinical perspective.

Pseudomonas aeruginosa is one of the most common nosocomial pathogens and part of the top emergent species associated with antimicrobial resistance that has become one of the greatest threat to public health in the twenty-first century. This bacterium is provided with a wide set of virulence factors that contribute to pathogenesis in acute and chronic infections. This review aims to summarize the impact of multidrug resistance on the virulence and fitness of P. aeruginosa. Although it is generally assumed that acquisition of resistant determinants is associated with a fitness cost, several studies support that resistance mutations may not be associated with a decrease in virulence and/or that certain compensatory mutations may allow multidrug resistance strains to recover their initial fitness. We discuss the interplay between resistance profiles and virulence from a microbiological perspective but also the clinical consequences in outcomes and the economic impact.

The balance between antibiotic resistance and fitness/virulence in Pseudomonas aeruginosa: an update on basic knowledge and fundamental research.

The interplay between antibiotic resistance and bacterial fitness/virulence has attracted the interest of researchers for decades because of its therapeutic implications, since it is classically assumed that resistance usually entails certain biological costs. Reviews on this topic revise the published data from a general point of view, including studies based on clinical strains or in vitro-evolved mutants in which the resistance phenotype is seen as a final outcome, i.e., a combination of mechanisms. However, a review analyzing the resistance/fitness balance from the basic research perspective, compiling studies in which the different resistance pathways and respective biological costs are individually approached, was missing. Here we cover this gap, specifically focusing on Pseudomonas aeruginosa, a pathogen that stands out because of its extraordinary capacity for resistance development and for which a considerable number of recent and particular data on the interplay with fitness/virulence have been released. The revised information, split into horizontally-acquired vs. mutation-driven resistance, suggests a great complexity and even controversy in the resistance-fitness/virulence balance in the acute infection context, with results ranging from high costs linked to certain pathways to others that are seemingly cost-free or even cases of resistance mechanisms contributing to increased pathogenic capacities. The elusive mechanistic basis for some enigmatic data, knowledge gaps, and possibilities for therapeutic exploitation are discussed. The information gathered suggests that resistance-fitness/virulence interplay may be a source of potential antipseudomonal targets and thus, this review poses the elementary first step for the future development of these strategies harnessing certain resistance-associated biological burdens.

Comparing biological and physical cost functions in VMAT planning for pediatric nasopharyngeal cancer.

BACKGROUND: Volumetric Modulated Arc Therapy (VMAT) is an option for the delivery of Radiotherapy treatment technique for pediatric nasopharyngeal cancer, VMAT is the most common treatment technique for pediatric nasopharyngeal cancer. The use of a combination of both biological and physical parameters in VMAT planning optimization may produce better target coverage and sparing of critical organs. This work was to compare Biological Cost Functions (BCFs) and Physical Cost Functions (PCFs) in the VMAT of pediatric nasopharyngeal cancer patients. METHOD: VMAT plans for 20 nasopharyngeal pediatric cancer patients were created using Monaco 5.11® treatment planning system (TPS). Three VMAT plans were retrospectively generated for each patient using BCFs, PCFs and mixed plan with a total dose of 61.2 Gy in 34 fractions to planning target volume (PTV). All plans were adjusted to deliver 95% of the prescribed dose to 95% of the PTV. The calculated plans were qualitatively and quantitatively evaluated using the dose-volume histogram (DVH). RESULTS: The coverage of the target and the maximum dose for the three plans were nearly the same, and better sparing was achieved in the serial organs (spinal cord and brain stem) with PCFs. On the contrary, more dose spring was observed using the BCFs in the organs at risk (OARs) that were not involved in the dose optimization, such as the optic nerve maximum dose, with a significant p-value (0.035 and 0.0001) respectively. Using the PCFs, both parotids received a lower mean dose, but not for the oral cavity, which had a lower mean dose using BCFs (p=<0.0001). The same values of tumor control probability (TCP) were found for both cost functions in PTVs and normal tissue complications probability (NTCP) (99%). The values reported were as follows: spinal cord = 0.5%, brain stem = 19.1%, and brain = 90.7% for BCFs, compared to spinal cord = 0.3%, brain stem = 14.9%, and brain = 90.7% for PCFs. The delivery time was found to be less in BCFs (p=0.005). CONCLUSION: The BCFs are superior to the PCFs in conformity index and time of radiation delivery. However, PCFs were better at dose sparing for the serial organs and achieving a sharper falloff dose around the involved volumes. A patient-specific clinical compromise is recommended to gain the best plan that meets the clinical goals.

Role of Enzymatic Activity in the Biological Cost Associated with the Production of AmpC ß-Lactamases in Pseudomonas aeruginosa.

In the current scenario of growing antibiotic resistance, understanding the interplay between resistance mechanisms and biological costs is crucial for designing therapeutic strategies. In this regard, intrinsic AmpC ß-lactamase hyperproduction is probably the most important resistance mechanism of Pseudomonas aeruginosa, proven to entail important biological burdens that attenuate virulence mostly under peptidoglycan recycling alterations. P. aeruginosa can acquire resistance to new ß-lactam-ß-lactamase inhibitor combinations (ceftazidime-avibactam and ceftolozane-tazobactam) through mutations affecting ampC and its regulatory genes, but the impact of these mutations on the associated biological cost and the role that ß-lactamase activity plays per se in contributing to the above-mentioned virulence attenuation are unknown. The same questions remain unsolved for plasmid-encoded AmpC-type ß-lactamases such as FOX enzymes, some of which also provide resistance to new ß-lactam-ß-lactamase inhibitor combinations. Here, we assessed from different perspectives the effects of changes in the active center and, thus, in the hydrolytic spectrum resistance to inhibitors of AmpC-type ß-lactamases on the fitness and virulence of P. aeruginosa, using site-directed mutagenesis; the previously described AmpC variants T96I, G183D, and ΔG229-E247; and, finally, blaFOX-4 versus blaFOX-8. Our results indicate the essential role of AmpC activity per se in causing the reported full virulence attenuation (in terms of growth, motility, cytotoxicity, and Galleria mellonella larvae killing), although the biological cost of the above-mentioned AmpC-type variants was similar to that of the wild-type enzymes. This suggests that there is not an important biological burden that may limit the selection/spread of these variants, which could progressively compromise the future effectiveness of the above-mentioned drug combinations. IMPORTANCE The growing antibiotic resistance of the top nosocomial pathogen Pseudomonas aeruginosa pushes research to explore new therapeutic strategies, for which the resistance-versus-virulence balance is a promising source of targets. While resistance often entails significant biological costs, little is known about the bases of the virulence attenuations associated with a resistance mechanism as extraordinarily relevant as ß-lactamase production. We demonstrate that besides potential energy and cell wall alterations, the enzymatic activity of the P. aeruginosa cephalosporinase AmpC is essential for causing the full attenuation associated with its hyperproduction by affecting different features related to pathogenesis, a fact exploitable from the antivirulence perspective. Less encouraging, we also show that the production of different chromosomal/plasmid-encoded AmpC derivatives conferring resistance to some of the newest antibiotic combinations causes no significantly increased biological burdens, which suggests a free way for the selection/spread of these types of variants, potentially compromising the future effectiveness of these antipseudomonal therapies.

Various cost functions evaluation of commercial biologically based treatment planning system for nasopharyngeal cancer.

The aim of this study was to evaluate various combinations of Equivalent Uniform Dose (EUD) based and Dose Volume based (DV) cost functions in terms of target coverage and organ sparing for Nasopharyngeal CA. Ten patients diagnosed with Nasopharyngeal CA were selected for this retrospective study. Different hybrid VMAT plans, including EUD- and DV-based cost functions, were generated for each patient to determine the optimum combination in terms of organ sparing and target coverage. The generated VMAT plans were evaluated based on physical and biological dose parameters. No statistical difference was observed among all plans in terms of target coverage. The p values were ≥ 0.005 for V95, Dmean, and tumor control probability (TCP). The MU efficiency was maximum (67%), and the number of segments (285 segments) was minimum in Hybrid plan. Hybrid plan showed a significant difference compared to others (p = 0.001) in terms of serial organs. Moreover, the combination of serial and parallel complication models provided better reduction of radiation dose in the parotid glands in Plan-3 (p = 0.001). In this study, better protection was obtained when DVH-based cost functions were defined for targets and a combination of EUD- and DVH-based cost functions were used for OARs.

Metabolic Shift of an Isogenic Strain of Enterococcus faecalis 14, Deficient in Its Own Bacteriocin Synthesis, as Revealed by a Transcriptomic Analysis.

The production of antimicrobial molecules often involves complex biological pathways. This study aimed at understanding the metabolic and physiological networks of enterocin EntDD14-associated function, in the bacteriocinogenic strain, Enterococcus faecalis 14. A global and comparative transcriptomic study was carried out on E. faecalis 14 and its isogenic mutant Δbac, inactivated in genes coding for EntDD14. The in vitro ability to form biofilm on polystyrene plates was assessed by the crystal violet method, while the cytotoxicity on human colorectal adenocarcinoma Caco-2 cells was determined by the Cell Counting Kit-8. Transcriptomic data revealed that 71 genes were differentially expressed in both strains. As expected, genes coding for EntDD14 were downregulated in the Δbac mutant, whereas the other 69 genes were upregulated. Upregulated genes were associated with phage-related chromosomal islands, biofilm formation capability, resistance to environmental stresses, and metabolic reprogramming. Interestingly, the Δbac mutant showed an improved bacterial growth, a high capacity to form biofilm on inanimate surfaces and a very weak cytotoxicity level. These multiple metabolic rearrangements delineate a new line of defense to counterbalance the loss of EntDD14.

Effect of Environment on the Evolutionary Trajectories and Growth Characteristics of Antibiotic-Resistant Escherichia coli Mutants.

The fitness cost to bacteria of acquisition of resistance determinants is critically under-investigated, and the identification and exploitation of these fitness costs may lead to novel therapeutic strategies that prevent the emergence of antimicrobial resistance. Here we used Escherichia coli and amoxicillin-clavulanic acid (AMC) resistance as a model to understand how the artificial environments utilized in studies of bacterial fitness could affect the emergence of resistance and associated fitness costs. Further, we explored the predictive value of this data when strains were grown in the more physiologically relevant environments of urine and urothelial organoids. Resistant E. coli isolates were selected for following 24-h exposure to sub-inhibitory concentrations of AMC in either M9, ISO, or LB, followed by growth on LB agar containing AMC. No resistant colonies emerged following growth in M9, whereas resistant isolates were detected from cultures grown in ISO and LB. We observed both within and between media-type variability in the levels of resistance and fitness of the resistant mutants grown in LB. MICs and fitness of these resistant strains in different media (M9, ISO, LB, human urine, and urothelial organoids) showed considerable variation. Media can therefore have a direct effect on the isolation of mutants that confer resistance to AMC and these mutants can exhibit unpredictable MIC and fitness profiles under different growth conditions. This preliminary study highlights the risks in relying on a single culture protocol as a model system to predict the behavior and treatment response of bacteria in vivo and highlights the importance of developing comprehensive experimental designs to ensure effective translation of diagnostic procedures to successful clinical outcomes.

Daptomycin Resistance in Clinical MRSA Strains Is Associated with a High Biological Fitness Cost.

Daptomycin remains as one of the main treatment options for Methicillin-Resistant Staphylococcus aureus (MRSA). Sporadic resistance cases reported in patients treated with either daptomycin or glycopeptides are a growing concern. In a previous study, we described a clinical case of a patient with a community-acquired MRSA infection resistant to daptomycin and with intermediate resistance to vancomycin who developed a recurrent infection with a susceptible isogenic strain. In the present work, we further investigated the sequential events to determine whether the switch from a daptomycin resistance to a susceptible phenotype was due to a phenomenon of resistance reversion or recurrent infection with a susceptible strain. Pairwise competition experiments showed that the susceptible clinical recurrent SA6850 strain had increased fitness when compared to the resistant counterpart SA6820 strain. In fact, although we have demonstrated that reversion of daptomycin resistance to daptomycin susceptible can occur in vitro after serial passages in drug-free media, phylogenetic analysis suggested that the in vivo process was the result of a recurrent infection with a previous susceptible isolate carried by the patient rather than a resistance reversion of the strain. Whole genome sequence of evolved strains showed that daptomycin resistance in MRSA is associated with a high fitness cost mediated by mutations in mprF gene, revealed as a key element of the biological cost. Moreover, we determined that daptomycin resistance-associated fitness cost was independent of vancomycin intermediate resistance phenotype, as demonstrated in additional clinical MRSA vancomycin susceptible strains. This study highlights important observations as, despite daptomycin offers a useful treatment option for the patients with persistent infections, it has to be carefully monitored. The high fitness cost associated to daptomycin resistance may explain the reduced dissemination of daptomycin resistance and the absence of daptomycin reported outbreaks.

Interplay between Colistin Resistance, Virulence and Fitness in Acinetobacter baumannii.

Acinetobacter baumannii is an important opportunistic nosocomial pathogen often resistant to multiple antibiotics classes. Colistin, an "old" antibiotic, is now considered a last-line treatment option for extremely resistant isolates. In the meantime, resistance to colistin has been reported in clinical A. baumannii strains. Colistin is a cationic peptide that disrupts the outer membrane (OM) of Gram-negative bacteria. Colistin resistance is primarily due to post-translational modification or loss of the lipopolysaccharide (LPS) molecules inserted into the outer leaflet of the OM. LPS modification prevents the binding of polymyxin to the bacterial surface and may lead to alterations in bacterial virulence. Antimicrobial pressure drives the evolution of antimicrobial resistance and resistance is often associated with a reduced bacterial fitness. Therefore, the alterations in LPS may induce changes in the fitness of A. baumannii. However, compensatory mutations in clinical A. baumannii may ameliorate the cost of resistance and may play an important role in the dissemination of colistin-resistant A. baumannii isolates. The focus of this review is to summarize the colistin resistance mechanisms, and understand their impact on the fitness and virulence of bacteria and on the dissemination of colistin-resistant A. baumannii strains.

Impact of multidrug resistance on the pathogenicity of Pseudomonas aeruginosa: in vitro and in vivo studies.

The biological cost of multidrug resistance in Pseudomonas aeruginosa (PA) remains unclear. This study aimed to evaluate the relationship between pathogenicity and the resistance profile of different PA strains, including the most common epidemic high-risk clones. Nine PA strains were studied, including two reference strains, PAO1 and PA14 [both susceptible to all antipseudomonals (multiS)], and seven clinical strains comprising three clinical multiS strains, a non-clonal multidrug-resistant (MDR) strain and the high-risk MDR clones ST111, ST235 and ST175. In vitro studies were performed to investigate growth rate, type III secretion system (TTSS) genotype, cytotoxicity and invasiveness. Additionally, a peritonitis/sepsis model was used in C57BL/6 mice. The in vitro bacterial duplication time was shorter in clinical multiS strains than in MDR-PA (0.42±0.08h vs. 0.55±0.14h; P=0.023). Among the clinical strains, exoU(+) genotype was observed only in the epidemic clone ST235. In the animal model, the probability of mortality at 48h was 70% for clinical multiS strains vs. 7.5% for clinical MDR-PA (P<0.001, log-rank). The high-risk clone ST235 was the only MDR strain that was able to cause mortality. Bacterial concentrations in peritoneal fluid were higher in mice inoculated with multiS strains compared with MDR-PA [log CFU/mL, 8.95 (IQR 3.42-9.32) vs. 1.98 (IQR 1.08-2.80); P<0.001]. These data indicate that MDR profiles are associated with a reduction in virulence of PA in a murine model. Further studies are needed to elucidate the clinical implications of these results.