Synergy between amikacin and Protium heptaphyllum essential oil against polymyxin resistance Klebsiella pneumoniae.

AIMS: We investigated the chemical composition and the in vitro and in vivo antibacterial effects of Protium heptaphyllum essential oil (PHEO) alone and in combination with antibiotics against polymyxin-resistant Klebsiella pneumoniae isolates. METHODS AND RESULTS: Hydrodistillation was used to obtain PHEO, and gas chromatography coupled with mass spectrometry revealed α-pinene, δ-3-carene, and ß-pinene as major components present in PHEO. Minimum inhibitory concentration was determined using the broth microdilution technique and ranged from 256 to 512 µg ml-1. The checkerboard method showed synergy with the combination of PHEO and amikacin (AMK) against the polymyxin-resistant K. pneumoniae isolates. In 8 of the 10 isolates tested, the fractional inhibitory concentration indexes (FICIs) ranged from 0.06 to 0.5, while in the remaining two isolates, the combination exerted an additive effect (FICI of 0.6 and 1.0), resulting in AMK dose reduce of range 2- to 16-fold, in the presence of PHEO. Analysis using zero interaction potency revealed high synergy score (63.9). In the in vivo assay, the survival of Caenorhabditis elegans was significantly improved in the presence of PHEO (1 µg ml-1) + AMK (µg ml-1) combination as compared to 32 µg ml-1 AMK alone. Furthermore, PHEO concentrations of 256 and 512 µg ml-1 were found to be non-toxic on the experimental model. CONCLUSION: To our knowledge, this is the first report of such type of synergism demonstrating an antimicrobial effect against polymyxin-resistant K. pneumoniae isolates.

Glucose Induces Resistance to Polymyxins in High-Alcohol-Producing Klebsiella pneumoniae via Increasing Capsular Polysaccharide and Maintaining Intracellular ATP.

High-alcohol-producing K. pneumoniae (HiAlc Kpn) causes nonalcoholic fatty liver disease (NAFLD) by producing excess endogenous alcohol in the gut of patients with NAFLD, using glucose as the main carbon source. The role of glucose in the response of HiAlc Kpn to environmental stresses such as antibiotics remains unclear. In this study, we found that glucose could enhance the resistance of HiAlc Kpn to polymyxins. First, glucose inhibited the expression of crp in HiAlc Kpn and promoted the increase of capsular polysaccharide (CPS), which promoted the drug resistance of HiAlc Kpn. Second, glucose maintained high ATP levels in HiAlc Kpn cells under the pressure of polymyxins, enhancing the resistance of the cells to the killing effect of antibiotics. Notably, the inhibition of CPS formation and the decrease of intracellular ATP levels could both effectively reverse glucose-induced polymyxins resistance. Our work demonstrated the mechanism by which glucose induces polymyxins resistance in HiAlc Kpn, thereby laying the foundation for developing effective treatments for NAFLD caused by HiAlc Kpn. IMPORTANCE HiAlc Kpn can use glucose to produce excess endogenous alcohol for promoting the development of NAFLD. Polymyxins are the last line of antibiotics and are commonly used to treat infections caused by carbapenem-resistant K. pneumoniae. In this study, we found that glucose increased bacterial resistance to polymyxins via increasing CPS and maintaining intracellular ATP; this increases the risk of failure to treat NAFLD caused by multidrug-resistant HiAlc Kpn infection. Further research revealed the important roles of glucose and the global regulator, CRP, in bacterial resistance and found that inhibiting CPS formation and decreasing intracellular ATP levels could effectively reverse glucose-induced polymyxins resistance. Our work reveals that glucose and the regulatory factor CRP can affect the resistance of bacteria to polymyxins, laying a foundation for the treatment of infections caused by multidrug-resistant bacteria.

Polymyxin Resistance and Heteroresistance Are Common in Clinical Isolates of Achromobacter Species and Correlate with Modifications of the Lipid A Moiety of Lipopolysaccharide.

The Achromobacter genus includes opportunistic pathogens that can cause chronic infections in immunocompromised patients, especially in people with cystic fibrosis (CF). Treatment of Achromobacter infections is complicated by antimicrobial resistance. In this study, a collection of Achromobacter clinical isolates, from CF and non-CF sources, was investigated for polymyxin B (PmB) resistance. Additionally, the effect of PmB challenge in a subset of isolates was examined and the presence of PmB-resistant subpopulations within the isolates was described. Further, chemical and mass spectrometry analyses of the lipid A of Achromobacter clinical isolates enabled the determination of the most common structures and showed that PmB challenge was associated with lipid A modifications that included the addition of glucosamine and palmitoylation and the concomitant loss of the free phosphate at the C-1 position. This study demonstrates that lipid A modifications associated with PmB resistance are prevalent in Achromobacter and that subresistant populations displaying the addition of positively charged residues and additional acyl chains to lipid A can be selected for and isolated from PmB-sensitive Achromobacter clinical isolates. IMPORTANCE Achromobacter species can cause chronic and potentially severe infections in immunocompromised patients, especially in those with cystic fibrosis. Bacteria cannot be eradicated due to Achromobacter's intrinsic multidrug resistance. We report that intrinsic resistance to polymyxin B (PmB), a last-resort antimicrobial peptide used to treat infections by multiresistant bacteria, is prevalent in Achromobacter clinical isolates; many isolates also display increased resistance upon PmB challenge. Analysis of the lipopolysaccharide lipid A moiety of several Achromobacter species reveals a penta-acylated lipid A, which in the PmB-resistant isolates was modified by the incorporation of glucosamine residues, an additional acyl chain, loss of phosphates, and hydroxylation of acyl chains, all of which can enhance PmB resistance in other bacteria. We conclude that PmB resistance, particularly in Achromobacter isolates from chronic respiratory infections, is a common phenomenon, and that Achromobacter lipid A displays modifications that may confer increased resistance to polymyxins and potentially other antimicrobial peptides.

Cooperative action of SP-A and its trimeric recombinant fragment with polymyxins against Gram-negative respiratory bacteria.

The exploration of therapies combining antimicrobial lung proteins and conventional antibiotics is important due to the growing problem of multidrug-resistant bacteria. The aim of this study was to investigate whether human SP-A and a recombinant trimeric fragment (rfhSP-A) have cooperative antimicrobial activity with antibiotics against pathogenic Gram-negative bacteria. We found that SP-A bound the cationic peptide polymyxin B (PMB) with an apparent dissociation constant (K D) of 0.32 ± 0.04 µM. SP-A showed synergistic microbicidal activity with polymyxin B and E, but not with other antibiotics, against three SP-A-resistant pathogenic bacteria: Klebsiella pneumoniae, non-typable Haemophilus influenzae (NTHi), and Pseudomonas aeruginosa. SP-A was not able to bind to K. pneumoniae, NTHi, or to mutant strains thereof expressing long-chain lipopolysaccharides (or lipooligosaccharides) and/or polysaccharide capsules. In the presence of PMB, SP-A induced the formation of SP-A/PMB aggregates that enhance PMB-induced bacterial membrane permeabilization. Furthermore, SP-A bound to a molecular derivative of PMB lacking the acyl chain (PMBN) with a K D of 0.26 ± 0.02 µM, forming SP-A/PMBN aggregates. PMBN has no bactericidal activity but can bind to the outer membrane of Gram-negative bacteria. Surprisingly, SP-A and PMBN showed synergistic bactericidal activity against Gram-negative bacteria. Unlike native supratrimeric SP-A, the trimeric rfhSP-A fragment had small but significant direct bactericidal activity against K. pneumoniae, NTHi, and P. aeruginosa. rfhSP-A did not bind to PMB under physiological conditions but acted additively with PMB and other antibiotics against these pathogenic bacteria. In summary, our results significantly improve our understanding of the antimicrobial actions of SP-A and its synergistic action with PMB. A peptide based on SP-A may aid the therapeutic use of PMB, a relatively cytotoxic antibiotic that is currently being reintroduced into clinics due to the global problem of antibiotic resistance.

Correlative proteomics identify the key roles of stress tolerance strategies in Acinetobacter baumannii in response to polymyxin and human macrophages.

The opportunistic pathogen Acinetobacter baumannii possesses stress tolerance strategies against host innate immunity and antibiotic killing. However, how the host-pathogen-antibiotic interaction affects the overall molecular regulation of bacterial pathogenesis and host response remains unexplored. Here, we simultaneously investigate proteomic changes in A. baumannii and macrophages following infection in the absence or presence of the polymyxins. We discover that macrophages and polymyxins exhibit complementary effects to disarm several stress tolerance and survival strategies in A. baumannii, including oxidative stress resistance, copper tolerance, bacterial iron acquisition and stringent response regulation systems. Using the spoT mutant strains, we demonstrate that bacterial cells with defects in stringent response exhibit enhanced susceptibility to polymyxin killing and reduced survival in infected mice, compared to the wild-type strain. Together, our findings highlight that better understanding of host-pathogen-antibiotic interplay is critical for optimization of antibiotic use in patients and the discovery of new antimicrobial strategy to tackle multidrug-resistant bacterial infections.

Overview of polymyxin resistance in Enterobacteriaceae.

Polymyxin antibiotics are disfavored owing to their potential clinical toxicity, especially nephrotoxicity. However, the dry antibiotic development pipeline, together with the increasing global prevalence of infections caused by multidrug-resistant (MDR) gram-negative bacteria, have renewed clinical interest in these polypeptide antibiotics. This review highlights the current information regarding the mechanisms of resistance to polymyxins and their molecular epidemiology. Knowledge of the resistance mechanisms and epidemiology of these pathogens is critical for the development of novel antibacterial agents and rapid treatment choices.

Label-free quantitative proteomics identifies unique proteomes of clinical isolates of the Liverpool Epidemic Strain of Pseudomonas aeruginosa and laboratory strain PAO1.

PURPOSE: Comparative genomics and phenotypic assays have shown that antibiotic resistance profiles differ among clinical isolates of Pseudomonas aeruginosa and that genotype-phenotype associations are difficult to establish for resistance phenotypes based on these comparisons alone. EXPERIMENTAL DESIGN: Here, we used label-free quantitative proteomics to compare two isolates of the Liverpool Epidemic Strain (LES) of P. aeruginosa, LESlike1 and LESB58, and the common laboratory strain P. aeruginosa PAO1 to more accurately predict functional differences between strains. RESULTS: Our results show that the proteomes of the LES isolates are more similar to each other than to PAO1; however, a number of differences were observed in the abundance of proteins involved in quorum sensing, virulence, and antibiotic resistance, including in the comparison of LESlike1 and LESB58. Additionally, the proteomic data revealed a higher abundance of proteins involved in polymyxin and aminoglycoside resistance in LESlike1. Minimum inhibitory concentration assays showed that LESlike1 had up to 128-fold higher resistance to antibiotics from these classes. CONCLUSIONS: These findings provide an example of the ability of proteomic data to complement genotypic and phenotypic studies to understand resistance in clinical isolates. CLINICAL RELEVANCE: P. aeruginosa is a predominant pathogen in chronic lung infections in individuals with cystic fibrosis (CF). LES isolates are capable of transferring between CF patients and have been associated with increased hospital visits and antibiotic treatments.

Polymyxin-Induced Metabolic Perturbations in Human Lung Epithelial Cells.

Inhaled polymyxins are associated with toxicity in human lung epithelial cells that involves multiple apoptotic pathways. However, the mechanism of polymyxin-induced pulmonary toxicity remains unclear. This study aims to investigate polymyxin-induced metabolomic perturbations in human lung epithelial A549 cells. A549 cells were treated with 0.5 or 1.0 mM polymyxin B or colistin for 1, 4, and 24 h. Cellular metabolites were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and significantly perturbed metabolites (log2 fold change [log2FC] ≥ 1; false-discovery rate [FDR] ≤ 0.2) and key pathways were identified relative to untreated control samples. At 1 and 4 h, very few significant changes in metabolites were observed relative to the untreated control cells. At 24 h, taurine (log2FC = -1.34 ± 0.64) and hypotaurine (log2FC = -1.20 ± 0.27) were significantly decreased by 1.0 mM polymyxin B. The reduced form of glutathione (GSH) was significantly depleted by 1.0 mM polymyxin B at 24 h (log2FC = -1.80 ± 0.42). Conversely, oxidized glutathione (GSSG) was significantly increased by 1.0 mM both polymyxin B (log2FC = 1.38 ± 0.13 at 4 h and 2.09 ± 0.20 at 24 h) and colistin (log2FC = 1.33 ± 0.24 at 24 h). l-Carnitine was significantly decreased by 1.0 mM of both polymyxins at 24 h, as were several key metabolites involved in biosynthesis and degradation of choline and ethanolamine (log2FC ≤ -1); several phosphatidylserines were also increased (log2FC ≥ 1). Polymyxins perturbed key metabolic pathways that maintain cellular redox balance, mitochondrial ß-oxidation, and membrane lipid biogenesis. These mechanistic findings may assist in developing new pharmacokinetic/pharmacodynamic strategies to attenuate the pulmonary toxicities of inhaled polymyxins and in the discovery of new-generation polymyxins.

Evolution of Polymyxin Resistance Regulates Colibactin Production in Escherichia coli.

The complex reservoir of metabolite-producing bacteria in the gastrointestinal tract contributes tremendously to human health and disease. Bacterial composition, and by extension gut metabolomic composition, is undoubtably influenced by the use of modern antibiotics. Herein, we demonstrate that polymyxin B, a last resort antibiotic, influences the production of the genotoxic metabolite colibactin from adherent-invasive Escherichia coli (AIEC) NC101. Colibactin can promote colorectal cancer through DNA double stranded breaks and interstrand cross-links. While the structure and biosynthesis of colibactin have been elucidated, chemical-induced regulation of its biosynthetic gene cluster and subsequent production of the genotoxin by E. coli are largely unexplored. Using a multiomic approach, we identified that polymyxin B stress enhances the abundance of colibactin biosynthesis proteins (Clb's) in multiple pks+ E. coli strains, including pro-carcinogenic AIEC, NC101; the probiotic strain, Nissle 1917; and the antibiotic testing strain, ATCC 25922. Expression analysis via qPCR revealed that increased transcription of clb genes likely contributes to elevated Clb protein levels in NC101. Enhanced production of Clb's by NC101 under polymyxin stress matched an increased production of the colibactin prodrug motif, a proxy for the mature genotoxic metabolite. Furthermore, E. coli with a heightened tolerance for polymyxin induced greater mammalian DNA damage, assessed by quantification of γH2AX staining in cultured intestinal epithelial cells. This study establishes a key link between the polymyxin B stress response and colibactin production in pks+ E. coli. Ultimately, our findings will inform future studies investigating colibactin regulation and the ability of seemingly innocuous commensal microbes to induce host disease.

Mechanisms of polymyxin resistance induced by Salmonella typhimurium in vitro.

The increase incidence of multi-drug resistant (MDR) Salmonella has become a major global health concern. Polymyxin, an ancient polypeptide antibiotic, has been given renewed attention over recent years, resulting in resistance of Gram-negative bacteria to polymyxin, but its resistance mechanism is not completely clear. Thus, it is important to study its resistance mechanisms. In this study, an in vitro induced polymyxin-resistant strain of Salmonella typhimurium in the laboratory were constructed to investigate the mechanism of resistance of Salmonella to polymyxin. Gradual induction of Salmonella typhimurium ATCC13311 (AT) by concentration increment was used to screen for a highly polymyxin-resistant strain AT-P128. The broth dilution technique was used to compare the sensitivity of the two strains to different antimicrobial drugs. Single nucleotide polymorphisms (SNPs) were then identified by whole genome sequencing, and differences in gene expression between the two strains were compared by transcriptome sequencing and reverse transcription-quantitative PCR (RT-qPCR). Finally, for the first time, the CRISPR/Cas9 gene-editing system was used to construct gene deletion mutants in Salmonella to knock out the phoP gene of AT-P128. The results showed that strain AT-P128 was significantly more resistant to amoxicillin, ceftiofur, ampicillin, fluphenazine, and chloramphenicol and significantly less resistant to sulfamethoxazole than the parental strain AT. The growth curve results showed no significant change in the growth rate between AT-P128 and AT. Motility and biofilm formation assays showed a significant decrease in AT-P128. Additionally, the WGS results showed that AT-P128 had mutations in 9 genes involving 14 SNPs. RNA-seq and RT-qPCR results showed increased expression of phoPQ. The loss of the phoP gene decreased AT-P128ΔphoP resistance to polymyxin by 32-fold. These results suggested that polymyxin resistance affected the biology, genome components, and gene expression levels of Salmonella and that the PhoPQ two-component system played a key role in polymyxin resistance in Salmonella, providing insights into the diversity and complexity of polymyxin resistance in Salmonella.

Polymyxins for the treatment of lower respiratory tract infections: lessons learned from the integration of clinical pharmacokinetic studies and clinical outcomes.

The global rise in nosocomial pneumonia caused by multidrug-resistant (MDR) Gram-negative pathogens and the increasingly limited antibiotic treatment options are growing threats to modern medicine. As a result, older antibiotics such as polymyxins are being used as last-resort drugs for MDR nosocomial pneumonia. Polymyxins are bactericidal against most aerobic Gram-negative bacilli. High-dose intravenous (IV) adminsitration of polymyxins, however, results in subtherapeutic concentrations at the site of infection making treatment challenging. Alternative forms of polymyxin delivery have been considered in order to better achieve the necessary concentrations at the site of infection. Several studies have evaluated the effectiveness of aerosolised polymyxins in patients with nosocomial pneumonia caused by MDR Gram-negative pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae. Here we evaluated the pharmacokinetic data supporting the use of inhaled polymyxins in nosocomial pneumonia and provide insight into the limitations and challenges that future studies should address. We have also reviewed the literature published between 2006 and 2020 on the use of aerosolised polymyxins for the treatment of nosocomial pneumonia, including ventilator-associated pneumonia, in patients without cystic fibrosis to evaluate their safety and efficacy as monotherapy or as an adjunct to IV antimicrobials. This review highlights the need for well-designed multicentre studies with standardised methodologies to further evaluate the effectiveness of inhaled polymyxins and to provide reliable pharmacokinetic/pharmacodynamic data in order to redefine appropriate dosing strategies.

Resistência aos antimicrobianos e diversidade genética de variantes fenotípicas de Pseudomonas aeruginosa não sensíveis aos carbapenêmicos recuperadas de pacientes com fibrose cística com infecção pulmonar crônica: uma análise temporal / Antimicrobial resistance and genetic diversity of phenotypic variants of carbapenem non susceptible Pseudomonas aeruginosa recovered from patients with cystic fibrosis with chronic lung infection: a temporal analysis

Pseudomonas aeruginosa, bactéria ubíqua e versátil, pode se comportar como um patógeno oportunista, com ampla capacidade adaptativa, por múltiplos fatores de virulência e resistência. Como agente patogênico nas infecções pulmonares em pacientes com fibrose cística (FC), é motivo de prognóstico ruim, aumento de hospitalizações e altas taxas de morbimortalidade, sendo quase impossível a sua erradicação, ao evoluírem para a cronicidade. Globalmente, é notável o aumento nos índices de amostras não sensíveis aos carbapenêmicos e a múltiplos antimicrobianos, essenciais à terapêutica. Assim, avaliamos temporalmente a susceptibilidade aos antimicrobianos e a presença de amostras hipermutáveis (HPM) em P. aeruginosa de diferentes morfotipos, não sensíveis aos carbapenêmicos (PANSC), obtidas de pacientes FC com infecção pulmonar crônica, acompanhados em dois centros de referência no Rio de Janeiro. De 2007 a 2016, a análise retrospectiva, através dos resultados obtidos no teste de disco-difusão (TDD), permitiu selecionar amostras de PANSC incluídas neste trabalho. Usando os resultados obtidos no TDD, foi definida a susceptibilidade a outros antimicrobianos, bem como os fenótipos de resistência, multi-(MDR), extensivo-(XDR) e pandroga resistentes (PDR). Adicionalmente, determinou-se a concentração inibitória mínima (CIM) para imipenem (IPM), meropenem (MEM), doripenem (DOR) e polimixina (POL). Através de teste fenotípico, foi calculada a frequência de mutação espontânea e as amostras hipermutáveis foram caracterizadas. O sequenciamento de genoma total (SGT) foi realizado em seis amostras de diferentes morfotipos, incluindo uma variante fenotípica rara, a small colony variant (SCV). Essas amostras foram recuperadas em dois episódios de exacerbação do paciente. Foram investigadas a clonalidade, resistência a antimicrobianos e virulência. Das 143 amostras, de 18 pacientes (9 pediátricos e 9 adultos), os resultados do TDD apontaram taxas de não susceptibilidade superiores a 44% para gentamicina, amicacina, tobramicina e ciprofloxacina, e maiores de 30 % para POL. Pela determinação da CIM, quase a totalidade (96%) das amostras foram não sensíveis a IMP, seguidos de 56% para MEM e 44% para DOR. Analisando-se a distribuição dos valores da CIM50 e CIM90 nos dois grupos de pacientes, os valores para IMP foram maiores entre as amostras dos pacientes pediátricos, equivalendo a 32 µg/mL e 64 µg/mL, respectivamente. Cerca de 25%, 37% e 6% eram MDR, XDR e PDR, respectivamente. Aproximadamente 12% eram HPM, e mais da metade destas foram XDR. Após o SGT, as seis amostras, recuperadas do caso clínico foram classificadas em um novo sequence type (ST2744), com a presença de genes de resistência adquiridos blaPAO, blaOXA-50, aph(3')-Iib, fosA, catB7 e crpP, apresentando mutações em genes codificadores de porinas e bombas de efluxo. Entretanto, não foram observados marcadores genéticos clássicos exclusivos para os fenótipos SCV e HPM. Este é o primeiro relato de P. aeruginosa SCV na FC, no Brasil. A vigilância epidemiológica de P. aeruginosa é crucial para a conduta terapêutica, bem como para o sucesso da resposta do paciente e erradicação da infecção pulmonar, justificando o uso de técnicas fenotípicas e moleculares na detecção dos mecanismos de resistência e virulência desse microrganismo na FC.

Pseudomonas aeruginosa, a ubiquitous and versatile bacterium, can behave as an opportunistic pathogen, with strong adaptive capacity, due to multiple virulence and resistance factors. As a pulmonary infection pathogen in patients with cystic fibrosis (CF), it is related with poor prognosis, increased hospitalizations and high rates of morbidity and mortality, and the eradication is almost impossible, especially after chronicity. The increase rates of isolates non-susceptible to carbapenem and multiple antimicrobials, essentials to therapy, have been observed worldwide. Therefore, we assessed the antimicrobial susceptibility and the presence of hypermutability (HPM) in non-susceptible to carbapenem P. aeruginosa (PANSC) isolates from different morphotypes, obtained from CF patients with chronic pulmonary infection, followed at two reference centers in Rio de Janeiro. Using the results obtained by disk-diffusion test (DDT) between 2007 to 2016, we select 143 PANSC and susceptibility to other antimicrobials was defined, as well as the resistance phenotypes, multi- (MDR), extensive- (XDR) and pandrug resistant (PDR). Additionally, the minimum inhibitory concentration (MIC) for imipenem (IPM), meropenem (MEM), doripenem (DOR) and polymyxin (POL) was determined. Hypermutable isolates were characterized by determination of mutation frequency. Whole genome sequencing (WGS) was performed in six morphotypes isolates, including the small colony variant (SCV), a rare variant phenotype. These isolates were recovered in two exacerbation episodes. Clonality, antimicrobial resistance and virulence were investigated. Of the total (143 isolates) isolated from 18 patients (9 pediatric and 9 adults), non-susceptibility rates above than 44% for gentamicin, amikacin, tobramycin and ciprofloxacin, and more than 30% for POL were observed. Almost all (96%) of the isolates were non-susceptible to IPM by MIC determination, followed by 56% for MEM and 44% for DOR. MIC50 (32 µg/mL) and MIC90 (64 µg/mL) rates for IPM were higher among pediatric patient isolates and 25%, 37% and 6% were MDR, XDR and PDR, respectively. 12% of all isolates were classified as HPM and more than half were categorized as XDR. Using WGS, the six isolates recovered from the clinical case, were identified as a new sequence type (ST2744). Acquired resistance genes blaPAO, blaOXA-50, aph (3')-Iib, fosA, catB7 and crpP and mutations in encoding genes for porins and efflux pumps, was annotated. None exclusive classic genetic markers related to SCV and HPM phenotypes were not observed. This is the first Brazilian report of P. aeruginosa SCV in CF. Our results highlight the importance of epidemiological surveillance in P. aeruginosa. The application of phenotypic and molecular techniques to investigate resistance and virulence mechanisms, can contribute to therapeutic success in CF.

Treatment of infections caused by Gram-negative pathogens: current status on the pharmacokinetics/pharmacodynamics of parenteral and inhaled polymyxins in patients.

Polymyxins are increasingly used as a last resort for the treatment of infections caused by multidrug-resistant Gram-negative bacteria in patients. Over the last decade, significant progress has been made in understanding the pharmacokinetics/pharmacodynamics/toxicodynamics (PK/PD/TD) of parenteral and inhaled polymyxins. This mini-review provides an overview of polymyxin chemistry, different dose definitions, and the latest research on their clinical use, toxicities, and PK/PD after intravenous and inhalation administration. Optimising the PK/PD/TD of polymyxins in patients is critical to maximise their efficacy while minimising toxicities and the emergence of resistance.

Inhibition of the ATP synthase sensitizes Staphylococcus aureus towards human antimicrobial peptides.

Antimicrobial peptides (AMPs) are an important part of the human innate immune system for protection against bacterial infections, however the AMPs display varying degrees of activity against Staphylococcus aureus. Previously, we showed that inactivation of the ATP synthase sensitizes S. aureus towards the AMP antibiotic class of polymyxins. Here we wondered if the ATP synthase similarly is needed for tolerance towards various human AMPs, including human ß-defensins (hBD1-4), LL-37 and histatin 5. Importantly, we find that the ATP synthase mutant (atpA) is more susceptible to killing by hBD4, hBD2, LL-37 and histatin 5 than wild type cells, while no changes in susceptibility was detected for hBD3 and hBD1. Administration of the ATP synthase inhibitor, resveratrol, sensitizes S. aureus towards hBD4-mediated killing. Neutrophils rely on AMPs and reactive oxygen molecules to eliminate bacteria and the atpA mutant is more susceptible to killing by neutrophils than the WT, even when the oxidative burst is inhibited.These results show that the staphylococcal ATP synthase enhance tolerance of S. aureus towards some human AMPs and this indicates that inhibition of the ATP synthase may be explored as a new therapeutic strategy that sensitizes S. aureus to naturally occurring AMPs of the innate immune system.

Polymyxin and Bacitracin in the Irrigation Solution Provide No Benefit for Bacterial Killing in Vitro.

BACKGROUND: Many surgeons add topical antibiotics to irrigation solutions assuming that this has a local effect and eliminates bacteria. However, prior studies have suggested that the addition of antibiotics to irrigation solution confers little benefit, adds cost, may potentiate anaphylactic reactions, and may contribute to antimicrobial resistance. We sought to compare the antimicrobial efficacy and cytotoxicity of an irrigation solution containing polymyxin-bacitracin with other commonly used irrigation solutions. METHODS: Staphylococcus aureus and Escherichia coli were exposed to irrigation solutions containing topical antibiotics (500,000-U/L polymyxin and 50,000-U/L bacitracin; 1-g/L vancomycin; or 80-mg/L gentamicin), as well as commonly used irrigation solutions (saline solution 0.9%; povidone-iodine 0.3%; chlorhexidine 0.05%; Castile soap 0.45%; and sodium hypochlorite 0.125%). Following 1 and 3 minutes of exposure, surviving bacteria were manually counted. Failure to eradicate all bacteria in any of the 3 replicates was considered not effective for that respective solution. Cytotoxicity analysis in human fibroblasts, osteoblasts, and chondrocytes exposed to the irrigation solutions was performed by visualization of cell structure and was quantified by lactate dehydrogenase (LDH) activity. Efficacy and cytotoxicity were assessed in triplicate experiments, with generalized linear mixed models. RESULTS: Polymyxin-bacitracin, saline solution, and Castile soap at both exposure times were not effective at eradicating S. aureus or E. coli. In contrast, povidone-iodine, chlorhexidine, and sodium hypochlorite irrigation were effective against both S. aureus and E. coli (p < 0.001). Vancomycin irrigation was effective against S. aureus but not against E. coli, whereas gentamicin irrigation showed partial efficacy against E. coli but none against S. aureus. Within fibroblasts, the greatest cytotoxicity was seen with chlorhexidine (mean [and standard error], 49.38% ± 0.80%; p < 0.0001), followed by Castile soap (33.57% ± 2.17%; p < 0.0001) and polymyxin-bacitracin (8.90% ± 1.40%). Povidone-iodine showed the least cytotoxicity of the efficacious solutions (5.00% ± 0.86%). Similar trends were seen at both exposure times and across fibroblasts, osteoblasts, and chondrocytes. CONCLUSIONS: Irrigation with polymyxin-bacitracin was ineffective at bacterial eradication, and statistically inferior to povidone-iodine. Chlorhexidine lavage conferred the greatest in vitro cytotoxicity. CLINICAL RELEVANCE: These data suggest that the addition of polymyxin-bacitracin to saline solution irrigation has little value. Given the cost and antimicrobial resistance implications, our findings, combined with prior clinical literature, provide adequate reason to avoid widespread use of antibiotics in irrigation solutions. Povidone-iodine may be a more effective and safer option.

Molecular mechanisms of polymyxin resistance and detection of mcr genes.

Antibiotic resistance is an ever-increasing global problem. Major commercial antibiotics often fail to fight common bacteria, and some pathogens have become multi-resistant. Polymyxins are potent bactericidal antibiotics against gram-negative bacteria. Known resistance to polymyxin includes intrinsic, mutational and adaptive mechanisms, with the recently described horizontally acquired resistance mechanisms. In this review, we present several strategies for bacteria to develop enhanced resistance to polymyxins, focusing on changes in the outer membrane, efflux and other resistance determinants. Better understanding of the genes involved in polymyxin resistance may pave the way for the development of new and effective antimicrobial agents. We also report novel in silico tested primers for PCR assay that may be able distinguish colistin-resistant isolates carrying the plasmid-encoded mcr genes and will assist in combating the spread of colistin resistance in bacteria.

In situ identification of Gram-negative bacteria in human lungs using a topical fluorescent peptide targeting lipid A.

Respiratory infections in mechanically ventilated patients caused by Gram-negative bacteria are a major cause of morbidity. Rapid and unequivocal determination of the presence, localization, and abundance of bacteria is critical for positive resolution of the infections and could be used for patient stratification and for monitoring treatment efficacy. Here, we developed an in situ approach to visualize Gram-negative bacterial species and cellular infiltrates in distal human lungs in real time. We used optical endomicroscopy to visualize a water-soluble optical imaging probe based on the antimicrobial peptide polymyxin conjugated to an environmentally sensitive fluorophore. The probe was chemically stable and nontoxic and, after in-human intrapulmonary microdosing, enabled the specific detection of Gram-negative bacteria in distal human airways and alveoli within minutes. The results suggest that pulmonary molecular imaging using a topically administered fluorescent probe targeting bacterial lipid A is safe and practical, enabling rapid in situ identification of Gram-negative bacteria in humans.

Novel synthetic polymyxins kill Gram-positive bacteria.

Background: Staphylococcus aureus, including 'superbug' MRSA, is a major cause of nosocomial infections. In the European Union, up to 171 200 new nosocomial MRSA infections are acquired annually, and in the USA S. aureus causes more deaths than HIV/AIDS and tuberculosis combined. MRSA is also the first group of pathogens that infect the pulmonary tract in young patients with cystic fibrosis. Objectives: We describe two newly developed and synthesized colistin (polymyxin E)-inspired molecules. Methods: A collection of several isolates of S. aureus [including MRSA and vancomycin-resistant S. aureus (VRSA)] was tested. To check the antimicrobial activity, we performed time-kill curves, growth curves, biofilm eradication, toxicity and isothermal titration calorimetry. Results: Both peptides showed high antimicrobial activities (MIC 4 mg/L) and low relative toxicities (selectivity index close to 23). Conclusions: Successful production of polymyxin-scaffold molecules active against S. aureus, both MRSA and VRSA, opens up new approaches to the treatment of these complicated infections.

Broad-Spectrum Adaptive Antibiotic Resistance Associated with Pseudomonas aeruginosa Mucin-Dependent Surfing Motility.

Surfing motility is a novel form of surface adaptation exhibited by the nosocomial pathogen Pseudomonas aeruginosa in the presence of the glycoprotein mucin, which is found in high abundance at mucosal surfaces, especially those of the lungs of cystic fibrosis and bronchiectasis patients. Here, we investigated the adaptive antibiotic resistance of P. aeruginosa under conditions in which surfing occurs compared that in to cells undergoing swimming. P. aeruginosa surfing cells were significantly more resistant to several classes of antibiotics, including aminoglycosides, carbapenems, polymyxins, and fluoroquinolones. This was confirmed by incorporation of antibiotics into growth medium, which revealed a concentration-dependent inhibition of surfing motility that occurred at concentrations much higher than those needed to inhibit swimming. To investigate the basis of resistance, transcriptome sequencing (RNA-Seq) was performed and revealed that surfing influenced the expression of numerous genes. Included among genes dysregulated under surfing conditions were multiple genes from the Pseudomonas resistome; these genes are known to affect antibiotic resistance when mutated. Screening transposon mutants in these surfing-dysregulated resistome genes revealed that several of these mutants exhibited changes in susceptibility to one or more antibiotics under surfing conditions, consistent with a contribution to the observed adaptive resistance. In particular, several mutants in resistome genes, including armR, recG, atpB, clpS, nuoB, and certain hypothetical genes, such as PA5130, PA3576, and PA4292, showed contributions to broad-spectrum resistance under surfing conditions and could be complemented by their respective cloned genes. Therefore, we propose that surfing adaption led to extensive multidrug adaptive resistance as a result of the collective dysregulation of diverse genes.

Interaction of Bacterial Phenazines with Colistimethate in Bronchial Epithelial Cells.

Multidrug-resistant bacterial infections are being increasingly treated in clinics with polymyxins, a class of antibiotics associated with adverse effects on the kidney, nervous system, or airways of a significant proportion of human and animal patients. Although many of the resistant pathogens display enhanced virulence, the hazard of cytotoxic interactions between polymyxin antibiotics and bacterial virulence factors (VFs) has not been assessed, to date. We report here the testing of paired combinations of four Pseudomonas aeruginosa VF phenazine toxins, pyocyanin (PYO), 1-hydroxyphenazine (1-HP), phenazine-1-carboxylic acid (PCA), and phenazine-1-carboxamide (PCN), and two commonly prescribed polymyxin drugs, colistin-colistimethate sodium (CMS) and polymyxin B, in three human airway cell lines, BEAS-2B, HBE-1, and CFT-1. Cytotoxicities of individual antibiotics, individual toxins, and their combinations were evaluated by the simultaneous measurement of mitochondrial metabolic, total transcriptional/translational, and Nrf2 stress response regulator activities in treated cells. Two phenazines, PYO and 1-HP, were cytotoxic at clinically relevant concentrations (100 to 150 µM) and prompted a significant increase in oxidative stress-induced transcriptional activity in surviving cells. The polymyxin antibiotics arrested cell proliferation at clinically achievable (<1 mM) concentrations as well, with CMS displaying surprisingly high cytotoxicity (50% effective dose [ED50] = 180 µM) in BEAS-2B cells. The dose-response curves were probed by a median-effect analysis, which established a synergistically enhanced cytotoxicity of the PYO-CMS combination in all three airway cell lines; a particularly strong effect on BEAS-2B cells was observed, with a combination index (CI) of 0.27 at the ED50 PCA, PCN, and 1-HP potentiated CMS cytotoxicity to a smaller extent. The cytotoxicity of CMS could be reduced with 10 mM N-acetyl-cysteine. Iron chelators, while ineffective against the polymyxins, could rescue all three bronchial epithelial cell lines treated with lethal PYO or CMS-PYO doses. These findings suggest that further evaluations of CMS safety are needed, along with a search for means to moderate potentially cytotoxic interactions.