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.

Compassionate use of a novel ß-lactam enhancer-based investigational antibiotic cefepime/zidebactam (WCK 5222) for the treatment of extensively-drug-resistant NDM-expressing Pseudomonas aeruginosa infection in an intra-abdominal infection-induced sepsis patient: a case report.

Infections in critically-ill patients caused by extensively-drug-resistant (XDR)-Pseudomonas aeruginosa are challenging to manage due to paucity of effective treatment options. Cefepime/zidebactam, which is currently in global Phase 3 clinical development (Clinical Trials Identifier: NCT04979806, registered on July 28, 2021) is a novel mechanism of action based ß-lactam/ ß-lactam-enhancer combination with a promising activity against a broad-range of Gram-negative pathogens including XDR P. aeruginosa. We present a case report of an intra-abdominal infection-induced sepsis patient infected with XDR P. aeruginosa and successfully treated with cefepime/zidebactam under compassionate use. The 50 year old female patient with past-history of bariatric surgery and recent elective abdominoplasty and liposuction developed secondary pneumonia and failed a prolonged course of polymyxins. The organism repeatedly isolated from the patient was a New-Delhi metallo ß-lactamase-producing XDR P. aeruginosa resistant to ceftazidime/avibactam, imipenem/relebactam and ceftolozane/tazobactam, susceptible only to cefepime/zidebactam. As polymyxins failed to rescue the patient, cefepime/zidebactam was administered under compassionate grounds leading to discharge of patient in stable condition. The present case highlights the prevailing precarious scenario of antimicrobial resistance and the need for novel antibiotics to tackle infections caused by XDR phenotype pathogens.

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.

Caracterização fenotípica e molecular de isolados de Salmonella spp. e Escherichia coli Patogênica Extraintestinal resistentes às polimixinas / Phenotypic and molecular characterization of Salmonella spp. and extraintestinal pathogenic Escherichia coli resistant to polymyxins

A resistência antimicrobiana está se tornando um grande desafio para saúde pública devido ao aumento da resistência aos beta-lactâmicos em geral. Os isolados de Salmonella spp. e Escherichia coli são os mais frequentes agentes causadores de doenças de transmissão hídrica e alimentar, mas também podem causar doenças invasivas graves, principalmente em imunodeprimidos, idosos e crianças. Ambos os patógenos vêm apresentando perfis de resistência as principais classes de antibióticos, nestes casos é necessária a busca de uma nova opção terapêutica, como por exemplo, as polimixinas. Em 2015, surgiu o primeiro relato da resistência às polimixinas mediado pelo gene mcr (mobile colistin resistance), que se disseminou por diversos continentes e ocasionou uma grande preocupação global em saúde pública. O objetivo deste trabalho foi identificar e caracterizar os mecanismos que medeiam à resistência à polimixinas em cepas de Salmonella spp. e E. coli Patogênica extraintestinal (ExPEC). Foi realizado a triagem pelo teste da gota e teste da CIM frente a colistina e polimixina B no total de 1026 isolados de Salmonella enterica e 159 isolados de ExPEC. Nos isolados resistentes foi verificado a presença de mutações nos genes (pmrA/B, phoP/Q) associados à resistência às polimixinas, e através da PCR foi feita a identificação dos genes de resistência plasmidial (mcr). Das 124 cepas de Salmonella resistentes a colistina e polimixina B, apenas um isolado foi positivo para o gene mcr-1, e este gene foi detectado em um plasmídeo do grupo IncX4. A cepa 2018.466 foi caracterizada como S. Choleraesuis proveniente de sangue de origem humana. Foram identificados 44 isolados de Salmonella spp. apresentando mutações em pmrA e pmrB. Dos 56 isolados de ExPEC resistentes a colistina, 21 isolados apresentaram o gene mcr-1. Este gene foi detectado em plasmídeos do grupo IncX4 (n=17) e em plasmídeos do grupo IncF (n=4). Cinco isolados de E.coli não apresentaram mutações nos genes estudados,sendo que três eram positivos para o gene mcr-1, enquanto as demais cepas apresentaram mutações em pmrA/B e phoP/Q. A tipagem pela PFGE foi realizada nos isolados de E.coli positivos para o gene mcr-1, com o objetivo de verificar a diversidade genética encontrada entre elas. Foram identificados 18 perfis genéticos, sem um clone principal...(AU)

Antimicrobial resistance is becoming a major public health challenge due to increasing resistance to beta-lactams in general. Salmonella spp. and Escherichia coli are the most frequent causative agents of diseases transmitted by water and food, but they can also cause serious invasive diseases, especially in immunosuppressed individuals, the elderly and children. Both pathogens have shown resistance profiles to the main classes of antibiotics, in these cases it is necessary to search for a new therapeutic option, such as polymyxins. In 2015, the first report of resistance to polymyxins mediated by the mcr gene (mobile colistin resistance) appeared, which spread across several continents and caused a major global concern in public health. The objective of this work was to identify and characterize the mechanisms that mediate resistance to polymyxins in strains of Salmonella spp. and extraintestinal pathogenic E. coli (ExPEC). Screening by drop test and MIC test against colistin and polymyxin B was performed on a total of 1026 Salmonella enterica isolates and 159 ExPEC isolates. In the resistant isolates, the presence of mutations in the genes (pmrA/B, phoP/Q) associated with resistance to polymyxins was verified, and through PCR the plasmid resistance genes (mcr) were identified. Of the 124 Salmonella strains resistant to colistin and polymyxin B, only one isolate was positive for the mcr-1 gene, and this gene was detected in a plasmid from the IncX4 group. Strain 2018.466 was characterized as S. Choleraesuis from blood of human origin. Forty-four Salmonella spp. showing mutations in pmrA and pmrB. Of the 56 colistin-resistant ExPEC isolates, 21 isolates harbored the mcr-1 gene. This gene was detected in plasmids from the IncX4 group (n=17) and in plasmids from the IncF group (n=4). Five E.coli isolates did not show mutations in the genes studied, three of which were positive for the mcr-1 gene, while the other strains showed mutations in pmrA/B and phoP/Q. Typing by PFGE was performed on E.coli isolates positive for the mcr-1 gene, with the objective of verifying the genetic diversity found among them. Eighteen genetic profiles were identified, without a main clone...(AU)

Self-Defensive Antimicrobial Surfaces Using Polymyxin-Loaded Poly(styrene sulfonate) Microgels.

Self-defensive antimicrobial surfaces are of interest because they can inhibit bacterial colonization while minimizing unnecessary antimicrobial release in the absence of a bacterial challenge. One self-defensive approach uses self-assembly to first deposit a submonolayer coating of polyelectrolyte microgels and subsequently load those microgels by complexation with small-molecule antimicrobials. The microgel/antimicrobial complexation strength is a key parameter that controls the ability of the antimicrobial both to remain sequestered within the microgels when exposed to medium and to release in response to a bacterial challenge. Here we study the relative complexation strengths of two FDA-approved cationic antibiotics─colistin (polymyxin E) and polymyxin B─with microgels of poly(styrene sulfonate) (PSS). These polymyxins are similar cyclic polypeptides with +5 charge at pH 7.4. However, polymyxin B substitutes an aromatic ring for a dimethyl moiety in colistin, and this aromaticity can influence complexation via π and hydrophobic interactions. Coarse-grained molecular dynamics shows that the free-energy change associated with polymyxin B/PSS complexation is more negative than that of colistin/PSS complexation. Experimentally, in situ optical microscopy of microgel deswelling shows that both antibiotics load quickly from low-ionic-strength phosphate buffer. The enhanced polymyxin B/PSS complexation strength is then manifested by subsequent exposure to flowing antibiotic-free buffer with varying NaCl concentration. Microgels loaded with polymyxin B remain stably deswollen to higher salt concentrations than do colistin/PSS microgels. Importantly, exposing loaded microgels to E. coli in nutrient-free-flowing phosphate buffer shows that bacteria are killed by physical contact with the loaded microgels consistent with the contact-transfer mechanism of self-defensiveness. In vitro culture experiments show that these same surfaces, nevertheless, support the adhesion, spreading and proliferation of human fetal osteoblasts. These findings suggest a pathway to create a self-defensive antimicrobial surface effective under physiological conditions based on the nonmetabolic bacteria-triggered release of FDA-approved antibiotics.

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.

Efficient side-chain deacylation of polymyxin B1 in recombinant Streptomyces strains.

OBJECTIVES: Polymyxins are antibacterial polypeptides used as "last resort" therapy option for multidrug-resistant Gram-negative bacteria. The expansion of polymyxin-resistant infections has inspired development of novel polymyxin derivatives, and deacylation is one of the critical steps in generating those antibiotics. Deacylase from Actinoplanes utahensis hydrolyze the acyl moieties of echinocandins, and also efficiently deacylates daptomycin, ramoplanin and other important antibiotics. Here, deacylase was studied considering its potential usefulness in deacylating polymyxin B1. RESULTS: All the six recombinant strains containing the deacylase gene catalyzed hydrolysis of polymyxin B1, yielding cyclic heptapeptides. The efficiency of recombinant S. albus (SAL701) was higher than that of the others, and deacylation was the most efficient at 40 °C in 0.2 M Tris buffer (pH 8.0) with 0.2 M Mg2+. The optimal substrate concentration of SAL701 was increased from 2.0 to 6.0 g/L. SAL701 was highly thermostable, showing no loss of activity at 50 °C for 12 h, and the mycelia could be recycled at least three times without loss of catalytic activity. SAL701 could not deacylate ß-lactam substrate such as penicillin G and cephalosporin C. Deacylase catalyzes the amide bond 1 closest to the nucleus of polymyxin B1 rather than the other bond, suggesting that it has high catalytic site specificity. Homology modeling and the docking results implied that Thr190 in deacylase could facilitate hydrolysis with high regioselectivity. CONCLUSIONS: These results show that SAL701 is effective in increasing the cyclic heptapeptide moiety of polymyxin B1. These properties of the biocatalyst may enable its development in the industrial production of polymyxins antibiotics.

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.

Infección de piel y tejidos blandos por Burkholderia stabilis en un niño con quemaduras graves / Skin and soft tissue infection due to Burkholderia stabilis in a severely burned child

Resumen Las heridas por quemadura representan un grave problema, sobre todo en la población pediátrica, dada la severidad de su presentación y la morbimortalidad asociada. La infección es la complicación más frecuente y grave en el paciente quemado. Las bacterias que conforman el complejo Burkholderia cepacia (CBc) son capaces de causar enfermedades en plantas, humanos y animales. En el hombre pueden establecer infecciones crónicas y frecuentemente graves, por lo general en pacientes con fibrosis quística y en inmunocomprometidos. El CBc está compuesto por al menos 22 especies filogenéticamente muy relacionadas. El objetivo de esta publicación fue describir el primer caso de una infección de piel y partes blandas por Burkholderia stabilis, una especie poco frecuente, en un niño con grandes quemaduras en la Argentina. Las especies del CBc son intrínsecamente resistentes a la mayoría de los antimicrobianos disponibles clínicamente, como aminoglucósidos, quinolonas, polimixinas y β-lactámicos. Esto representa un serio problema en el momento de tratar las infecciones por las escasas opciones terapéuticas.

Abstract Burn wounds represent a serious problem, especially in the pediatric population, given the severity of their presentation and the associated morbidity and mortality. Infection is the most frequent and serious complication in the burned patient. Burkholderia cepacia (CBc) complex bacteria are capable of causing disease in plants, humans, and animals. In human beings they can establish chronic and frequently serious infections, generally in patients with cystic fibrosis and in immunocompromised patients. The CBc is composed of 22 phylogenetically closely related species. The objective of this publication was to describe the first report of a skin and soft tissue infection by Burkholderia stabilis, a rare species, in a child with extensive burns in Argentina. CBc species are inherently resistant to most clinically available antimicrobials, such as aminoglycosides, quinolones, polymyxins, and β-lactams. This represents a serious problem when treating infections, due to the limited therapeutic options.

Resumo As feridas por queimadura representam um grave problema, principalmente na população pediátrica, devido à gravidade de sua apresentação e morbimortalidade associada. A infecção é a complicação mais frequente e grave do paciente queimado. As bactérias que compõem o complexo Burkholderia cepacia (CBc) são capazes de causar doenças em plantas, humanos e animais. No homem, podem estabelecer infecções crônicas e freqüentemente graves, geralmente em pacientes com fibrose cística e imunocomprometidos. O CBc é composto, no mínimo, por 22 espécies filogeneticamente muito relacionadas. O objetivo desta publicação é descrever o primeiro caso de uma infecção de pele e tecidos moles por Burkholderia stabilis, uma espécie rara, em uma criança com queimaduras extensas na Argentina. As espécies do CBc são inerentemente resistentes à maioria dos antimicrobianos disponíveis clinicamente, como aminoglicosídeos, quinolonas, polimixinas e β-lactâmicos. Isso representa um problema sério na hora de tratar as infecções devido às opções terapêuticas limitadas.

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.

Effects of sub-lethal antimicrobial photodynamic therapy mediated by haematoporphyrin monomethyl ether on polymyxin-resistant Escherichia coli clinical isolate.

BACKGROUND AND AIM: It is generally believed that bacteria can not develop resistance to antimicrobial photodynamic therapy (aPDT). This work employed a polymyxin-resistant Escherichia coli clinical isolate (E15017) to study whether it could become resistant to aPDT mediated by haematoporphyrin monomethyl ether (HMME) via consecutive photodynamic treatments at sub-lethal condition. METHODS: The sub-lethal and lethal photodynamic treatment conditions for E15017 were determined by colony forming units (CFU) assay. Bacterial cells of E15017 were treated with 20 cycles of repeated sub-lethal HMME-mediated aPDT, and subsequently subjected to aPDT at lethal condition. The antibiotic susceptibility, zeta-potential and membrane integrity of sub-lethal aPDT treated E15017 cells were also investigated. RESULTS: After 20 cycles of repeated HMME-mediated aPDT treatments at sub-lethal condition, E15017 cells didn't become more resistant to aPDT. Sub-lethal HMME-mediated aPDT decreased the MIC values of E15017 to ceftazidime and polymyxin E by 4 and 2-fold, respectively, and increased the electronegativity of bacterial surface and affected the bacterial membrane integrity. CONCLUSIONS: The results obtained in this study confirmed that antibiotic-resistant bacteria could not develop resistance to aPDT, and HMME-mediated aPDT is an attractive potential treatment for MDR E. coli caused infections.

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.

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.

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.

Antibacterial activity of Cinnamomum cassia L. essential oil in a carbapenem- and polymyxin-resistant Klebsiella aerogenes strain.

INTRODUCTION: Essential oils can serve as novel sources of antibiotics for multidrug-resistant bacteria. METHODS: The multidrug-resistance profile of a Klebsiella aerogenes strain was assessed by PCR and sequencing. The antibacterial activity of Cinnamomum cassia essential oil (CCeo) against K. aerogenes was assessed by broth microdilution and time-kill methods. RESULTS: K. aerogenes showed high antibiotic resistance. The genes bla KPC-2, ampC, bla CTX-M-15, bla OXA-1, and bla TEM were present. CCeo exhibited an inhibitory effect with a minimum inhibitory concentration of 17.57 µg/mL. CONCLUSIONS: The antibacterial activity of CCeo makes it a potential candidate for treating carbapenem- and polymyxin-resistant K. aerogenes strains.