Intermittent alternating magnetic fields diminish metal-associated biofilm in vivo.

Prosthetic joint infection (PJI) is a complication of arthroplasty that results in significant morbidity. The presence of biofilm makes treatment difficult, and removal of the prosthesis is frequently required. We have developed a non-invasive approach for biofilm eradication from metal implants using intermittent alternating magnetic fields (iAMF) to generate targeted heating at the implant surface. The goal of this study was to determine whether iAMF demonstrated efficacy in an in vivo implant biofilm infection model. iAMF combined with antibiotics led to enhanced reduction of biofilm on metallic implants in vivo compared to antibiotics or untreated control. iAMF-antibiotic combinations resulted in a > 1 - log further reduction in biofilm burden compared to antibiotics or iAMF alone. This combination effect was seen in both S. aureus and P. aeruginosa and seen with multiple antibiotics used to treat infections with these pathogens. In addition, efficacy was temperature dependent with increasing temperatures resulting in a greater reduction of biofilm. Tissue damage was limited (< 1 mm from implant-tissue interface). This non-invasive approach to eradicating biofilm could serve as a new paradigm in treating PJI.

Ceftolozane/tazobactam heteroresistance in cystic fibrosis-related Pseudomonas aeruginosa infections.

Objectives: Cystic fibrosis (CF) patients are often colonized with Pseudomonas aeruginosa. During treatment, P. aeruginosa can develop subpopulations exhibiting variable in vitro antimicrobial (ABX) susceptibility patterns. Heteroresistance (HR) may underlie reported discrepancies between in vitro susceptibility results and clinical responses to various ABXs. Here, we sought to examine the presence and nature of P. aeruginosa polyclonal HR (PHR) and monoclonal HR (MHR) to ceftolozane/tazobactam in isolates originating from CF pulmonary exacerbations. Methods: This was a single-centre, non-controlled study. Two hundred and forty-six P. aeruginosa isolates from 26 adult CF patients were included. PHR was defined as the presence of different ceftolozane/tazobactam minimum inhibitory concentration (MIC) values among P. aeruginosa isolates originating from a single patient specimen. Population analysis profiles (PAPs) were performed to assess the presence of MHR, defined as ≥4-fold change in the ceftolozane/tazobactam MIC from a single P. aeruginosa colony. Results: Sixteen of 26 patient specimens (62%) contained PHR P. aeruginosa populations. Of these 16 patients, 6 (23%) had specimens in which PHR P. aeruginosa isolates exhibited ceftolozane/tazobactam MICs with categorical differences (i.e. susceptible versus resistant) compared to results reported as part of routine care. One isolate, PSA 1311, demonstrated MHR. Canonical ceftolozane/tazobactam resistance genes were not found in the MHR isolates (MHR PSA 1311 or PHR PSA 6130). Conclusions: Ceftolozane/tazobactam PHR exists among P. aeruginosa isolates in this work, and approximately a quarter of these populations contained isolates with ceftolozane/tazobactam susceptibiilty interpretations different from what was reported clinically, supporting concerns surrounding the utility of traditional susceptibility testing methodology in the setting of CF specimens. Genome sequencing of isolates with acquired MHR to ceftolozane/tazobactam revealed variants of unknown significance. Future work will be centred on determining the significance of these mutations to better understand these data in clinical context.

Clinically undetected polyclonal heteroresistance among Pseudomonas aeruginosa isolated from cystic fibrosis respiratory specimens.

BACKGROUND: Pseudomonas aeruginosa infection is the leading cause of death among patients with cystic fibrosis (CF) and a common cause of difficult-to-treat hospital-acquired infections. P. aeruginosa uses several mechanisms to resist different antibiotic classes and an individual CF patient can harbour multiple resistance phenotypes. OBJECTIVES: To determine the rates and distribution of polyclonal heteroresistance (PHR) in P. aeruginosa by random, prospective evaluation of respiratory cultures from CF patients at a large referral centre over a 1 year period. METHODS: We obtained 28 unique sputum samples from 19 CF patients and took multiple isolates from each, even when morphologically similar, yielding 280 unique isolates. We performed antimicrobial susceptibility testing (AST) on all isolates and calculated PHR on the basis of variability in AST in a given sample. We then performed whole-genome sequencing on 134 isolates and used a machine-learning association model to interrogate phenotypic PHR from genomic data. RESULTS: PHR was identified in most sampled patients (n = 15/19; 79%). Importantly, resistant phenotypes were not detected by routine AST in 26% of patients (n = 5/19). The machine-learning model, using the extended sampling, identified at least one genetic variant associated with phenotypic resistance in 94.3% of isolates (n = 1392/1476). CONCLUSION: PHR is common among P. aeruginosa in the CF lung. While traditional microbiological methods often fail to detect resistant subpopulations, extended sampling of isolates and conventional AST identified PHR in most patients. A machine-learning tool successfully identified at least one resistance variant in almost all resistant isolates by leveraging this extended sampling and conventional AST.

Alternating magnetic fields and antibiotics eradicate biofilm on metal in a synergistic fashion.

Hundreds of thousands of human implant procedures require surgical revision each year due to infection. Infections are difficult to treat with conventional antibiotics due to the formation of biofilm on the implant surface. We have developed a noninvasive method to eliminate biofilm on metal implants using heat generated by intermittent alternating magnetic fields (iAMF). Here, we demonstrate that heat and antibiotics are synergistic in biofilm elimination. For Pseudomonas aeruginosa biofilm, bacterial burden was reduced >3 log with iAMF and ciprofloxacin after 24 h compared with either treatment alone (p < 0.0001). This effect was not limited by pathogen or antibiotic as similar biofilm reductions were seen with iAMF and either linezolid or ceftriaxone in Staphylococcus aureus. iAMF and antibiotic efficacy was seen across various iAMF settings, including different iAMF target temperatures, dose durations, and dosing intervals. Initial mechanistic studies revealed membrane disruption as one factor important for AMF enhanced antibacterial activity in the biofilm setting. This study demonstrates the potential of utilizing a noninvasive approach to reduce biofilm off of metallic implants.

Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers Retain Activity against Multidrug-Resistant Pseudomonas aeruginosa In Vitro and In Vivo.

Most antimicrobials currently in the clinical pipeline are modifications of existing classes of antibiotics and are considered short-term solutions due to the emergence of resistance. Pseudomonas aeruginosa represents a major challenge for new antimicrobial drug discovery due to its versatile lifestyle, ability to develop resistance to most antibiotic classes, and capacity to form robust biofilms on surfaces and in certain hosts such as those living with cystic fibrosis (CF). A precision antibiotic approach to treating Pseudomonas could be achieved with an antisense method, specifically by using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs). Here, we demonstrate that PPMOs targeting acpP (acyl carrier protein), lpxC (UDP-(3-O-acyl)-N-acetylglucosamine deacetylase), and rpsJ (30S ribosomal protein S10) inhibited the in vitro growth of several multidrug-resistant clinical P. aeruginosa isolates at levels equivalent to those that were effective against sensitive strains. Lead PPMOs reduced established pseudomonal biofilms alone or in combination with tobramycin or piperacillin-tazobactam. Lead PPMO dosing alone or combined with tobramycin in an acute pneumonia model reduced lung bacterial burden in treated mice at 24 h and reduced morbidity up to 5 days postinfection. PPMOs reduced bacterial burden of extensively drug-resistant P. aeruginosa in the same model and resulted in superior survival compared to conventional antibiotics. These data suggest that lead PPMOs alone or in combination with clinically relevant antibiotics represent a promising therapeutic approach for combating P. aeruginosa infections.IMPORTANCE Numerous Gram-negative bacteria are becoming increasingly resistant to multiple, if not all, classes of existing antibiotics. Multidrug-resistant Pseudomonas aeruginosa bacteria are a major cause of health care-associated infections in a variety of clinical settings, endangering patients who are immunocompromised or those who suffer from chronic infections, such as people with cystic fibrosis (CF). Herein, we utilize antisense molecules that target mRNA of genes essential to bacterial growth, preventing the formation of the target proteins, including acpP, rpsJ, and lpxC We demonstrate here that antisense molecules targeted to essential genes, alone or in combination with clinically relevant antibiotics, were effective in reducing biofilms and protected mice in a lethal model of acute pneumonia.

Cefiderocol Retains Antibiofilm Activity in Multidrug-Resistant Gram-Negative Pathogens.

Cefiderocol is a siderophore cephalosporin with potent antibacterial activity against a broad range of Gram-negative pathogens, including multidrug-resistant strains. Siderophore antibiotics bind ferric iron and utilize iron transporters to cross the cell membrane. In the biofilm setting, where antibiotic resistance is high but iron scavenging is important, cefiderocol may have advantageous antimicrobial properties. In this study, we compared the antimicrobial activity of cefiderocol to that of seven commonly used antibiotics in well-characterized multidrug-resistant pathogens and then determined their efficacy in the biofilm setting. MIC90 values for cefiderocol were consistently lower than those of other antibiotics (ceftolozane-tazobactam, ceftazidime-avibactam, ceftazidime, piperacillin-tazobactam, imipenem, and tobramycin) in all strains tested. Cefiderocol treatment displayed a reduction in the levels of Pseudomonas aeruginosa biofilm (93%, P < 0.0001) superior to that seen with the other antibiotics (49% to 82%). Cefiderocol was generally as effective as or superior to the other antibiotics, depending on the pathogen-antibiotic combination, in reducing biofilm in other pathogens. There was a trend toward greater biofilm reduction seen with increased antibiotic dose or with increased frequency of antibiotic treatment. We conclude that cefiderocol effectively reduces biofilm and is a potent inhibitor of planktonic growth across a range of Gram-negative medically important pathogens.

AcrAB-TolC Inhibition by Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers Restores Antibiotic Activity in Vitro and in Vivo.

Overexpression of bacterial efflux pumps is a driver of increasing antibiotic resistance in Gram-negative pathogens. The AcrAB-TolC efflux pump has been implicated in resistance to a number of important antibiotic classes including fluoroquinolones, macrolides, and ß-lactams. Antisense technology, such as peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), can be utilized to inhibit expression of efflux pumps and restore susceptibility to antibiotics. Targeting of the AcrAB-TolC components with PPMOs revealed a sequence for acrA, which was the most effective at reducing antibiotic efflux. This acrA-PPMO enhances the antimicrobial effects of the levofloxacin and azithromycin in a panel of clinical Enterobacteriaceae strains. Additionally, acrA-PPMO enhanced azithromycin in vivo in a K. pneumoniae septicemia model. PPMOs targeting the homologous resistance-nodulation-division (RND)-efflux system in P. aeruginosa, MexAB-OprM, also enhanced potency to several classes of antibiotics in a panel of strains and in a cell culture infection model. These data suggest that PPMOs can be used as an adjuvant in antibiotic therapy to increase the efficacy or extend the spectrum of useful antibiotics against a variety of Gram-negative infections.

Fabrication and Microscopic and Spectroscopic Characterization of Cytocompatible Self-Assembling Antimicrobial Nanofibers.

The discovery of antimicrobial peptides (AMPs) has brought tremendous promise and opportunities to overcome the prevalence of bacterial resistance to commonly used antibiotics. However, their widespread use and translation into clinical application is hampered by the moderate to severe hemolytic activity and cytotoxicity. Here, we presented and validated a supramolecular platform for the construction of hemo- and cytocompatible AMP-based nanomaterials, termed self-assembling antimicrobial nanofibers (SAANs). SAANs, the "nucleus" of our antimicrobial therapeutic platform, are supramolecular assemblies of de novo designed AMPs that undergo programmed self-assembly into nanostructured fibers to "punch holes" in the bacterial membrane, thus killing the bacterial pathogen. In this study, we performed solid-state NMR spectroscopy showing predominant antiparallel ß-sheet assemblies rather than monomers to interact with liposomes. We investigated the mode of antimicrobial action of SAANs using transmission electron microscopy and provided compelling microscopic evidence that self-assembled nanofibers were physically in contact with bacterial cells causing local membrane deformation and rupture. While effectively killing bacteria, SAANs, owing to their nanoparticulate nature, were found to cross mammalian cell membranes harmlessly with greatly reduced membrane accumulation and possess exceptional cytocompatibility and hemocompatibility compared to natural AMPs. Through these systematic investigations, we expect to establish this new paradigm for the customized design of SAANs that will provide exquisite, tunable control of both bactericidal activity and cytocompatibility and can potentially overcome the drawbacks of traditional AMPs.

Morpholino oligomers tested in vitro, in biofilm and in vivo against multidrug-resistant Klebsiella pneumoniae.

Background: Klebsiella pneumoniae is an opportunistic pathogen and many strains are multidrug resistant. KPC is one of the most problematic resistance mechanisms, as it confers resistance to most ß-lactams, including carbapenems. A promising platform technology for treating infections caused by MDR pathogens is the nucleic acid-like synthetic oligomers that silence bacterial gene expression by an antisense mechanism. Objectives: To test a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) in a mouse model of K. pneumoniae infection. Methods: PPMOs were designed to target various essential genes of K. pneumoniae and screened in vitro against a panel of diverse strains. The most potent PPMOs were further tested for their bactericidal effects in broth cultures and in established biofilms. Finally, a PPMO was used to treat mice infected with a KPC-expressing strain. Results: The most potent PPMOs targeted acpP, rpmB and ftsZ and had MIC75s of 0.5, 4 and 4 µM, respectively. AcpP PPMOs were bactericidal at 1-2 × MIC and reduced viable cells and biofilm mass in established biofilms. In a mouse pneumonia model, therapeutic intranasal treatment with ∼30 mg/kg AcpP PPMO improved survival by 89% and reduced bacterial burden in the lung by ∼3 logs. Survival was proportional to the dose of AcpP PPMO. Delaying treatment by 2, 8 or 24 h post-infection improved survival compared with control groups treated with PBS or scrambled sequence (Scr) PPMOs. Conclusions: PPMOs have the potential to be effective therapeutic agents against KPC-expressing, MDR K. pneumoniae.

A Moraxella catarrhalis two-component signal transduction system necessary for growth in liquid media affects production of two lysozyme inhibitors.

There are a paucity of data concerning gene products that could contribute to the ability of Moraxella catarrhalis to colonize the human nasopharynx. Inactivation of a gene (mesR) encoding a predicted response regulator of a two-component signal transduction system in M. catarrhalis yielded a mutant unable to grow in liquid media. This mesR mutant also exhibited increased sensitivity to certain stressors, including polymyxin B, SDS, and hydrogen peroxide. Inactivation of the gene (mesS) encoding the predicted cognate sensor (histidine) kinase yielded a mutant with the same inability to grow in liquid media as the mesR mutant. DNA microarray and real-time reverse transcriptase PCR analyses indicated that several genes previously shown to be involved in the ability of M. catarrhalis to persist in the chinchilla nasopharynx were upregulated in the mesR mutant. Two other open reading frames upregulated in the mesR mutant were shown to encode small proteins (LipA and LipB) that had amino acid sequence homology to bacterial adhesins and structural homology to bacterial lysozyme inhibitors. Inactivation of both lipA and lipB did not affect the ability of M. catarrhalis O35E to attach to a human bronchial epithelial cell line in vitro. Purified recombinant LipA and LipB fusion proteins were each shown to inhibit human lysozyme activity in vitro and in saliva. A lipA lipB deletion mutant was more sensitive than the wild-type parent strain to killing by human lysozyme in the presence of human apolactoferrin. This is the first report of the production of lysozyme inhibitors by M. catarrhalis.

Identification of an outer membrane lipoprotein involved in nasopharyngeal colonization by Moraxella catarrhalis in an animal model.

Colonization of the human nasopharynx by Moraxella catarrhalis is presumed to involve attachment of this bacterium to the mucosa. DNA microarray analysis was used to determine whether attachment of M. catarrhalis to human bronchial epithelial (HBE) cells in vitro affected gene expression in this bacterium. Attachment affected expression of at least 454 different genes, with 163 being upregulated and 291 being downregulated. Among the upregulated genes was one (ORF113) previously annotated as encoding a protein with some similarity to outer membrane protein A (OmpA). The protein encoded by ORF113 was predicted to have a signal peptidase II cleavage site, and globomycin inhibition experiments confirmed that this protein was indeed a lipoprotein. The ORF113 protein also contained a predicted peptidoglycan-binding domain in its C-terminal half. The use of mutant and recombinant M. catarrhalis strains confirmed that the ORF113 protein was present in outer membrane preparations, and this protein was also shown to be at least partially exposed on the bacterial cell surface. A mutant unable to produce the ORF113 protein showed little or no change in its growth rate in vitro, in its ability to attach to HBE cells in vitro, or in its autoagglutination characteristics, but it did exhibit a reduced ability to survive in the chinchilla nasopharynx. This is the first report of a lipoprotein essential to the ability of M. catarrhalis to persist in an animal model.

Development of a LacZ-based transcriptional reporter system for use with Moraxella catarrhalis.

The lack of a transcriptional reporter system for use in Moraxella catarrhalis has hindered studies of gene regulation in this pathogen. PCR and recombinant DNA methods were used to insert a multicloning site (MCS) and promoterless full-length Escherichia coli lacZ gene, flanked by transcriptional terminators both immediately upstream and downstream, into the M. catarrhalis recombinant plasmid pWW115. Insertion into the MCS in the newly constructed plasmid pASE222 of M. catarrhalis promoter regions controlled by either a repressor (i.e., NsrR) or activator (i.e., PhoB) yielded transcriptional fusion constructs that were appropriately responsive to signal inputs dependent on the host strain genotype, as measured quantitatively by means of a Miller ß-galactosidase assay. The transcriptional reporter plasmid pASE222 should prove to be a useful tool for rapid screening of factors affecting gene expression in M. catarrhalis.

Use of the chinchilla model for nasopharyngeal colonization to study gene expression by Moraxella catarrhalis.

Young adult chinchillas were atraumatically inoculated with Moraxella catarrhalis via the nasal route. Detailed histopathologic examination of nasopharyngeal tissues isolated from these M. catarrhalis-infected animals revealed the presence of significant inflammation within the epithelium. Absence of similar histopathologic findings in sham-inoculated animals confirmed that M. catarrhalis was exposed to significant host-derived factors in this environment. Twenty-four hours after inoculation, viable M. catarrhalis organisms were recovered from the nasal cavity and nasopharynx of the animals in numbers sufficient for DNA microarray analysis. More than 100 M. catarrhalis genes were upregulated in vivo, including open reading frames (ORFs) encoding proteins that are involved in a truncated denitrification pathway or in the oxidative stress response, as well as several putative transcriptional regulators. Additionally, 200 M. catarrhalis genes were found to be downregulated when this bacterium was introduced into the nasopharynx. These downregulated genes included ORFs encoding several well-characterized M. catarrhalis surface proteins including Hag, McaP, and MchA1. Real-time reverse transcriptase PCR (RT-PCR) was utilized as a stringent control to validate the results of in vivo gene expression patterns as measured by DNA microarray analysis. Inactivation of one of the genes (MC ORF 1550) that was upregulated in vivo resulted in a decrease in the ability of M. catarrhalis to survive in the chinchilla nasopharynx over a 3-day period. This is the first evaluation of global transcriptome expression by M. catarrhalis cells in vivo.

Identification of gene products involved in the oxidative stress response of Moraxella catarrhalis.

Moraxella catarrhalis is subjected to oxidative stress from both internal and environmental sources. A previous study (C. D. Pericone, K. Overweg, P. W. Hermans, and J. N. Weiser, Infect. Immun. 68:3990-3997, 2000) indicated that a wild-type strain of M. catarrhalis was very resistant to killing by exogenous hydrogen peroxide (H2O2). The gene encoding OxyR, a LysR family transcriptional regulator, was identified and inactivated in M. catarrhalis strain O35E, resulting in an increase in sensitivity to killing by H2O2 in disk diffusion assays and a concomitant aerobic serial dilution effect. Genes encoding a predicted catalase (KatA) and an alkyl hydroperoxidase (AhpCF) showed dose-dependent upregulation in wild-type cells exposed to H2O2. DNA microarray and real-time reverse transcription-PCR (RT-PCR) analyses identified M. catarrhalis genes whose expression was affected by oxidative stress in an OxyR-dependent manner. Testing of M. catarrhalis O35E katA and ahpC mutants for their abilities to scavenge exogenous H2O2 showed that the KatA catalase was responsible for most of this activity in the wild-type parent strain. The introduction of the same mutations into M. catarrhalis strain ETSU-4 showed that the growth of a ETSU-4 katA mutant was markedly inhibited by the addition of 50 mM H2O2 but that this mutant could still form a biofilm equivalent to that produced by its wild-type parent strain.

Transcription-associated mutation in Bacillus subtilis cells under stress.

Adaptive (stationary phase) mutagenesis is a phenomenon by which nondividing cells acquire beneficial mutations as a response to stress. Although the generation of adaptive mutations is essentially stochastic, genetic factors are involved in this phenomenon. We examined how defects in a transcriptional factor, previously reported to alter the acquisition of adaptive mutations, affected mutation levels in a gene under selection. The acquisition of mutations was directly correlated to the level of transcription of a defective leuC allele placed under selection. To further examine the correlation between transcription and adaptive mutation, we placed a point-mutated allele, leuC427, under the control of an inducible promoter and assayed the level of reversion to leucine prototrophy under conditions of leucine starvation. Our results demonstrate that the level of Leu(+) reversions increased significantly in parallel with the induced increase in transcription levels. This mutagenic response was not observed under conditions of exponential growth. Since transcription is a ubiquitous biological process, transcription-associated mutagenesis may influence evolutionary processes in all organisms.

Novel role of mfd: effects on stationary-phase mutagenesis in Bacillus subtilis.

Previously, using a chromosomal reversion assay system, we established that an adaptive mutagenic process occurs in nongrowing Bacillus subtilis cells under stress, and we demonstrated that multiple mechanisms are involved in generating these mutations (41, 43). In an attempt to delineate how these mutations are generated, we began an investigation into whether or not transcription and transcription-associated proteins influence adaptive mutagenesis. In B. subtilis, the Mfd protein (transcription repair coupling factor) facilitates removal of RNA polymerase stalled at transcriptional blockages and recruitment of repair proteins to DNA lesions on the transcribed strand. Here we demonstrate that the loss of Mfd has a depressive effect on stationary-phase mutagenesis. An association between Mfd mutagenesis and aspects of transcription is discussed.

The Escherichia coli gabDTPC operon: specific gamma-aminobutyrate catabolism and nonspecific induction.

Nitrogen limitation induces the nitrogen-regulated (Ntr) response, which includes proteins that assimilate ammonia and scavenge nitrogen. Nitrogen limitation also induces catabolic pathways that degrade four metabolically related compounds: putrescine, arginine, ornithine, and gamma-aminobutyrate (GABA). We analyzed the structure, function, and regulation of the gab operon, whose products degrade GABA, a proposed intermediate in putrescine catabolism. We showed that the gabDTPC gene cluster constitutes an operon based partially on coregulation of GabT and GabD activities and the polarity of an insertion in gabT on gabC. A DeltagabDT mutant grew normally on all of the nitrogen sources tested except GABA. The unexpected growth with putrescine resulted from specific induction of gab-independent enzymes. Nac was required for gab transcription in vivo and in vitro. Ntr induction did not require GABA, but various nitrogen sources did not induce enzyme activity equally. A gabC (formerly ygaE) mutant grew faster with GABA and had elevated levels of gab operon products, which suggests that GabC is a repressor. GabC is proposed to reduce nitrogen source-specific modulation of expression. Unlike a wild-type strain, a gabC mutant utilized GABA as a carbon source and such growth required sigma(S). Previous studies showing sigma(S)-dependent gab expression in stationary phase involved gabC mutants, which suggests that such expression does not occur in wild-type strains. The seemingly narrow catabolic function of the gab operon is contrasted with the nonspecific (nitrogen source-independent) induction. We propose that the gab operon and the Ntr response itself contribute to putrescine and polyamine homeostasis.