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The type VI secretion system (T6SS) is a molecular machine utilised by many Gram-negative bacteria to deliver antibacterial toxins into adjacent cells. Here we present the structure of Tse15, a T6SS Rhs effector from the nosocomial pathogen Acinetobacter baumannii. Tse15 forms a triple layered β-cocoon Rhs domain with an N-terminal α-helical clade domain and an unfolded C-terminal toxin domain inside the Rhs cage. Tse15 is cleaved into three domains, through independent auto-cleavage events involving aspartyl protease activity for toxin self-cleavage and a nucleophilic glutamic acid for N-terminal clade cleavage. Proteomic analyses identified that significantly more peptides from the N-terminal clade and toxin domains were secreted than from the Rhs cage, suggesting toxin delivery often occurs without the cage. We propose the clade domain acts as an internal chaperone to mediate toxin tethering to the T6SS machinery. Conservation of the clade domain in other Gram-negative bacteria suggests this may be a common mechanism for delivery., (© 2024. The Author(s).)

Objective: Hypermutable Pseudomonas aeruginosa strains are highly prevalent in chronic lung infections of patients with cystic fibrosis (CF). Acute exacerbations of these infections have limited treatment options. This study aimed to investigate inhaled aztreonam and tobramycin against clinical hypermutable P. aeruginosa strains using the CDC dynamic in vitro biofilm reactor (CBR), mechanism-based mathematical modelling (MBM) and genomic studies., Methods: Two CF multidrug-resistant strains were investigated in a 168 h CBR (n = 2 biological replicates). Regimens were inhaled aztreonam (75 mg 8-hourly) and tobramycin (300 mg 12-hourly) in monotherapies and combination. The simulated pharmacokinetic profiles of aztreonam and tobramycin (t 1/2 = 3 h) were based on published lung fluid concentrations in patients with CF. Total viable and resistant counts were determined for planktonic and biofilm bacteria. MBM of total and resistant bacterial counts and whole genome sequencing were completed., Results: Both isolates showed reproducible bacterial regrowth and resistance amplification for the monotherapies by 168 h. The combination performed synergistically, with minimal resistant subpopulations compared to the respective monotherapies at 168 h. Mechanistic synergy appropriately described the antibacterial effects of the combination regimen in the MBM. Genomic analysis of colonies recovered from monotherapy regimens indicated noncanonical resistance mechanisms were likely responsible for treatment failure., Conclusion: The combination of aztreonam and tobramycin was required to suppress the regrowth and resistance of planktonic and biofilm bacteria in all biological replicates of both hypermutable multidrug-resistant P. aeruginosa CF isolates. The developed MBM could be utilised for future investigations of this promising inhaled combination., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pasteurella multocida is an upper respiratory tract commensal in several mammal and bird species but can also cause severe disease in humans and in production animals such as poultry, cattle, and pigs. In this study, we performed whole-genome sequencing of P. multocida isolates recovered from a range of human infections, from the mouths of cats, and from wounds on dogs. Together with publicly available P. multocida genome sequences, we performed phylogenetic and comparative genomic analyses. While isolates from cats and dogs were spread across the phylogenetic tree, human infections were caused almost exclusively by subsp. septica strains. Most of the human isolates were capsule type A and LPS type L1 and L3; however, some strains lacked a capsule biosynthesis locus, and some strains contained a novel LPS outer-core locus, distinct from the eight LPS loci that can currently be identified using an LPS multiplex PCR. In addition, the P. multocida strains isolated from human infections contained novel mobile genetic elements. We compiled a curated database of known P. multocida virulence factor and antibiotic resistance genes (PastyVRDB) allowing for detailed characterization of isolates. The majority of human P. multocida isolates encoded a reduced range of iron receptors and contained only one filamentous hemagglutinin gene. Finally, gene-trait analysis identified a putative L-fucose uptake and utilization pathway that was over-represented in subsp. septica strains and may represent a novel host predilection mechanism in this subspecies. Together, these analyses have identified pathogenic mechanisms likely important for P. multocida zoonotic infections.IMPORTANCE Pasteurella multocida can cause serious infections in humans, including skin and wound infections, pneumonia, peritonitis, meningitis, and bacteraemia. Cats and dogs are known vectors of human pasteurellosis, transmitting P. multocida via bite wounds or contact with animal saliva. The mechanisms that underpin P. multocida human predilection and pathogenesis are poorly understood. With increasing identification of antibiotic-resistant P. multocida strains, understanding these mechanisms is vital for developing novel treatments and control strategies to combat P. multocida human infection. Here, we show that a narrow range of P. multocida strains cause disease in humans, while cats and dogs, common vectors for zoonotic infections, can harbor a wide range of P. multocida strains. We also present a curated P. multocida -specific database, allowing quick and detailed characterization of newly sequenced P. multocida isolates., Competing Interests: The authors declare no conflict of interest.

Objective: Acute exacerbations of biofilm-associated Pseudomonas aeruginosa infections in cystic fibrosis (CF) have limited treatment options. Ceftolozane/tazobactam (alone and with a second antibiotic) has not yet been investigated against hypermutable clinical P. aeruginosa isolates in biofilm growth. This study aimed to evaluate, using an in vitro dynamic biofilm model, ceftolozane/tazobactam alone and in combination with tobramycin at simulated representative lung fluid pharmacokinetics against free-floating (planktonic) and biofilm states of two hypermutable P. aeruginosa epidemic strains (LES-1 and CC274) from adolescents with CF., Methods: Regimens were intravenous ceftolozane/tazobactam 4.5 g/day continuous infusion, inhaled tobramycin 300 mg 12-hourly, intravenous tobramycin 10 mg/kg 24-hourly, and both ceftolozane/tazobactam-tobramycin combinations. The isolates were susceptible to both antibiotics. Total and less-susceptible free-floating and biofilm bacteria were quantified over 120-168 h. Ceftolozane/tazobactam resistance mechanisms were investigated by whole-genome sequencing. Mechanism-based modelling of bacterial viable counts was performed., Results: Monotherapies of ceftolozane/tazobactam and tobramycin did not sufficiently suppress emergence of less-susceptible subpopulations, although inhaled tobramycin was more effective than intravenous tobramycin. Ceftolozane/tazobactam resistance development was associated with classical (AmpC overexpression plus structural modification) and novel (CpxR mutations) mechanisms depending on the strain. Against both isolates, combination regimens demonstrated synergy and completely suppressed the emergence of ceftolozane/tazobactam and tobramycin less-susceptible free-floating and biofilm bacterial subpopulations., Conclusion: Mechanism-based modelling incorporating subpopulation and mechanistic synergy well described the antibacterial effects of all regimens against free-floating and biofilm bacterial states. These findings support further investigation of ceftolozane/tazobactam in combination with tobramycin against biofilm-associated P. aeruginosa infections in adolescents with CF., (Copyright © 2023 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Pseudomonas aeruginosa remains a challenge in chronic respiratory infections in cystic fibrosis (CF). Ceftolozane-tazobactam has not yet been evaluated against multidrug-resistant hypermutable P. aeruginosa isolates in the hollow-fiber infection model (HFIM). Isolates CW41, CW35, and CW44 (ceftolozane-tazobactam MICs of 4, 4, and 2 mg/L, respectively) from adults with CF were exposed to simulated representative epithelial lining fluid pharmacokinetics of ceftolozane-tazobactam in the HFIM. Regimens were continuous infusion (CI; 4.5 g/day to 9 g/day, all isolates) and 1-h infusions (1.5 g every 8 hours and 3 g every 8 hours, CW41). Whole-genome sequencing and mechanism-based modeling were performed for CW41. CW41 (in four of five biological replicates) and CW44 harbored preexisting resistant subpopulations; CW35 did not. For replicates 1 to 4 of CW41 and CW44, 9 g/day CI decreased bacterial counts to <3 log 10 CFU/mL for 24 to 48 h, followed by regrowth and resistance amplification. Replicate 5 of CW41 had no preexisting subpopulations and was suppressed below ~3 log 10 CFU/mL for 120 h by 9 g/day CI, followed by resistant regrowth. Both CI regimens reduced CW35 bacterial counts to <1 log 10 CFU/mL by 120 h without regrowth. These results corresponded with the presence or absence of preexisting resistant subpopulations and resistance-associated mutations at baseline. Mutations in ampC , algO , and mexY were identified following CW41 exposure to ceftolozane-tazobactam at 167 to 215 h. Mechanism-based modeling well described total and resistant bacterial counts. The findings highlight the impact of heteroresistance and baseline mutations on the effect of ceftolozane-tazobactam and limitations of MIC to predict bacterial outcomes. The resistance amplification in two of three isolates supports current guidelines that ceftolozane-tazobactam should be utilized together with another antibiotic against P. aeruginosa in CF., Competing Interests: The authors declare no conflict of interest.

Pasteurella multocida is a Gram-negative capsulated bacterium responsible for a range of diseases that cause severe morbidity and mortality in livestock animals. The hyaluronic acid (HA) capsule produced by P. multocida serogroup A strains is a critical virulence factor. In this study, we utilized transposon-directed insertion site sequencing (TraDIS) to identify genes essential for in vitro growth of P. multocida and combined TraDIS with discontinuous density gradients (TraDISort) to identify genes required for HA capsule production and regulation in this pathogen. Analysis of mutants with a high cell density phenotype, indicative of the loss of extracellular capsule, led to the identification of 69 genes important for capsule production. These genes included all previously characterized genes in the capsule biosynthesis locus and fis and hfq , which encode known positive regulators of P. multocida capsule. Many of the other capsule-associated genes identified in this study were involved in regulation or activation of the stringent response, including spoT and relA , which encode proteins that regulate the concentration of guanosine alarmones. Disruption of the autoregulatory domains in the C-terminal half of SpoT using insertional mutagenesis resulted in reduced expression of capsule biosynthesis genes and an acapsular phenotype. Overall, these findings have greatly increased the understanding of hyaluronic acid capsule production and regulation in P. multocida. IMPORTANCE The bacterial pathogen P. multocida can cause serious disease in production animals, including fowl cholera in poultry, hemorrhagic septicemia in cattle and buffalo, atrophic rhinitis in pigs, and respiratory diseases in a range of livestock. P. multocida produces a capsule that is essential for systemic disease, but the complete mechanisms underlying synthesis and regulation of capsule production are not fully elucidated. A whole-genome analysis using TraDIS was undertaken to identify genes essential for growth in rich media and to obtain a comprehensive characterization of capsule production. Many of the capsule-associated genes identified in this study were involved in the stringent response to stress, a novel finding for this important animal pathogen.

The Gram-negative pathogen Pasteurella multocida is the causative agent of many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. One mechanism by which bacteria regulate transcript abundance and protein production is riboregulation, which involves the interaction of a small RNA (sRNA) with a target mRNA to alter transcript stability and/or translational efficiency. This interaction often requires stabilization by an RNA-binding protein such as ProQ or Hfq. In Escherichia coli and a small number of other species, ProQ has been shown to play a critical role in stabilizing sRNA-mRNA interactions and preferentially binds to the 3' stem-loop regions of the mRNA transcripts, characteristic of intrinsic transcriptional terminators. The aim of this study was to determine the role of ProQ in regulating P. multocida transcript abundance and identify the RNA targets to which it binds. We assessed differentially expressed transcripts in a proQ mutant and identified sites of direct ProQ-RNA interaction using in vivo UV-cross-linking and analysis of cDNA (CRAC). These analyses demonstrated that ProQ binds to, and stabilizes, ProQ-dependent sRNAs and transfer RNAs in P. multocida via adenosine-enriched, highly structured sequences. The binding of ProQ to two RNA molecules was characterized, and these analyses showed that ProQ bound within the coding sequence of the transcript PmVP161&#95;1121, encoding an uncharacterized protein, and within the 3' region of the putative sRNA Prrc13. IMPORTANCE Regulation in P. multocida involving the RNA-binding protein Hfq is required for hyaluronic acid capsule production and virulence. This study further expands our understanding of riboregulation by examining the role of a second RNA-binding protein, ProQ, in transcript regulation and abundance in P. multocida.

Acinetobacter baumannii is a problematic nosocomial pathogen owing to its increasing resistance to antibiotics and its great ability to survive in the hospital environment, which is linked to its capacity to form biofilms. Structural and functional investigations of post-translational modifications, such as phosphorylations, may lead to identification of candidates for therapeutic targets against this pathogen. Here, we present the first S/T/Y phosphosecretome of two A. baumannii strains, the reference strain ATCC 17978 and the virulent multi-drug resistant strain AB0057, cultured in two modes of growth (planktonic and biofilm) using TiO 2 chromatography followed by high resolution mass spectrometry. In ATCC 17978, we detected a total of 137 (97 phosphoproteins) and 52 (33 phosphoproteins) phosphosites in biofilm and planktonic modes of growth, respectively. Similarly, in AB0057, 155 (119 phosphoproteins) and 102 (74 phosphoproteins) phosphosites in biofilm and planktonic modes of growth were identified, respectively. Both strains in the biofilm mode of growth showed a higher number of phosphosites and phosphoproteins compared to planktonic growth. Several phosphorylated sites are localized in key regions of proteins involved in either drug resistance (β-lactamases), adhesion to host tissues (pilins), or protein secretion (Hcp). Site-directed mutagenesis of the Hcp protein, essential for type VI secretion system-mediated interbacterial competition, showed that four of the modified residues are essential for type VI secretion system activity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Massier, Robin, Mégroz, Wright, Harper, Hayes, Cosette, Broutin, Boyce, Dé and Hardouin.)

Objectives: Hypermutable Pseudomonas aeruginosa strains are a major challenge in cystic fibrosis. We investigated bacterial killing and resistance emergence for approved ceftazidime and tobramycin regimens, alone and in combination., Methods: Pseudomonas aeruginosa PAOΔmutS and six hypermutable clinical isolates were examined using 48-h static concentration time-kill (SCTK) studies (inoculum ~10 7.5 CFU/mL); four strains were also studied in a dynamic in vitro model (IVM) (inoculum ~10 8 CFU/mL). The IVM simulated concentration-time profiles in epithelial lining fluid following intravenous administration of ceftazidime (3 g/day and 9 g/day continuous infusion), tobramycin (5 mg/kg and 10 mg/kg via 30-min infusion 24-hourly; half-life 3.5 h), and their combinations. Time courses of total and less-susceptible populations were determined., Results: Ceftazidime plus tobramycin demonstrated synergistic killing in SCTK studies for all strains, although to a lesser extent for ceftazidime-resistant strains. In the IVM, ceftazidime and tobramycin monotherapies provided ≤5.4 and ≤3.4 log 10 initial killing, respectively; however, re-growth with resistance occurred by 72 h. Against strains susceptible to one or both antibiotics, high-dose combination regimens provided >6 log 10 initial killing, which was generally synergistic from 8-24 h, and marked suppression of re-growth and resistance at 72 h. The time course of bacterial density in the IVM was well described by mechanism-based models, enabling Monte Carlo simulations (MCSs) to predict likely effectiveness of the combination in patients., Conclusion: Results of the IVM and MCS suggested antibacterial effect depends both on the strain's susceptibility and hypermutability. Further investigation of the combination against hypermutable P. aeruginosa strains is warranted., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Carbapenem-resistant Klebsiella pneumoniae has been classified as an Urgent Threat by the Centers for Disease Control and Prevention (CDC). The combination of two "old" antibiotics, polymyxin and chloramphenicol, displays synergistic killing against New Delhi metallo-β-lactamase (NDM)-producing K. pneumoniae . However, the mechanism(s) underpinning their synergistic killing are not well studied. We employed an in vitro pharmacokinetic/pharmacodynamic model to mimic the pharmacokinetics of the antibiotics in patients and examined bacterial killing against NDM-producing K. pneumoniae using a metabolomic approach. Metabolomic analysis was integrated with an isolate-specific genome-scale metabolic network (GSMN). Our results show that metabolic responses to polymyxin B and/or chloramphenicol against NDM-producing K. pneumoniae involved the inhibition of cell envelope biogenesis, metabolism of arginine and nucleotides, glycolysis, and pentose phosphate pathways. Our metabolomic and GSMN modeling results highlight the novel mechanisms of a synergistic antibiotic combination at the network level and may have a significant potential in developing precision antimicrobial chemotherapy in patients.

We describe the structural characterization of the capsular polysaccharides (CPSs) of Pasteurella multocida serotypes B and E. CPS was isolated following organic solvent precipitation of the supernatant from flask grown cells. Structural analysis utilizing nuclear magnetic resonance spectroscopy enabled the determination of the CPS structures and revealed significant structural similarities between the two serotypes, but also provided an explanation for the serological distinction. This observation was extended by the development of polyclonal sera to the glycoconjugate of serotype B CPS that corroborated the structural likenesses and differences. Finally, identification of these structures enabled a more comprehensive interrogation of the genetic loci and prediction of roles for some of the encoded proteins in repeat unit biosynthesis., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Multidrug-resistant bacteria are causing a serious global health crisis. A dramatic decline in antibiotic discovery and development investment by pharmaceutical industry over the last decades has slowed the adoption of new technologies. It is imperative that we create new mechanistic insights based on latest technologies, and use translational strategies to optimize patient therapy. Although drug development has relied on minimal inhibitory concentration testing and established in vitro and mouse infection models, the limited understanding of outer membrane permeability in Gram-negative bacteria presents major challenges. Our team has developed a platform using the latest technologies to characterize target site penetration and receptor binding in intact bacteria that inform translational modeling and guide new discovery. Enhanced assays can quantify the outer membrane permeability of β-lactam antibiotics and β-lactamase inhibitors using multiplex liquid chromatography tandem mass spectrometry. While β-lactam antibiotics are known to bind to multiple different penicillin-binding proteins (PBPs), their binding profiles are almost always studied in lysed bacteria. Novel assays for PBP binding in the periplasm of intact bacteria were developed and proteins identified via proteomics. To characterize bacterial morphology changes in response to PBP binding, high-throughput flow cytometry and time-lapse confocal microscopy with fluorescent probes provide unprecedented mechanistic insights. Moreover, novel assays to quantify cytosolic receptor binding and intracellular drug concentrations inform target site occupancy. These mechanistic data are integrated by quantitative and systems pharmacology modeling to maximize bacterial killing and minimize resistance in in vitro and mouse infection models. This translational approach holds promise to identify antibiotic combination dosing strategies for patients with serious infections., (© 2021 The Authors. Clinical Pharmacology & Therapeutics © 2021 American Society for Clinical Pharmacology and Therapeutics. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Multidrug-resistant Acinetobacter baumannii is a top-priority Gram-negative pathogen and polymyxins are a last-line therapeutic option. Previous systems pharmacological studies examining polymyxin killing and resistance usually focused on individual strains, and the derived knowledge could be limited by strain-specific genomic context. In this study, we examined the gene expression of five A. baumannii strains (34654, 1207552, 1428368, 1457504 and ATCC 19606) to determine the common differentially expressed genes in response to polymyxin treatments. A pan-genome containing 6061 genes was identified for 89 A. baumannii genomes from RefSeq database which included the five strains examined in this study; 2822 of the 6061 genes constituted the core genome. After 2 mg L -1 or 0.75 × MIC polymyxin treatments for 15 min, 41 genes were commonly up-regulated, including those involved in membrane biogenesis and homeostasis, lipoprotein and phospholipid trafficking, efflux pump and poly-N-acetylglucosamine biosynthesis; six genes were commonly down-regulated, three of which were related to fatty acid biosynthesis. Additionally, comparison of the gene expression at 15 and 60 min in ATCC 19606 revealed that polymyxin treatment resulted in a rapid change in amino acid metabolism at 15 min and perturbations on envelope biogenesis at both time points. This is the first pan-transcriptomic study for polymyxin-treated A. baumannii and our results identified that the remodelled outer membrane, up-regulated efflux pumps and down-regulated fatty acid biosynthesis might be essential for early responses to polymyxins in A. baumannii. Our findings provide important mechanistic insights into bacterial responses to polymyxin killing and may facilitate the optimisation of polymyxin therapy against this problematic 'superbug'.

The combination of polymyxins and chloramphenicol possesses synergistic killing activity against New Delhi metallo-β-lactamase (NDM)-producing Klebsiella pneumoniae. This systems study examined the transcriptomic responses to the polymyxin/chloramphenicol combination in clinical NDM-producing K. pneumoniae isolate S01. Klebsiella pneumoniae S01 (initial inoculum ~10 8 CFU/mL) was treated with polymyxin B (1 mg/L, continuous infusion) or chloramphenicol [maximum concentration (C max ) = 8 mg/L, half-life (t 1/2 ) = 4 h], alone or in combination, using an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model to mimic their pharmacokinetics in patients. Transcriptomic profiles of bacterial samples collected at 0, 0.25, 1, 4 and 24 h were examined using RNA sequencing (RNA-Seq). Chloramphenicol monotherapy significantly increased the expression of genes involved in ribosomal synthesis across the entire 24-h treatment, reflective of chloramphenicol-mediated inhibition of protein synthesis. The effect of polymyxin B was rapid and no major pathways were perturbed at later time points (4 h and 24 h). Combination treatment yielded the highest number of differentially expressed genes, including a large number observed following chloramphenicol monotherapy, in particular carbohydrate, nucleotide, amino acid and cell wall metabolism. Notably, chloramphenicol alone and in combination with polymyxin B significantly inhibited the expression of the arn operon that is responsible for lipid A modification and polymyxin resistance. These results indicate that the polymyxin/chloramphenicol combination displayed persistent transcriptomic responses over 24 h mainly on cell envelope synthesis and metabolism of carbohydrates, nucleotides and amino acids., (Copyright © 2020 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Multidrug-resistant Acinetobacter baumannii is a top-priority pathogen globally and polymyxins are a last-line therapy. Polymyxin dependence in A. baumannii (i.e., nonculturable on agar without polymyxins) is a unique and highly-resistant phenotype with a significant potential to cause treatment failure in patients. The present study discovers that a polymyxin-dependent A. baumannii strain possesses mutations in both lpxC (lipopolysaccharide biosynthesis) and katG (reactive oxygen species scavenging) genes. Correlative multiomics analyses show a significantly remodeled cell envelope and remarkably abundant phosphatidylglycerol in the outer membrane (OM). Molecular dynamics simulations and quantitative membrane lipidomics reveal that polymyxin-dependent growth emerges only when the lipopolysaccharide-deficient OM distinctively remodels with ≥ 35% phosphatidylglycerol, and with "patch" binding on the OM by the rigid polymyxin molecules containing strong intramolecular hydrogen bonding. Rather than damaging the OM, polymyxins bind to the phosphatidylglycerol-rich OM and strengthen the membrane integrity, thereby protecting bacteria from external reactive oxygen species. Dependent growth is observed exclusively with polymyxin analogues, indicating a critical role of the specific amino acid sequence of polymyxins in forming unique structures for patch-binding to bacterial OM. Polymyxin dependence is a novel antibiotic resistance mechanism and the current findings highlight the risk of 'invisible' polymyxin-dependent isolates in the evolution of resistance., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Published by WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Two-component signal transduction systems (TCSTS) are abundant among prokaryotes and regulate important functions, including drug resistance and virulence. The Gram-negative bacterium Burkholderia pseudomallei, which causes the severe infectious disease melioidosis, encodes 136 putative TCSTS components. In silico analyses of these TCSTS indicated that the predicted BbeR-BbeS system (BPSL1036-BPSL1037) displayed significant amino acid sequence similarity to the Shigella flexneri virulence-associated OmpR-EnvZ osmoregulator. To assess the function of the B. pseudomallei BbeR-BbeS system, we constructed by allelic exchange a ΔbbeRS double mutant strain lacking both genes, and single ΔbbeR and ΔbbeS mutants. All three mutant strains caused disease in the BALB/c acute melioidosis model at the same rate as the wild-type strain, displayed unchanged swarming motility on semi-solid medium, and were unaffected for viability on high-osmolarity media. However, when cultured at 37 °C for at least 14 days, ΔbbeS and ΔbbeR colonies developed a distinct, hypermucoid morphology absent in similarly-cultured wild-type colonies. At both 30 °C and 37 °C, these hypermucoid strains produced wild-type levels of type I capsule but released increased quantities of extracellular DNA (eDNA). Upon static growth in liquid medium, all B. pseudomallei strains produced pellicle biofilms that contained DNA in close association with bacterial cells; however, the ΔbbeS and ΔbbeR strains produced increased biofilms with altered microscopic architecture compared to the wild-type. Unusually, while the ΔbbeS and ΔbbeR single-deletion mutants displayed clear phenotypes, the ΔbbeRS double-deletion mutant was indistinguishable from the wild-type strain. We propose that BbeR-BbeS indirectly affects eDNA secretion and biofilm formation through cross-talk with one or more other TCSTS., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While β-lactam antibiotics are commonly used against Klebsiella pneumoniae and Enterobacter cloacae , there are limited data on OM permeability especially in K. pneumoniae Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five β-lactams in carbapenem-resistant K. pneumoniae and E. cloacae Both clinical isolates harbored a bla KPC-2 and several other β-lactamases. The OM permeability of each antibiotic was studied separately ("discrete assay") and simultaneously ("cassette assay") by determining the degradation of extracellular β-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses. Our K. pneumoniae isolate was polymyxin resistant, whereas the E. cloacae was polymyxin susceptible. Imipenem penetrated the OM at least 7-fold faster than meropenem for both isolates. Imipenem penetrated E. cloacae at least 258-fold faster and K. pneumoniae 150-fold faster compared to aztreonam, cefepime, and ceftazidime. For our β-lactams, OM permeability was substantially higher in the E. cloacae compared to the K. pneumoniae isolate (except for aztreonam). This correlated with a higher OmpC porin production in E. cloacae , as determined by proteomics. The cassette and discrete assays showed comparable results, suggesting limited or no competition during influx through OM porins. This cassette assay allowed us, for the first time, to efficiently quantify the OM permeability of multiple β-lactams in carbapenem-resistant K. pneumoniae and E. cloacae Characterizing the OM permeability presents a critical contribution to combating the antimicrobial resistance crisis and enables us to rationally optimize the use of β-lactam antibiotics. IMPORTANCE Antimicrobial resistance is causing a global human health crisis and is affecting all antibiotic classes. While β-lactams have been commonly used against susceptible isolates of Klebsiella pneumoniae and Enterobacter cloacae , carbapenem-resistant isolates are spreading worldwide and pose substantial clinical challenges. Rapid penetration of β-lactams leads to high drug concentrations at their periplasmic target sites, allowing β-lactams to more completely inactivate their target receptors. Despite this, there are limited tangible data on the permeability of β-lactams through the outer membranes of many Gram-negative pathogens. This study presents a novel, cassette assay, which can simultaneously characterize the permeability of five β-lactams in multidrug-resistant clinical isolates. We show that carbapenems, and especially imipenem, penetrate the outer membrane of K. pneumoniae and E. cloacae substantially faster than noncarbapenem β-lactams. The ability to efficiently characterize the outer membrane permeability is critical to optimize the use of β-lactams and combat carbapenem-resistant isolates.

Many Gram-negative bacteria use a type VI secretion system (T6SS) for microbial warfare and/or host manipulation. Acinetobacter baumannii is an important nosocomial pathogen and many A. baumannii strains utilize a T6SS to deliver toxic effector proteins to surrounding bacterial cells. These toxic effectors are usually delivered together with VgrG proteins, which form part of the T6SS tip complex. All previously identified A. baumannii T6SS effectors are encoded within a three- or four-gene locus that also encodes a cognate VgrG and immunity protein, and sometimes a chaperone. In order to characterize the diversity and distribution of T6SS effectors and immunity proteins in this species, we first identified all vgrG genes in 97 A. baumannii strains via the presence of the highly conserved VgrG domain. Most strains encoded between two and four different VgrG proteins. We then analyzed the regions downstream of the identified vgrG genes and identified more than 240 putative effectors. The presence of conserved domains in these effectors suggested a range of functions, including peptidoglycan hydrolases, lipases, nucleases, and nucleic acid deaminases. However, 10 of the effector groups had no functionally characterized domains. Phylogenetic analysis of these putative effectors revealed that they clustered into 32 distinct groups that appear to have been acquired from a diverse set of ancestors. Corresponding immunity proteins were identified for all but two of the effector groups. Effectors from eight of the 32 groups contained N-terminal rearrangement hotspot (RHS) domains. The C-terminal regions of these RHS proteins, which are predicted to confer the toxic effector function, were very diverse, but the N-terminal RHS domains clustered into just two groups. While the majority of A. baumannii strains contained an RHS type effector, no strains encoded two RHS effectors with similar N-terminal sequences, suggesting that the presence of similar N-terminal RHS domains leads to competitive exclusion. Together, these analyses define the extreme diversity of T6SS effectors within A. baumannii and, as many have unknown functions, future detailed characterization of these effectors may lead to the identification of proteins with novel antibacterial properties., (Copyright © 2019 Lewis, Deveson Lucas, Harper and Boyce.)

There is a great need for efficacious therapies against Gram-negative bacteria. Double β-lactam combination(s) (DBL) are relatively safe, and preclinical data are promising; however, their clinical role has not been well defined. We conducted a metaanalysis of the clinical and microbiological efficacy of DBL compared to β-lactam plus aminoglycoside combinations (BLAG). PubMed, Embase, ISI Web of Knowledge, and Cochrane Controlled Trials Register database were searched through July 2018. We included randomized controlled clinical trials that compared DBL with BLAG combinations. Clinical response was used as the primary outcome and microbiological response in Gram-negative bacteria as the secondary outcome; sensitivity analyses were performed for Pseudomonas aeruginosa , Klebsiella spp., and Escherichia coli Heterogeneity and risk of bias were assessed. Safety results were classified by systems and organs. Thirteen studies evaluated 2,771 cases for clinical response and 665 cases for microbiological response in various Gram-negative species. DBL achieved slightly, but not significantly, better clinical response (risk ratio, 1.05; 95% confidence interval [CI], 0.99 to 1.11) and microbiological response in Gram-negatives (risk ratio, 1.11; 95% CI, 0.99 to 1.25) compared with BLAG. Sensitivity analyses by pathogen showed the same trend. No significant heterogeneity across studies was found. DBL was significantly safer than BLAG regarding renal toxicity (6.6% versus 8.8%, P =  0.0338) and ototoxicity (0.7 versus 3.1%, P =  0.0137). Other adverse events were largely comparable. Overall, empirically designed DBL showed comparable clinical and microbiological responses across different Gram-negative species, and were significantly safer than BLAG. Therefore, DBL should be rationally optimized via the latest translational approaches, leveraging mechanistic insights and newer β-lactams for future evaluation in clinical trials., (Copyright © 2019 American Society for Microbiology.)

Hypermutable Pseudomonas aeruginosa isolates (hypermutators) have been identified in patients with cystic fibrosis (CF) and are associated with reduced lung function. Hypermutators display a greatly increased mutation rate and an enhanced ability to become resistant to antibiotics during treatment. Their prevalence has been established among patients with CF, but it has not been determined for patients with CF in Australia. This study aimed to determine the prevalence of hypermutable P. aeruginosa isolates from adult patients with CF from a health care institution in Australia and to characterize the genetic diversity and antibiotic susceptibility of these isolates. A total of 59  P. aeruginosa clinical isolates from patients with CF were characterized. For all isolates, rifampin (RIF) mutation frequencies and susceptibility to a range of antibiotics were determined. Of the 59 isolates, 13 (22%) were hypermutable. Whole-genome sequences were determined for all hypermutable isolates. Core genome polymorphisms were used to assess genetic relatedness of the isolates, both to each other and to a sample of previously characterized P. aeruginosa strains. Phylogenetic analyses showed that the hypermutators were from divergent lineages and that hypermutator phenotype was mostly the result of mutations in mutL or, less commonly, in mutS Hypermutable isolates also contained a range of mutations that are likely associated with adaptation of P. aeruginosa to the CF lung environment. Multidrug resistance was more prevalent in hypermutable than nonhypermutable isolates (38% versus 22%). This study revealed that hypermutable P. aeruginosa strains are common among isolates from patients with CF in Australia and are implicated in the emergence of antibiotic resistance., (Copyright © 2019 American Society for Microbiology.)

Polymyxin antibiotics are increasingly being used as last-line therapeutic options against a number of multidrug resistant bacteria. These antibiotics show strong bactericidal activity against a range of Gram-negative bacteria, but with the increased use of these antibiotics resistant strains are emerging at an alarming rate. Furthermore, some Gram-negative species, such as Neisseria meningitidis, Proteus mirabilis and Burkholderia spp., are intrinsically resistant to the action of polymyxins. Most identified polymyxin resistance mechanisms in Gram-negative bacteria involve changes to the lipopolysaccharide (LPS) structure, as polymyxins initially interact with the negatively charged lipid A component of LPS. The controlled addition of positively charged residues such as 4-amino- L -arabinose, phosphoethanolamine and/or galactosamine to LPS results in a reduced negative charge on the bacterial surface and therefore reduced interaction between the polymyxin and the LPS. Polymyxin resistant species produce LPS that intrinsically contains one or more of these additions. While the genes necessary for most of these additions are chromosomally encoded, plasmid-borne phosphoethanolamine transferases (mcr-1 to mcr-8) have recently been identified and these plasmids threaten to increase the rate of dissemination of clinically relevant colistin resistance. Uniquely, Acinetobacter baumannii can also become highly resistant to polymyxins via spontaneous mutations in the lipid A biosynthesis genes lpxA, lpxC or lpxD such that they produce no LPS or lipid A. A range of other non-LPS-dependent polymyxin resistance mechanisms has also been identified in bacteria, but these generally result in only low levels of resistance. These include increased anionic capsular polysaccharide production in Klebsiella pneumoniae, expression of efflux systems such as MtrCDE in N. meningitidis, and altered expression of outer membrane proteins in a small number of species.

Hypermutable Pseudomonas aeruginosa organisms are prevalent in chronic respiratory infections and have been associated with reduced lung function in cystic fibrosis (CF); these isolates can become resistant to all antibiotics in monotherapy. This study aimed to evaluate the time course of bacterial killing and resistance of meropenem and ciprofloxacin in combination against hypermutable and nonhypermutable P. aeruginosa Static concentration time-kill experiments over 72 h assessed meropenem and ciprofloxacin in mono- and combination therapies against PAO1 (nonhypermutable), PAOΔ mutS (hypermutable), and hypermutable isolates CW8, CW35, and CW44 obtained from CF patients with chronic respiratory infections. Meropenem (1 or 2 g every 8 h [q8h] as 3-h infusions and 3 g/day as a continuous infusion) and ciprofloxacin (400 mg q8h as 1-h infusions) in monotherapies and combinations were further evaluated in an 8-day hollow-fiber infection model study (HFIM) against CW44. Concentration-time profiles in lung epithelial lining fluid reflecting the pharmacokinetics in CF patients were simulated and counts of total and resistant bacteria determined. All data were analyzed by mechanism-based modeling (MBM). In the HFIM, all monotherapies resulted in rapid regrowth with resistance at 48 h. The maximum daily doses of 6 g meropenem (T >MIC of 80% to 88%) and 1.2 g ciprofloxacin (area under the concentration-time curve over 24 h in the steady state divided by the MIC [AUC/MIC], 176), both given intermittently, in monotherapy failed to suppress regrowth and resulted in substantial emergence of resistance (≥7.6 log 10 CFU/ml resistant populations). The combination of these regimens achieved synergistic killing and suppressed resistance. MBM with subpopulation and mechanistic synergy yielded unbiased and precise curve fits. Thus, the combination of 6 g/day meropenem plus ciprofloxacin holds promise for future clinical evaluation against infections by susceptible hypermutable P. aeruginosa ., (Copyright © 2018 American Society for Microbiology.)

The type VI secretion system (T6SS) is a macromolecular machine that delivers protein effectors into host cells and/or competing bacteria. The effectors may be delivered as noncovalently bound cargo of T6SS needle proteins (VgrG/Hcp/PAAR) or as C-terminal extensions of these proteins. Many Acinetobacter baumannii strains produce a T6SS, but little is known about the specific effectors or how they are delivered. In this study, we show that A. baumannii AB307-0294 encodes three vgrG loci, each containing a vgrG gene, a T6SS toxic effector gene, and an antitoxin/immunity gene. Each of the T6SS toxic effectors could kill Escherichia coli when produced in trans unless the cognate immunity protein was coproduced. To determine the role of each VgrG in effector delivery, we performed interbacterial competitive killing assays using A. baumannii AB307-0294 vgrG mutants, together with Acinetobacter baylyi prey cells expressing pairs of immunity genes that protected against two toxic effectors but not a third. Using this approach, we showed that AB307-0294 produces only three T6SS toxic effectors capable of killing A. baylyi and that each VgrG protein is specific for the carriage of one effector. Finally, we analyzed a number of A. baumannii genomes and identified significant diversity in the range of encoded T6SS VgrG and effector proteins, with correlations between effector types and A. baumannii global clone lineages., (Copyright © 2018 American Society for Microbiology.)

Colistin is a crucial last-line drug used for the treatment of life-threatening infections caused by multidrug-resistant strains of the Gram-negative bacterium Acinetobacter baumannii However, colistin-resistant A. baumannii isolates can still be isolated following failed colistin therapy. Resistance is most often mediated by the addition of phosphoethanolamine (pEtN) to lipid A by PmrC, following missense mutations in the pmrCAB operon encoding PmrC and the two-component signal transduction system PmrA/PmrB. We recovered a pair of A. baumannii isolates from a single patient before (6009-1) and after (6009-2) failed colistin treatment. These strains displayed low and very high levels of colistin resistance (MICs, 8 to 16 μg/ml and 128 μg/ml), respectively. To understand how increased colistin resistance arose, we sequenced the genome of each isolate, which revealed that 6009-2 had an extra copy of the insertion sequence element IS Aba125 within a gene encoding an H-NS family transcriptional regulator. To confirm the role of H-NS in colistin resistance, we generated an hns deletion mutant in 6009-1 and showed that colistin resistance increased upon the deletion of hns We also provided 6009-2 with an intact copy of hns and showed that the strain was no longer resistant to high concentrations of colistin. Transcriptomic analysis of the clinical isolates identified more than 150 genes as being differentially expressed in the colistin-resistant hns mutant 6009-2. Importantly, the expression of eptA , encoding a second lipid A-specific pEtN transferase but not pmrC , was increased in the hns mutant. This is the first time an H-NS family transcriptional regulator has been associated with a pEtN transferase and colistin resistance., (Copyright © 2018 American Society for Microbiology.)

Pasteurella multocida is a Gram-negative bacterium responsible for many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. Small RNA (sRNA) molecules are critical regulators that act by binding to specific mRNA targets, often in association with the RNA chaperone protein Hfq. In this study, transcriptomic analysis of the P. multocida strain VP161 revealed a putative sRNA with high identity to GcvB from Escherichia coli and Salmonella enterica serovar Typhimurium. High-throughput quantitative liquid proteomics was used to compare the proteomes of the P. multocida VP161 wild-type strain, a gcvB mutant, and a GcvB overexpression strain. These analyses identified 46 proteins that displayed significant differential production after inactivation of gcvB , 36 of which showed increased production. Of the 36 proteins that were repressed by GcvB, 27 were predicted to be involved in amino acid biosynthesis or transport. Bioinformatic analyses of putative P. multocida GcvB target mRNAs identified a strongly conserved 10 nucleotide consensus sequence, 5'-AACACAACAT-3', with the central eight nucleotides identical to the seed binding region present within GcvB mRNA targets in E. coli and S. Typhimurium. Using a defined set of seed region mutants, together with a two-plasmid reporter system that allowed for quantification of sRNA-mRNA interactions, this sequence was confirmed to be critical for the binding of the P. multocida GcvB to the target mRNA, gltA ., (© 2018 Gulliver et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

We aimed to prospectively validate an optimized combination dosage regimen against a clinical carbapenem-resistant Acinetobacter baumannii (CRAB) isolate (imipenem MIC, 32 mg/liter; tobramycin MIC, 2 mg/liter). Imipenem at constant concentrations (7.6, 13.4, and 23.3 mg/liter, reflecting a range of clearances) was simulated in a 7-day hollow-fiber infection model (inoculum, ∼10 7.2 CFU/ml) with and without tobramycin (7 mg/kg q24h, 0.5-h infusions). While monotherapies achieved no killing or failed by 24 h, this rationally optimized combination achieved >5 log 10 bacterial killing and suppressed resistance., (Copyright © 2018 American Society for Microbiology.)

Hypermutable Pseudomonas aeruginosa strains are prevalent in patients with cystic fibrosis and rapidly become resistant to antibiotic monotherapies. Combination dosage regimens have not been optimized against such strains using mechanism-based modeling (MBM) and the hollow-fiber infection model (HFIM). The PAO1 wild-type strain and its isogenic hypermutable PAOΔ mutS strain (MIC meropenem of 1.0 mg/liter and MIC tobramycin of 0.5 mg/liter for both) were assessed using 96-h static-concentration time-kill studies (SCTK) and 10-day HFIM studies (inoculum, ∼10 8.4 CFU/ml). MBM of SCTK data were performed to predict expected HFIM outcomes. Regimens studied in the HFIM were meropenem at 1 g every 8 h (0.5-h infusion), meropenem at 3 g/day with continuous infusion, tobramycin at 10 mg/kg of body weight every 24 h (1-h infusion), and both combinations. Meropenem regimens delivered the same total daily dose. Time courses of total and less susceptible populations and MICs were determined. For the PAOΔ mutS strain in the HFIM, all monotherapies resulted in rapid regrowth to >10 8.7 CFU/ml with near-complete replacement by less susceptible bacteria by day 3. Meropenem every 8 h with tobramycin caused >7-log 10 bacterial killing followed by regrowth to >6 log 10 CFU/ml by day 5 and high-level resistance (MIC meropenem , 32 mg/liter; MIC tobramycin , 8 mg/liter). Continuous infusion of meropenem with tobramycin achieved >8-log 10 bacterial killing without regrowth. For PAO1, meropenem monotherapies suppressed bacterial growth to <4 log 10 over 7 to 9 days, with both combination regimens achieving near eradication. An MBM-optimized meropenem plus tobramycin regimen achieved synergistic killing and resistance suppression against a difficult-to-treat hypermutable P. aeruginosa strain. For the combination to be maximally effective, it was critical to achieve the optimal shape of the concentration-time profile for meropenem., (Copyright © 2018 American Society for Microbiology.)

The lipopolysaccharide (LPS) produced by the Gram-negative bacterial pathogen Pasteurella multocida has phosphoethanolamine (PEtn) residues attached to lipid A, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and galactose. In this report, we show that PEtn is transferred to lipid A by the P. multocida EptA homologue, PetL, and is transferred to galactose by a novel PEtn transferase that is unique to P. multocida called PetG. Transcriptomic analyses indicated that petL expression was positively regulated by the global regulator Fis and negatively regulated by an Hfq-dependent small RNA. Importantly, we have identified a novel PEtn transferase called PetK that is responsible for PEtn addition to the single Kdo molecule (Kdo 1 ), directly linked to lipid A in the P. multocida glycoform A LPS. In vitro assays showed that the presence of a functional petL and petK , and therefore the presence of PEtn on lipid A and Kdo 1 , was essential for resistance to the cationic, antimicrobial peptide cathelicidin-2. The importance of PEtn on Kdo 1 and the identification of the transferase responsible for this addition have not previously been shown. Phylogenetic analysis revealed that PetK is the first representative of a new family of predicted PEtn transferases. The PetK family consists of uncharacterized proteins from a range of Gram-negative bacteria that produce LPS glycoforms with only one Kdo molecule, including pathogenic species within the genera Vibrio , Bordetella , and Haemophilus We predict that many of these bacteria will require the addition of PEtn to Kdo for maximum protection against host antimicrobial peptides., (Copyright © 2017 American Society for Microbiology.)

Pasteurella multocida is a heterogeneous species that is a primary pathogen of many different vertebrates. This Gram-negative bacterium can cause a range of diseases, including fowl cholera in birds, haemorrhagic septicaemia in ungulates, atrophic rhinitis in swine, and lower respiratory tract infections in cattle and pigs. One of the primary virulence factors of P. multocida is lipopolysaccharide (LPS). Recent work has shown that this crucial surface molecule shows significant structural variability across different P. multocida strains, with many producing LPS structures that are highly similar to the carbohydrate component of host glycoproteins. It is likely that this LPS mimicry of host molecules plays a major role in the survival of P. multocida in certain host niches. P. multocida LPS also plays a significant role in resisting the action of chicken cathelicidins, and is a strong stimulator of host immune responses. The inflammatory response to the endotoxic lipid A component is a major contributor to the pathogenesis of certain infections. Recent work has shown that vaccines containing killed bacteria give protection only against other strains with identical, or nearly identical, surface LPS structures. Conversely, live attenuated vaccines give protection that is broadly protective, and their efficacy is independent of LPS structure., Competing Interests: The authors declare no conflict of interest.

Background: Acinetobacter baumannii is a pathogen of major importance in intensive care units worldwide, with the potential to cause problematic outbreaks and acquire high-level resistance to antibiotics. There is an urgent need to understand the mechanisms of A. baumannii pathogenesis for the future development of novel targeted therapies. In this study we performed an in vivo transcriptomic analysis of A. baumannii isolated from a mammalian host with bacteremia., Methods: Mice were infected with A. baumannii American Type Culture Collection 17978 using an intraperitoneal injection, and blood was extracted at 8 hours to purify bacterial RNA for RNA-Seq with an Illumina platform., Results: Approximately one-quarter of A. baumannii protein coding genes were differentially expressed in vivo compared with in vitro (false discovery rate, ≤0.001; 2-fold change) with 557 showing decreased and 329 showing increased expression. Gene groups with functions relating to translation and RNA processing were overrepresented in genes with increased expression, and those relating to chaperone and protein turnover were overrepresented in the genes with decreased expression. The most strongly up-regulated genes corresponded to the 3 recognized siderophore iron uptake clusters, reflecting the iron-restrictive environment in vivo. Metabolic changes in vivo included reduced expression of genes involved in amino acid and fatty acid transport and catabolism, indicating metabolic adaptation to a different nutritional environment. Genes encoding types I and IV pili, quorum sensing components, and proteins involved in biofilm formation all showed reduced expression. Many genes that have been reported as essential for virulence showed reduced or unchanged expression in vivo., Conclusion: This study provides the first insight into A. baumannii gene expression profiles during a life-threatening mammalian infection. Analysis of differentially regulated genes highlights numerous potential targets for the design of novel therapeutics., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Background: Clostridium perfringens causes toxin-mediated diseases, including gas gangrene (clostridial myonecrosis) and food poisoning in humans. The production of the toxins implicated in gas gangrene, α-toxin and perfringolysin O, is regulated by the VirSR two-component regulatory system. In addition, RevR, an orphan response regulator, has been shown to affect virulence in the mouse myonecrosis model. RevR positively regulates the expression of genes that encode hydrolytic enzymes, including hyaluronidases and sialidases., Results: To further characterize the VirSR and RevR regulatory networks, comparative transcriptomic analysis was carried out with strand-specific RNA-seq on C. perfringens strain JIR325 and its isogenic virR and revR regulatory mutants. Using the edgeR analysis package, 206 genes in the virR mutant and 67 genes in the revR mutant were found to be differentially expressed. Comparative analysis revealed that VirR acts as a global negative regulator, whilst RevR acts as a global positive regulator. Therefore, about 95 % of the differentially expressed genes were up-regulated in the virR mutant, whereas 81 % of the differentially expressed genes were down-regulated in the revR mutant. Importantly, we identified 23 genes that were regulated by both VirR and RevR, 18 of these genes, which included the sporulation-specific spoIVA, sigG and sigF genes, were regulated positively and negatively by RevR and VirR, respectively. Furthermore, analysis of the mapped RNA-seq reads visualized as depth of coverage plots showed that there were 93 previously unannotated transcripts in intergenic regions. These transcripts potentially encode small RNA molecules., Conclusion: In conclusion, using strand-specific RNA-seq analysis, this study has identified differentially expressed chromosomal and pCP13 native plasmid-encoded genes, antisense transcripts, and transcripts within intergenic regions that are controlled by the VirSR or RevR regulatory systems.

Polymyxins are often last-line therapeutic agents used to treat infections caused by multidrug-resistant A. baumannii. Recent reports of polymyxin-resistant A. baumannii highlight the urgent need for research into mechanisms of polymyxin resistance. This study employed genomic and transcriptomic analyses to investigate the mechanisms of polymyxin resistance in A. baumannii AB307-0294 using an in vitro dynamic model to mimic four different clinically relevant dosage regimens of polymyxin B and colistin over 96 h. Polymyxin B dosage regimens that achieved peak concentrations above 1 mg/L within 1 h caused significant bacterial killing (~5 log10CFU/mL), while the gradual accumulation of colistin resulted in no bacterial killing. Polymyxin resistance was observed across all dosage regimens; partial reversion to susceptibility was observed in 6 of 8 bacterial samples during drug-free passaging. Stable polymyxin-resistant samples contained a mutation in pmrB. The transcriptomes of stable and non-stable polymyxin-resistant samples were not substantially different and featured altered expression of genes associated with outer membrane structure and biogenesis. These findings were further supported via integrated analysis of previously published transcriptomics data from strain ATCC19606. Our results provide a foundation for understanding the mechanisms of polymyxin resistance following exposure to polymyxins and the need to explore effective combination therapies.

Background: Burkholderia pseudomallei is the causative agent of melioidosis, a severe invasive disease of humans and animals. Initial screening of a B. pseudomallei signature-tagged mutagenesis library identified an attenuated mutant with a transposon insertion in a gene encoding the sensor component of an uncharacterised two-component signal transduction system (TCSTS), which we designated BprRS., Results: Single gene inactivation of either the response regulator gene (bprR) or the sensor histidine kinase gene (bprS) resulted in mutants with reduced swarming motility and reduced virulence in mice. However, a bprRS double mutant was not attenuated for virulence and displayed wild-type levels of motility. The transcriptomes of the bprS, bprR and bprRS mutants were compared with the transcriptome of the parent strain K96243. Inactivation of the entire BprRS TCSTS (bprRS double mutant) resulted in altered expression of only nine genes, including both bprR and bprS, five phage-related genes and bpss0686, encoding a putative 5, 10-methylene tetrahydromethanopterin reductase involved in one carbon metabolism. In contrast, the transcriptomes of each of the bprR and bprS single gene mutants revealed more than 70 differentially expressed genes common to both mutants, including regulatory genes and those required for flagella assembly and for the biosynthesis of the cytotoxic polyketide, malleilactone., Conclusions: Inactivation of the entire BprRS TCSTS did not alter virulence or motility and very few genes were differentially expressed indicating that the definitive BprRS regulon is relatively small. However, loss of a single component, either the sensor histidine kinase BprS or its cognate response regulator BprR, resulted in significant transcriptomic and phenotypic differences from the wild-type strain. We hypothesize that the dramatically altered phenotypes of these single mutants are the result of cross-regulation with one or more other TCSTSs and concomitant dysregulation of other key regulatory genes.

The Gram-negative bacterium Pasteurella multocida is the causative agent of a number of economically important animal diseases, including avian fowl cholera. Numerous P. multocida virulence factors have been identified, including capsule, lipopolysaccharide (LPS), and filamentous hemagglutinin, but little is known about how the expression of these virulence factors is regulated. Hfq is an RNA-binding protein that facilitates riboregulation via interaction with small noncoding RNA (sRNA) molecules and their mRNA targets. Here, we show that a P. multocida hfq mutant produces significantly less hyaluronic acid capsule during all growth phases and displays reduced in vivo fitness. Transcriptional and proteomic analyses of the hfq mutant during mid-exponential-phase growth revealed altered transcript levels for 128 genes and altered protein levels for 78 proteins. Further proteomic analyses of the hfq mutant during the early exponential growth phase identified 106 proteins that were produced at altered levels. Both the transcript and protein levels for genes/proteins involved in capsule biosynthesis were reduced in the hfq mutant, as were the levels of the filamentous hemagglutinin protein PfhB2 and its secretion partner LspB2. In contrast, there were increased expression levels of three LPS biosynthesis genes, encoding proteins involved in phosphocholine and phosphoethanolamine addition to LPS, suggesting that these genes are negatively regulated by Hfq-dependent mechanisms. Taken together, these data provide the first evidence that Hfq plays a crucial role in regulating the global expression of P. multocida genes, including the regulation of key P. multocida virulence factors, capsule, LPS, and filamentous hemagglutinin., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)