Redox Coenzyme F420 Biosynthesis in Thermomicrobia Involves Reduction by Stand-Alone Nitroreductase Superfamily Enzymes

ABSTRACT Coenzyme F420 is a redox cofactor involved in hydride transfer reactions in archaea and bacteria. Since F420-dependent enzymes are attracting increasing interest as tools in biocatalysis, F420 biosynthesis is being revisited. While it was commonly accepted for a long time that the 2-phospho-l-lactate (2-PL) moiety of F420 is formed from free 2-PL, it was recently shown that phosphoenolpyruvate is incorporated in Actinobacteria and that the C-terminal domain of the FbiB protein, a member of the nitroreductase (NTR) superfamily, converts dehydro-F420 into saturated F420. Outside the Actinobacteria, however, the situation is still unclear because FbiB is missing in these organisms and enzymes of the NTR family are highly diversified. Here, we show by heterologous expression and in vitro assays that stand-alone NTR enzymes from Thermomicrobia exhibit dehydro-F420 reductase activity. Metabolome analysis and proteomics studies confirmed the proposed biosynthetic pathway in Thermomicrobium roseum. These results clarify the biosynthetic route of coenzyme F420 in a class of Gram-negative bacteria, redefine functional subgroups of the NTR superfamily, and offer an alternative for large-scale production of F420 in Escherichia coli in the future. IMPORTANCE Coenzyme F420 is a redox cofactor of Archaea and Actinobacteria, as well as some Gram-negative bacteria. Its involvement in processes such as the biosynthesis of antibiotics, the degradation of xenobiotics, and asymmetric enzymatic reductions renders F420 of great relevance for biotechnology. Recently, a new biosynthetic step during the formation of F420 in Actinobacteria was discovered, involving an enzyme domain belonging to the versatile nitroreductase (NTR) superfamily, while this process remained blurred in Gram-negative bacteria. Here, we show that a similar biosynthetic route exists in Thermomicrobia, although key biosynthetic enzymes show different domain architectures and are only distantly related. Our results shed light on the biosynthesis of F420 in Gram-negative bacteria and refine the knowledge about sequence-function relationships within the NTR superfamily of enzymes. Appreciably, these results offer an alternative route to produce F420 in Gram-negative model organisms and unveil yet another biochemical facet of this pathway to be explored by synthetic microbiologists.

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AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions

Journal of Bacteriology ◽

10.1128/jb.00314-15 ◽

2015 ◽

Vol 197 (24) ◽

pp. 3788-3796 ◽

Cited By ~ 3

Author(s):

Takayuki Kuge ◽

Haruhiko Teramoto ◽

Masayuki Inui

Keyword(s):

Corynebacterium Glutamicum ◽

Binding Sites ◽

Large Scale ◽

Transcriptional Regulator ◽

Scale Production ◽

Primary Role ◽

Promoter Regions ◽

Independent Manner ◽

Content Type

ABSTRACTInCorynebacterium glutamicumATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression ofl-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BSB) between the divergent operons (araBDAandgalM-araR) and two (BSE1and BSE2) upstream ofaraE.l-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the expression of the AraR regulon. BSBmutation resulted in derepression of botharaBDAandgalM-araRoperons. The effects of BSE1and/or BSE2mutation onaraEexpression revealed that the two sites independently function as theciselements, but BSE1plays the primary role. However, AraR was shown to bind to these sites with almost the same affinityin vitro. Taken together, the expression ofaraBDAandaraEis strongly repressed by binding of AraR to a single site immediately downstream of the respective transcriptional start sites, whereas the binding site overlapping the −10 or −35 region of thegalM-araRandaraEpromoters is less effective in repression. Furthermore, downregulation ofaraBDAandaraEdependent onl-arabinose catabolism observed in the BSBmutant and the AraR-independentaraRpromoter identified withingalM-araRadd complexity to regulation of the AraR regulon derepressed byl-arabinose.IMPORTANCECorynebacterium glutamicumhas a long history as an industrial workhorse for large-scale production of amino acids. An important aspect of industrial microorganisms is the utilization of the broad range of sugars for cell growth and production process. MostC. glutamicumstrains are unable to use a pentose sugarl-arabinose as a carbon source. However, genes forl-arabinose utilization and its regulation have been recently identified inC. glutamicumATCC 31831. This study elucidates the roles of the multiple binding sites of the transcriptional repressor AraR in the derepression byl-arabinose and thereby highlights the complex regulatory feedback loops in combination withl-arabinose catabolism-dependent repression of the AraR regulon in an AraR-independent manner.

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Membrane Protein Complex ExbB4-ExbD1-TonB1from Escherichia coli Demonstrates Conformational Plasticity

Journal of Bacteriology ◽

10.1128/jb.00069-15 ◽

2015 ◽

Vol 197 (11) ◽

pp. 1873-1885 ◽

Cited By ~ 17

Author(s):

Aleksandr Sverzhinsky ◽

Jacqueline W. Chung ◽

Justin C. Deme ◽

Lucien Fabre ◽

Kristian T. Levey ◽

...

Keyword(s):

Escherichia Coli ◽

Iron Acquisition ◽

Proton Translocation ◽

Three Dimensional ◽

Structural Data ◽

Structural Features ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type

ABSTRACTIron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His6-taggedexbB-exbDand S-taggedtonB, we purified detergent-solubilized complexes of ExbB-ExbD-TonB fromEscherichia coli. Protein-detergent complexes of ∼230 kDa with a hydrodynamic radius of ∼6.0 nm were similar to previously purified ExbB4-ExbD2complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB4-ExbD1-TonB1. Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB4-ExbD1-TonB1. Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbB-ExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization.IMPORTANCEReceptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstratein vitroevidence of ExbB4-ExbD1-TonB1complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.

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Clinical Experience of Colistin-Glycopeptide Combination in Critically Ill Patients Infected with Gram-Negative Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00871-13 ◽

2013 ◽

Vol 58 (2) ◽

pp. 851-858 ◽

Cited By ~ 62

Author(s):

Nicola Petrosillo ◽

Maddalena Giannella ◽

Massimo Antonelli ◽

Mario Antonini ◽

Bruno Barsic ◽

...

Keyword(s):

Protective Factor ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Carbapenem Resistant ◽

Causative Agents ◽

Treatment Onset ◽

Cox Analysis

ABSTRACTA colistin-glycopeptide combination (CGC) has been shownin vitroto be synergistic against multidrug-resistant Gram-negative bacteria (MDR GNB), especiallyAcinetobacter baumannii, and to prevent further resistance. However, clinical data are lacking. We carried out a retrospective multicenter study of patients hospitalized in intensive care units (ICUs) who received colistin for GNB infection over a 1-year period, to assess the rates of nephrotoxicity and 30-day mortality after treatment onset among patients treated with and without CGC for ≥48 h. Of the 184 patients treated with colistin, GNB infection was documented for 166. The main causative agents were MDRA. baumannii(59.6%), MDRPseudomonas aeruginosa(18.7%), and carbapenem-resistantKlebsiella pneumoniae(14.5%); in 16.9% of patients, a Gram-positive bacterium (GPB) coinfection was documented. Overall, 68 patients (40.9%) received CGC. Comparison of patients treated with and without CGC showed significant differences for respiratory failure (39.7% versus 58.2%), ventilator-associated pneumonia (54.4% versus 71.4%), MDRA. baumanniiinfection (70.6% versus 52%), and GPB coinfection (41.2% versus 0%); there were no differences for nephrotoxicity (11.8% versus 13.3%) and 30-day mortality (33.8% versus 29.6%). Cox analysis performed on patients who survived for ≥5 days after treatment onset showed that the Charlson index (hazard ratio [HR], 1.26; 95% confidence interval [CI], 1.01 to 1.44;P= 0.001) and MDRA. baumanniiinfection (HR, 2.51; 95% CI, 1.23 to 5.12;P= 0.01) were independent predictors of 30-day mortality, whereas receiving CGC for ≥5 days was a protective factor (HR, 0.42; 95% CI, 0.19 to 0.93;P= 0.03). We found that CGC was not associated with higher nephrotoxicity and was a protective factor for mortality if administered for ≥5 days.

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Efficacy of Humanized Exposures of Cefiderocol (S-649266) against a Diverse Population of Gram-Negative Bacteria in a Murine Thigh Infection Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01022-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 35

Author(s):

Marguerite L. Monogue ◽

Masakatsu Tsuji ◽

Yoshinori Yamano ◽

Roger Echols ◽

David P. Nicolau

Keyword(s):

Antibacterial Activity ◽

Multidrug Resistant ◽

Infection Model ◽

Gram Negative Bacteria ◽

Diverse Population ◽

Gram Negative ◽

Content Type ◽

Log Reduction

ABSTRACT Cefiderocol (S-649266) is a novel siderophore cephalosporin with potent in vitro activity against clinically encountered multidrug-resistant (MDR) Gram-negative isolates; however, its spectrum of antibacterial activity against these difficult-to-treat isolates remains to be fully explored in vivo. Here, we evaluated the efficacy of cefiderocol humanized exposures in a neutropenic murine thigh model to support a suitable MIC breakpoint. Furthermore, we compared cefiderocol's efficacy with humanized exposures of meropenem and cefepime against a subset of these phenotypically diverse isolates. Ninety-five Gram-negative isolates were studied. Efficacy was determined as the change in log10 CFU at 24 h compared with 0-h controls. Bacterial stasis or ≥1 log reduction in 67 isolates with MICs of ≤4 μg/ml was noted in 77, 88, and 85% of Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa, respectively. For isolates with MICs of ≥8 μg/ml, bacterial stasis or ≥1 log10 reduction was observed in only 2 of 28 (8 Enterobacteriaceae, 19 A. baumannii, and 1 P. aeruginosa) strains. Against highly resistant meropenem and cefepime organisms, cefiderocol maintained its in vivo efficacy. Overall, humanized exposures of cefiderocol produced similar reductions in bacterial density for organisms with MICs of ≤4 μg/ml, whereas isolates with MICs of ≥8 μg/ml generally displayed bacterial growth in the presence of the compound. Data derived in the current study will assist with the delineation of MIC susceptibility breakpoints for cefiderocol against these important nosocomial Gram-negative pathogens; however, additional clinical data are required to substantiate these observations.

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In VitroActivity of Ceftazidime-Avibactam against 338 Molecularly Characterized Gentamicin-Nonsusceptible Gram-Negative Clinical Isolates Obtained from Patients in Canadian Hospitals

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00364-15 ◽

2015 ◽

Vol 59 (6) ◽

pp. 3623-3626 ◽

Cited By ~ 6

Author(s):

Andrew J. Denisuik ◽

James A. Karlowsky ◽

Tyler Denisuik ◽

Wright W. Nichols ◽

Thomas A. Keating ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

16S Rrna ◽

Klebsiella Pneumoniae ◽

Gram Negative Bacteria ◽

Aminoglycoside Resistance ◽

Single Strain ◽

Gram Negative ◽

Content Type ◽

16S Rrna Methylase

ABSTRACTThe mechanism of aminoglycoside resistance among 338 gentamicin-nonsusceptible Gram-negative bacteria (207Enterobacteriaceaeand 131Pseudomonas aeruginosa) was assessed, and thein vitroactivity of ceftazidime-avibactam against these isolates was determined. Aminoglycoside-modifying enzymes were detected in 91.8% ofEnterobacteriaceaeand 13.7% ofP. aeruginosaisolates. A single strain ofKlebsiella pneumoniaeharbored a 16S rRNA methylase (ArmA). The ceftazidime-avibactam MIC90values were 0.5 μg/ml (MIC, ≤8 μg/ml for 100% of isolates) and 16 μg/ml (MIC, ≤8 μg/ml for 87.8% of isolates) against gentamicin-nonsusceptibleEnterobacteriaceaeandP. aeruginosaisolates, respectively.

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Outer Membrane Targeting of Pseudomonas aeruginosa Proteins Shows Variable Dependence on the Components of Bam and Lol Machineries

mBio ◽

10.1128/mbio.00246-11 ◽

2011 ◽

Vol 2 (6) ◽

Cited By ~ 31

Author(s):

Hanh H. Hoang ◽

Nicholas N. Nickerson ◽

Vincent T. Lee ◽

Anastasia Kazimirova ◽

Mohamed Chami ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Protein Translocation ◽

Lipid Vesicles ◽

Structural Features ◽

Gram Negative Bacteria ◽

Variable Effect ◽

Gram Negative ◽

Content Type

ABSTRACT In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting of three secretins that are functionally related OM proteins was less affected (PilQ and PscC) or not at all affected (XcpQ). Depletion of LolB affected all lipoproteins examined and had a variable effect on the nonlipidated proteins. While the levels of OprF, PilQ, and PscC were significantly reduced by LolB depletion, XcpQ was unaffected and was correctly localized to the OM. These results suggest that certain β-barrel proteins such as OprF primarily utilize the complete Bam machinery. The Lol machinery participates in the OM targeting of secretins to variable degrees, likely through its involvement in the assembly of lipidated Bam components. XcpQ, but not PilQ or PscC, was shown to assemble spontaneously into liposomes as multimers. This work raises the possibility that there is a gradient of utilization of Bam and Lol insertion and targeting machineries. Structural features of individual proteins, including their β-barrel content, may determine the propensity of these proteins for folding (or misfolding) during periplasmic transit and OM insertion, thereby influencing the extent of utilization of the Bam targeting machinery, respectively. IMPORTANCE Targeting of lipidated and nonlipidated proteins to the outer membrane (OM) compartment in Gram-negative bacteria involves the transfer across the periplasm utilizing the Lol and Bam machineries, respectively. We show that depletion of Bam and Lol components in Pseudomonas aeruginosa does not lead to a general OM protein translocation defect, but the severity (and therefore, Lol and Bam dependence), varies with individual proteins. XcpQ, the secretin component of the type II secretion apparatus, is translocated into the OM without the assistance of Bam or Lol machineries. The hypothesis that XcpQ, after secretion across the cytoplasmic membrane, does not utilize the OM targeting machineries was supported by demonstrating that in vitro-synthesized XcpQ (but not the other P. aeruginosa secretins) can spontaneously incorporate into lipid vesicles. Therefore, the requirement for ancillary factors appears to be, in certain instances, dictated by the intrinsic properties of individual OM proteins, conceivably reflecting their propensities to misfold during periplasmic transit.

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Evaluation of the Revogene Carba C Assay for Detection and Differentiation of Carbapenemase-Producing Gram-Negative Bacteria

Journal of Clinical Microbiology ◽

10.1128/jcm.01927-19 ◽

2020 ◽

Vol 58 (4) ◽

Cited By ~ 2

Author(s):

Delphine Girlich ◽

Marine Laguide ◽

Laurent Dortet ◽

Thierry Naas

Keyword(s):

Pseudomonas Aeruginosa ◽

Nucleic Acid ◽

Sensitivity And Specificity ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Microbiology Laboratory ◽

Bacterial Host ◽

Gram Negative ◽

Content Type

ABSTRACT The Revogene Carba C assay (formerly GenePOC Carba assay) is a multiplex nucleic acid-based in vitro diagnostic test intended for the detection of carbapenemase-producing Enterobacterales (CPE) from cultured colonies. This assay was evaluated directly on colonies of 118 well-characterized Enterobacterales with reduced susceptibility to carbapenems and on 49 multidrug-resistant (MDR) Pseudomonas aeruginosa and 40 MDR Acinetobacter baumannii isolates. The Revogene Carba C assay’s performance was high, as it was able to detect the five major carbapenemases (NDM, VIM, IMP, KPC, and OXA-48). In Enterobacterales, sensitivity and specificity were 100%. When extrapolating the results to the French CPE epidemiology between 2012 and 2018, this assay would have detected 99.28% of the 9,624 CPE isolates sent to the French NRC, missing 69 CPE isolates (2 GES-5, 10 OXA-23, 2 TMB-1, 1 SME-4, 53 IMI, and 1 FRI). The overall sensitivity and specificity for CP P. aeruginosa were 93.7 and 100%, respectively, as two rare IMP variants (IMP-31 and -46) were not detected. Extrapolating these results to the French epidemiology of CP P. aeruginosa in 2017, 93.3% would have been identified, missing only 1 DIM and 10 GES variants. The Revogene Carba C assay accurately identified the targeted carbapenemase genes in A. baumannii, but when extrapolating these results to the French CP A. baumannii epidemiology of 2017, only 12.50% of them could be detected, as OXA-23 is the most prevalent carbapenemase in CP A. baumannii. The Revogene Carba C assay showed excellent sensitivity and specificity for the five most common carbapenemases regardless of the bacterial host. It is well adapted to the CPE and CP P. aeruginosa epidemiology of many countries worldwide, which makes it suitable for use in the routine microbiology laboratory, with a time to result of ca. 85 min for eight isolates simultaneously.

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Aztreonam plus Clavulanate, Tazobactam, or Avibactam for Treatment of Infections Caused by Metallo-β-Lactamase-Producing Gram-Negative Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00010-19 ◽

2019 ◽

Vol 63 (5) ◽

Cited By ~ 31

Author(s):

Cécile Emeraud ◽

Lelia Escaut ◽

Athénaïs Boucly ◽

Nicolas Fortineau ◽

Rémy A. Bonnin ◽

...

Keyword(s):

Superposition Method ◽

Gram Negative Bacteria ◽

Drug Resistant ◽

Simple Method ◽

Gram Negative ◽

Content Type ◽

Dead End ◽

Amoxicillin Clavulanate

ABSTRACT Metallo-β-lactamase (MBL)-producing Gram-negative bacteria are often extremely resistant, leading to a real therapeutic dead end. Here, we evaluated the in vitro and in vivo efficacy of aztreonam in combination with ceftazidime-avibactam, ceftolozane-tazobactam, or amoxicillin-clavulanate for the treatment of infections caused by MBL-producing Enterobacteriaceae, MBL-producing Pseudomonas aeruginosa, and extremely drug-resistant Stenotrophomonas maltophilia. First, we report two clinical cases, namely, a urinary tract infection caused by an NDM-5-producing Escherichia coli isolate and a pulmonary infection caused by a S. maltophilia isolate efficiently treated with the association of aztreonam-ceftazidime-avibactam and aztreonam-amoxicillin-clavulanate, respectively. Then, a total of 50 MBL-producing Enterobacteriaceae isolates, 3 MBL-producing P. aeruginosa isolates, and 5 extremely drug-resistant S. maltophilia isolates were used to test aztreonam susceptibility in combination with ceftolozane-tazobactam, ceftazidime-avibactam, or amoxicillin-clavulanate. The Etest strip superposition method was used to determine the MICs of the aztreonam/inhibitor combinations. According to CLSI breakpoints, aztreonam susceptibility was fully restored for 86%, 20%, and 50% of the MBL-producing Enterobacteriaceae isolates when combined with ceftazidime-avibactam, ceftolozane-tazobactam, and amoxicillin-clavulanate, respectively. In P. aeruginosa, the aztreonam-ceftazidime-avibactam combination was the most potent, even though the reduction in MICs was at most 2-fold. With the 5 S. maltophilia isolates, aztreonam-ceftazidime-avibactam and aztreonam-amoxicillin-clavulanate were found to be equal (100% susceptibility). Overall, aztreonam-ceftazidime-avibactam was the most potent combination to treat infections caused by MBL producers compared with aztreonam-amoxicillin-clavulanate and aztreonam-ceftolozane-tazobactam. However, in many cases aztreonam-amoxicillin-clavulanate was found to be as efficient as aztreonam-ceftazidime-avibactam, offering the main advantage to be markedly cheaper. We also confirmed the validity of Etest superpositions as a very simple method to determine MICs of aztreonam combinations.

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In VitroEfficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and anIn VivoModel of Infectious Endometritis Utilizing Isolates from the Equine Uterus

Journal of Clinical Microbiology ◽

10.1128/jcm.02861-15 ◽

2015 ◽

Vol 54 (3) ◽

pp. 631-639 ◽

Cited By ~ 21

Author(s):

Ryan A. Ferris ◽

Patrick M. McCue ◽

Grace I. Borlee ◽

Kristen D. Loncar ◽

Margo L. Hennet ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Biofilm Disruption ◽

Infectious Endometritis ◽

Clinical Cases

In this study, we evaluated the ability of the equine clinical treatmentsN-acetylcysteine, EDTA, and hydrogen peroxide to disruptin vitrobiofilms and kill equine reproductive pathogens (Escherichia coli,Pseudomonas aeruginosa, orKlebsiella pneumoniae) isolated from clinical cases.N-acetylcysteine (3.3%) decreased biofilm biomass and killed bacteria within the biofilms ofE. coliisolates. The CFU of recoverableP. aeruginosaandK. pneumoniaeisolates were decreased, but the biofilm biomass was unchanged. Exposure to hydrogen peroxide (1%) decreased the biofilm biomass and reduced the CFU ofE. coliisolates,K. pneumoniaeisolates were observed to have a reduction in CFU, and minimal effects were observed forP. aeruginosaisolates. Chelating agents (EDTA formulations) reducedE. coliCFU but were ineffective at disrupting preformed biofilms or decreasing the CFU ofP. aeruginosaandK. pneumoniaewithin a biofilm. No single nonantibiotic treatment commonly used in equine veterinary practice was able to reduce the CFU and biofilm biomass of all three Gram-negative species of bacteria evaluated. Anin vivoequine model of infectious endometritis was also developed to monitor biofilm formation, utilizing bioluminescence imaging with equineP. aeruginosaisolates from this study. Following infection, the endometrial surface contained focal areas of bacterial growth encased in a strongly adherent “biofilm-like” matrix, suggesting that biofilms are present during clinical cases of infectious equine endometritis. Our results indicate that Gram-negative bacteria isolated from the equine uterus are capable of producing a biofilmin vitro, andP. aeruginosais capable of producing biofilm-like materialin vivo.

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In Vitro Activity of Cefiderocol, a Siderophore Cephalosporin, against Multidrug-Resistant Gram-Negative Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01582-20 ◽

2020 ◽

Vol 64 (12) ◽

Author(s):

Shazad Mushtaq ◽

Zahra Sadouki ◽

Anna Vickers ◽

David M. Livermore ◽

Neil Woodford

Keyword(s):

Dipicolinic Acid ◽

Carbapenem Resistance ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

In Vitro Activity ◽

Gram Negative ◽

Content Type ◽

Mueller Hinton ◽

Mueller Hinton Broth

ABSTRACT Cefiderocol is a parenteral siderophore cephalosporin with a catechol-containing 3′ substituent. We evaluated its MICs against Gram-negative bacteria, using iron-depleted Mueller-Hinton broth. The panel comprised 305 isolates of Enterobacterales, 111 of Pseudomonas aeruginosa, and 99 of Acinetobacter baumannii, all selected for carbapenem resistance and multidrug resistance to other agents. At 2 and 4 μg/ml, cefiderocol inhibited 78.7 and 92.1%, respectively, of all Enterobacterales isolates tested, with rates of 80 to 100% for isolates with all modes of carbapenem resistance except NDM enzymes (41.0% inhibited at 2 μg/ml and 72.1% at 4 μg/ml) or combinations of extended-spectrum β-lactamase (ESBL) and porin loss (61.5% inhibited at 2 μg/ml and 88.5% at 4 μg/ml). Cefiderocol also inhibited 81.1 and 86.5% of all P. aeruginosa isolates at 2 and 4 μg/ml, respectively, with rates of 80 to 100% for isolates with VIM, IMP, GES, or VEB β-lactamases and slightly lower rates for those with NDM (45.5% at 2 μg/ml and 72.7% at 4 μg/ml) and PER (66.7% at 2 μg/ml and 73.3% at 4 μg/ml) enzymes; 63.3% of P. aeruginosa isolates were inhibited at the FDA’s 1-μg/ml breakpoint. Lastly, cefiderocol at 2 and 4 μg/ml inhibited 80.8 and 88.9% of the A. baumannii isolates, respectively, with rates of >85% for isolates with OXA-51-like, -23, -24, or -58 enzymes and 50% at 2 μg/ml and 80% at 4 μg/ml for those with NDM carbapenemases. Dipicolinic acid and avibactam weakly potentiated cefiderocol against Enterobacterales isolates with metallo-β-lactamases (MBLs) and serine carbapenemase, respectively, indicating incomplete β-lactamase stability.

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