Evidence for Rho-dependent control of a virulence switch in Acinetobacter baumannii.

IMPORTANCE: Acinetobacter baumannii is a significant cause of infections in the healthcare setting. More recently, A. baumannii has been a leading cause of secondary bacterial pneumonia in patients infected with SARS-CoV-2 and the overall frequency of A. baumannii infection increased 78% during the COVID-19 pandemic. A. baumannii can exist in virulent or avirulent subpopulations and this interconversion is mediated by the expression of a family of TetR-type transcriptional regulators. In this study, we demonstrate that Rho is a key regulatory component in the expression of these TetR regulators. Overall, this study is the first to address a role for Rho in A. baumannii and provides additional evidence for the role of Rho in regulating diversity in bacterial subpopulations.

The Acinetobacter baumannii website (Ab-web): a multidisciplinary knowledge hub, communication platform, and workspace.

Acinetobacter baumannii is a Gram-negative bacterium increasingly implicated in hospital-acquired infections and outbreaks. Effective prevention and control of such infections are commonly challenged by the frequent emergence of multidrug-resistant strains. Here we introduce Ab-web (https://www.acinetobacterbaumannii.no), the first online platform for sharing expertise on A. baumannii. Ab-web is a species-centric knowledge hub, initially with 10 articles organized into two main sections, 'Overview' and 'Topics', and three themes, 'epidemiology', 'antibiotic resistance', and 'virulence'. The 'workspace' section provides a spot for colleagues to collaborate, build, and manage joint projects. Ab-web is a community-driven initiative amenable to constructive feedback and new ideas.

Collateral Changes in Cell Physiology Associated with ADC-7 ß-Lactamase Expression in Acinetobacter baumannii.

The ADC (AmpC) ß-lactamase is universally present in the Acinetobacter baumannii chromosome, suggesting it may have a yet-to-be-identified cellular function. Using peptidoglycan composition analysis, we show that overexpressing the ADC-7 ß-lactamase in A. baumannii drives changes consistent with altered l,d-transpeptidase activity. Based on this, we tested whether cells overexpressing ADC-7 would exhibit new vulnerabilities. As proof of principle, a screen of transposon insertions revealed that an insertion in the distal 3' end of canB, encoding carbonic anhydrase, resulted in a significant loss of viability when the adc-7 gene was overexpressed. A canB deletion mutant exhibited a more pronounced loss of viability than the transposon insertion, and this became amplified when cells overexpressed ADC-7. Interestingly, overexpression of the OXA-23 or TEM-1 ß-lactamases also led to a pronounced loss of viability in cells with reduced carbonic anhydrase activity. In addition, we demonstrate that reduced CanB activity led to increased sensitivity to peptidoglycan synthesis inhibitors and to the carbonic anhydrase inhibitor ethoxzolamide. Furthermore, this strain exhibited a synergistic interaction with the peptidoglycan inhibitor fosfomycin and ethoxzolamide. Our results highlight the impact of ADC-7 overexpression on cell physiology and reveal that the essential carbonic anhydrase CanB may represent a novel target for antimicrobial agents that would exhibit increased potency against ß-lactamase-overexpressing A. baumannii. IMPORTANCE Acinetobacter baumannii has become resistant to all classes of antibiotics, with ß-lactam resistance responsible for the majority of treatment failures. New classes of antimicrobials are needed to treat this high-priority pathogen. This study had uncovered a new genetic vulnerability in ß-lactamase-expressing A. baumannii, where reduced carbonic anhydrase activity becomes lethal. Inhibitors of carbonic anhydrase could represent a new method for treating A. baumannii infections.

Sulfonamidoboronic Acids as "Cross-Class" Inhibitors of an Expanded-Spectrum Class C Cephalosporinase, ADC-33, and a Class D Carbapenemase, OXA-24/40: Strategic Compound Design to Combat Resistance in Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative organism listed as an urgent threat pathogen by the World Health Organization (WHO). Carbapenem-resistant A. baumannii (CRAB), especially, present therapeutic challenges due to complex mechanisms of resistance to ß-lactams. One of the most important mechanisms is the production of ß-lactamase enzymes capable of hydrolyzing ß-lactam antibiotics. Co-expression of multiple classes of ß-lactamases is present in CRAB; therefore, the design and synthesis of "cross-class" inhibitors is an important strategy to preserve the efficacy of currently available antibiotics. To identify new, nonclassical ß-lactamase inhibitors, we previously identified a sulfonamidomethaneboronic acid CR167 active against Acinetobacter-derived class C ß-lactamases (ADC-7). The compound demonstrated affinity for ADC-7 with a Ki = 160 nM and proved to be able to decrease MIC values of ceftazidime and cefotaxime in different bacterial strains. Herein, we describe the activity of CR167 against other ß-lactamases in A. baumannii: the cefepime-hydrolysing class C extended-spectrum ß-lactamase (ESAC) ADC-33 and the carbapenem-hydrolyzing OXA-24/40 (class D). These investigations demonstrate CR167 as a valuable cross-class (C and D) inhibitor, and the paper describes our attempts to further improve its activity. Five chiral analogues of CR167 were rationally designed and synthesized. The structures of OXA-24/40 and ADC-33 in complex with CR167 and select chiral analogues were obtained. The structure activity relationships (SARs) are highlighted, offering insights into the main determinants for cross-class C/D inhibitors and impetus for novel drug design.

Characterization of Virulent T4-Like Acinetobacter baumannii Bacteriophages DLP1 and DLP2.

The world is currently facing a global health crisis due to the rapid increase in antimicrobial-resistant bacterial infections. One of the most concerning pathogens is Acinetobacter baumannii, which is listed as a Priority 1 pathogen by the World Health Organization. This Gram-negative bacterium has many intrinsic antibiotic resistance mechanisms and the ability to quickly acquire new resistance determinants from its environment. A limited number of effective antibiotics against this pathogen complicates the treatment of A. baumannii infections. A potential treatment option that is rapidly gaining interest is "phage therapy", or the clinical application of bacteriophages to selectively kill bacteria. The myoviruses DLP1 and DLP2 (vB_AbaM-DLP_1 and vB_AbaM-DLP_2, respectively) were isolated from sewage samples using a capsule minus variant of A. baumannii strain AB5075. Host range analysis of these phages against 107 A. baumannii strains shows a limited host range, infecting 15 and 21 for phages DLP1 and DLP2, respectively. Phage DLP1 has a large burst size of 239 PFU/cell, a latency period of 20 min, and virulence index of 0.93. In contrast, DLP2 has a smaller burst size of 24 PFU/cell, a latency period of 20 min, and virulence index of 0.86. Both phages show potential for use as therapeutics to combat A. baumannii infections.

Boronic Acid Transition State Inhibitors as Potent Inactivators of KPC and CTX-M ß-Lactamases: Biochemical and Structural Analyses.

Design of novel ß-lactamase inhibitors (BLIs) is one of the currently accepted strategies to combat the threat of cephalosporin and carbapenem resistance in Gram-negative bacteria. Boronic acid transition state inhibitors (BATSIs) are competitive, reversible BLIs that offer promise as novel therapeutic agents. In this study, the activities of two α-amido-ß-triazolylethaneboronic acid transition state inhibitors (S02030 and MB_076) targeting representative KPC (KPC-2) and CTX-M (CTX-M-96, a CTX-M-15-type extended-spectrum ß-lactamase [ESBL]) ß-lactamases were evaluated. The 50% inhibitory concentrations (IC50s) for both inhibitors were measured in the nanomolar range (2 to 135 nM). For S02030, the k2/K for CTX-M-96 (24,000 M-1 s-1) was twice the reported value for KPC-2 (12,000 M-1 s-1); for MB_076, the k2/K values ranged from 1,200 M-1 s-1 (KPC-2) to 3,900 M-1 s-1 (CTX-M-96). Crystal structures of KPC-2 with MB_076 (1.38-Å resolution) and S02030 and the in silico models of CTX-M-96 with these two BATSIs show that interaction in the CTX-M-96-S02030 and CTX-M-96-MB_076 complexes were overall equivalent to that observed for the crystallographic structure of KPC-2-S02030 and KPC-2-MB_076. The tetrahedral interaction surrounding the boron atom from S02030 and MB_076 creates a favorable hydrogen bonding network with S70, S130, N132, N170, and S237. However, the changes from W105 in KPC-2 to Y105 in CTX-M-96 and the missing residue R220 in CTX-M-96 alter the arrangement of the inhibitors in the active site of CTX-M-96, partially explaining the difference in kinetic parameters. The novel BATSI scaffolds studied here advance our understanding of structure-activity relationships (SARs) and illustrate the importance of new approaches to ß-lactamase inhibitor design.

Spatial regulation of cell motility and its fitness effect in a surface-attached bacterial community.

On a surface, microorganisms grow into a multi-cellular community. When a community becomes densely populated, cells migrate away to expand the community's territory. How microorganisms regulate surface motility to optimize expansion remains poorly understood. Here, we characterized surface motility of Proteus mirabilis. P. mirabilis is well known for its ability to expand its colony rapidly on a surface. Cursory visual inspection of an expanding colony suggests partial migration, i.e., one fraction of a population migrates while the other is sessile. Quantitative microscopic imaging shows that this migration pattern is determined by spatially inhomogeneous regulation of cell motility. Further analyses reveal that this spatial regulation is mediated by the Rcs system, which represses the expression of the motility regulator (FlhDC) in a nutrient-dependent manner. Alleviating this repression increases the colony expansion speed but results in a rapid drop in the number of viable cells, lowering population fitness. These findings collectively demonstrate how Rcs regulates cell motility dynamically to increase the fitness of an expanding bacterial population, illustrating a fundamental trade-off underlying bacterial colonization of a surface.

Stochastic activation of a family of TetR type transcriptional regulators controls phenotypic heterogeneity in Acinetobacter baumannii.

Phenotypic heterogeneity is an important mechanism for regulating bacterial virulence, where a single regulatory switch is typically activated to generate virulent and avirulent subpopulations. The opportunistic pathogen Acinetobacter baumannii can transition at high frequency between virulent opaque (VIR-O) and avirulent translucent subpopulations, distinguished by cells that form opaque or translucent colonies. We demonstrate that expression of 11 TetR-type transcriptional regulators (TTTRs) can drive cells from the VIR-O opaque subpopulation to cells that form translucent colonies. Remarkably, in a subpopulation of VIR-O cells, four of these TTTRs were stochastically activated in different combinations to drive cells to the translucent state. The resulting translucent subvariants exhibited unique phenotypic differences and the majority were avirulent. Due to their functional redundancy, a quadruple mutant with all four of these TTTRs inactivated was required to observe a loss of switching from the VIR-O state. Further, we demonstrate a small RNA, SrvS, acts as a "rheostat," where the levels of SrvS expression influences both the VIR-O to translucent switching frequency, and which TTTR is activated when VIR-O cells switch. In summary, this work has revealed a new paradigm for phenotypic switching in bacteria, where an unprecedented number of related transcriptional regulators are activated in different combinations to control virulence and generate unique translucent subvariants with distinct phenotypic properties.

OXA-23 ß-Lactamase Overexpression in Acinetobacter baumannii Drives Physiological Changes Resulting in New Genetic Vulnerabilities.

ß-Lactamase expression is the major mechanism of resistance to penicillins, cephalosporins, and carbapenems in the multidrug-resistant (MDR) bacterium Acinetobacter baumannii. In fact, stable high-level expression of at least one ß-lactamase has been rapidly increasing and reported to occur in up to 98.5% of modern A. baumannii isolates recovered in the clinic. Moreover, the OXA-51 ß-lactamase is universally present in the A. baumannii chromosome, suggesting it may have a cellular function beyond antibiotic resistance. However, the consequences associated with OXA ß-lactamase overexpression on A. baumannii physiology are not well understood. Using peptidoglycan composition analysis, we show that overexpressing the OXA-23 ß-lactamase in A. baumannii drives significant collateral changes with alterations consistent with increased amidase activity. Consequently, we predicted that these changes create new cellular vulnerabilities. As proof of principle, a small screen of random transposon insertions revealed three genes, where mutations resulted in a greater than 19-fold loss of viability when OXA-23 was overexpressed. The identified genes remained conditionally essential even when a catalytically inactive OXA-23 ß-lactamase was overexpressed. In addition, we demonstrated a synergistic lethal relationship between OXA-23 overexpression and a CRISPR interference (CRISPRi) knockdown of the essential peptidoglycan synthesis enzyme MurA. Last, OXA-23 overexpression sensitized cells to two inhibitors of peptidoglycan synthesis, d-cycloserine and fosfomycin. Our results highlight the impact of OXA-23 hyperexpression on peptidoglycan integrity and reveal new genetic vulnerabilities, which may represent novel targets for antimicrobial agents specific to MDR A. baumannii and other OXA ß-lactamase-overexpressing Enterobacteriaceae, while having no impact on the normal flora. IMPORTANCE Acinetobacter baumannii has become a serious pathogen in both hospital and community settings. The ß-lactam class of antibiotics is a primary treatment option for A. baumannii infections, and expression of ß-lactamases is the most frequent mechanism of resistance in this bacterium. New approaches to treating multidrug-resistant A. baumannii strains are needed. In this study, we demonstrate that overexpressing the OXA-23 ß-lactamase leads to significant collateral changes, where peptidoglycan structure is altered. We have identified genes that become selectively essential in OXA-23-expressing strains and confirmed the relationship between altered peptidoglycan and OXA-23 expression by demonstrating that OXA-23 overexpression sensitizes cells to genetic and chemical inhibition of peptidoglycan synthesis. This work paves the way for the identification of new antimicrobial targets, where inhibitors would selectively kill ß-lactamase-expressing strains.

A LysR-Type Transcriptional Regulator Controls Multiple Phenotypes in Acinetobacter baumannii.

Acinetobacter baumannii is a multidrug-resistant, Gram-negative nosocomial pathogen that exhibits phenotypic heterogeneity resulting in virulent opaque (VIR-O) and avirulent translucent (AV-T) colony variants. Each variant has a distinct gene expression profile resulting in multiple phenotypic differences. Cells interconvert between the VIR-O and AV-T variants at high frequency under laboratory conditions, suggesting that the genetic mechanism underlying the phenotypic switch could be manipulated to attenuate virulence. Therefore, our group has focused on identifying and characterizing genes that regulate this switch, which led to the investigation of ABUW_1132 (1132), a highly conserved gene predicted to encode a LysR-type transcriptional regulator. ABUW_1132 was shown to be a global regulator as the expression of 74 genes was altered ≥ 2-fold in an 1132 deletion mutant. The 1132 deletion also resulted in a 16-fold decrease in VIR-O to AV-T switching, loss of 3-OH-C12-HSL secretion, and reduced surface-associated motility. Further, the deletion of 1132 in the AV-T background caused elevated capsule production, which increased colony opacity and altered the typical avirulent phenotype of translucent cells. These findings distinguish 1132 as a global regulatory gene and advance our understanding of A. baumannii's opacity-virulence switch.

Broad Spectrum Antibiotic Xanthocillin X Effectively Kills Acinetobacter baumannii via Dysregulation of Heme Biosynthesis.

Isonitrile natural products exhibit promising antibacterial activities. However, their mechanism of action (MoA) remains largely unknown. Based on the nanomolar potency of xanthocillin X (Xan) against diverse difficult-to-treat Gram-negative bacteria, including the critical priority pathogen Acinetobacter baumannii, we performed in-depth studies to decipher its MoA. While neither metal binding nor cellular protein targets were detected as relevant for Xan's antibiotic effects, sequencing of resistant strains revealed a conserved mutation in the heme biosynthesis enzyme porphobilinogen synthase (PbgS). This mutation caused impaired enzymatic efficiency indicative of reduced heme production. This discovery led to the validation of an untapped mechanism, by which direct heme sequestration of Xan prevents its binding into cognate enzyme pockets resulting in uncontrolled cofactor biosynthesis, accumulation of porphyrins, and corresponding stress with deleterious effects for bacterial viability. Thus, Xan represents a promising antibiotic displaying activity even against multidrug resistant strains, while exhibiting low toxicity to human cells.

A comprehensive and contemporary "snapshot" of ß-lactamases in carbapenem resistant Acinetobacter baumannii.

Successful treatment of Acinetobacter baumannii infections require early and appropriate antimicrobial therapy. One of the first steps in this process is understanding which ß-lactamase (bla) alleles are present and in what combinations. Thus, we performed WGS on 98 carbapenem-resistant A. baumannii (CR Ab). In most isolates, an acquired blaOXA carbapenemase was found in addition to the intrinsic blaOXA allele. The most commonly found allele was blaOXA-23 (n = 78/98). In some isolates, blaOXA-23 was found in addition to other carbapenemase alleles: blaOXA-82 (n = 12/78), blaOXA-72 (n = 2/78) and blaOXA-24/40 (n = 1/78). Surprisingly, 20% of isolates carried carbapenemases not routinely assayed for by rapid molecular diagnostic platforms, i.e., blaOXA-82 and blaOXA-172; all had ISAba1 elements. In 8 CR Ab, blaOXA-82 or blaOXA-172 was the only carbapenemase. Both blaOXA-24/40 and its variant blaOXA-72 were each found in 6/98 isolates. The most prevalent ADC variants were blaADC-30 (21%), blaADC-162 (21%), and blaADC-212 (26%). Complete combinations are reported.

Copy Number of an Integron-Encoded Antibiotic Resistance Locus Regulates a Virulence and Opacity Switch in Acinetobacter baumannii AB5075.

We describe a novel genetic mechanism in which tandem amplification of a plasmid-borne integron regulates virulence, opacity variation, and global gene expression by altering levels of a putative small RNA (sRNA) in Acinetobacter baumannii AB5075. Copy number of this amplified locus correlated with the rate of switching between virulent opaque (VIR-O) and avirulent translucent (AV-T) cells. We found that prototypical VIR-O colonies, which exhibit high levels of switching and visible sectoring with AV-T cells by 24 h of growth, harbor two copies of this locus. However, a subset of opaque colonies that did not form AV-T sectors within 24 h were found to harbor only one copy. The colonies with decreased sectoring to AV-T were designated low-switching opaque (LSO) variants and were found to exhibit a 3-log decrease in switching relative to that of the VIR-O. Overexpression studies revealed that the element regulating switching was localized to the 5' end of the aadB gene within the amplified locus. Northern blotting indicated that an sRNA of approximately 300 nucleotides (nt) is encoded in this region and is likely responsible for regulating switching to AV-T. Copy number of the ∼300-nt sRNA was also found to affect virulence, as the LSO variant exhibited decreased virulence during murine lung infections. Global transcriptional profiling revealed that >100 genes were differentially expressed between VIR-O and LSO variants, suggesting that the ∼300-nt sRNA may act as a global regulator. Several virulence genes exhibited decreased expression in LSO cells, potentially explaining their decreased virulence.IMPORTANCEAcinetobacter baumannii remains a leading cause of hospital-acquired infections. Widespread multidrug resistance in this species has prompted the WHO to name carbapenem-resistant A. baumannii as its top priority for research and development of new antibiotics. Many strains of A. baumannii undergo a high-frequency virulence switch, which is an attractive target for new therapeutics targeting this pathogen. This study reports a novel mechanism controlling the frequency of switching in strain AB5075. The rate of switching from the virulent opaque (VIR-O) to the avirulent translucent (AV-T) variant is positively influenced by the copy number of an antibiotic resistance locus encoded on a plasmid-borne composite integron. Our data suggest that this locus encodes a small RNA that regulates opacity switching. Low-switching opaque variants, which harbor a single copy of this locus, also exhibit decreased virulence. This study increases our understanding of this critical phenotypic switch, while also identifying potential targets for virulence-based A. baumannii treatments.

Insights Into Mechanisms of Biofilm Formation in Acinetobacter baumannii and Implications for Uropathogenesis.

Multidrug resistant Acinetobacter baumannii is a serious healthcare threat. In fact, the Center for Disease Control recently reported that carbapenem-resistant A. baumannii is responsible for more than 8,500 infections, 700 deaths, and $281 million in healthcare costs annually in the United States with few, if any, treatment options available, leading to its designation as a pathogen of urgent concern and a priority for novel antimicrobial development. It is hypothesized that biofilms are, at least in part, responsible for the high prevalence of A. baumannii nosocomial and recurrent infections because they frequently contaminate hospital surfaces and patient indwelling devices; therefore, there has been a recent push for mechanistic understanding of biofilm formation, maturation and dispersal. However, most research has focused on A. baumannii pneumonia and bloodstream infections, despite a recent retrospective study showing that 17.1% of A. baumannii isolates compiled from clinical studies over the last two decades were obtained from urinary samples. This highlights that A. baumannii is an underappreciated uropathogen. The following minireview will examine our current understanding of A. baumannii biofilm formation and how this influences urinary tract colonization and pathogenesis.

1,2,3-Triazolylmethaneboronate: A Structure Activity Relationship Study of a Class of ß-Lactamase Inhibitors against Acinetobacter baumannii Cephalosporinase.

Boronic acid transition state inhibitors (BATSIs) are known reversible covalent inhibitors of serine ß-lactamases. The selectivity and high potency of specific BATSIs bearing an amide side chain mimicking the ß-lactam's amide side chain are an established and recognized synthetic strategy. Herein, we describe a new class of BATSIs where the amide group is replaced by a bioisostere triazole; these compounds were designed as molecular probes. To this end, a library of 26 α-triazolylmethaneboronic acids was synthesized and tested against the clinically concerning Acinetobacter-derived cephalosporinase, ADC-7. In steady state analyses, these compounds demonstrated Ki values ranging from 90 nM to 38 µM (±10%). Five compounds were crystallized in complex with ADC-7 ß-lactamase, and all the crystal structures reveal the triazole is in the putative amide binding site, thus confirming the triazole-amide bioisosterism. The easy synthetic access of these new inhibitors as prototype scaffolds allows the insertion of a wide range of chemical groups able to explore the enzyme binding site and provides insights on the importance of specific residues in recognition and catalysis. The best inhibitor identified, compound 6q (Ki 90 nM), places a tolyl group near Arg340, making favorable cation-π interactions. Notably, the structure of 6q does not resemble the natural substrate of the ß-lactamase yet displays a pronounced inhibition activity, in addition to lowering the minimum inhibitory concentration (MIC) of ceftazidime against three bacterial strains expressing class C ß-lactamases. In summary, these observations validate the α-triazolylboronic acids as a promising template for further inhibitor design.

Characterization of RelA in Acinetobacter baumannii.

In response to nutrient depletion, the RelA and SpoT proteins generate the signaling molecule (p)ppGpp, which then controls a number of downstream effectors to modulate cell physiology. In Acinetobacter baumannii strain AB5075, a relA ortholog (ABUW_3302) was identified by a transposon insertion that conferred an unusual colony phenotype. An in-frame deletion in relA (ΔrelA) failed to produce detectable levels of ppGpp when amino acid starvation was induced with serine hydroxamate. The ΔrelA mutant was blocked from switching from the virulent opaque colony variant (VIR-O) to the avirulent translucent colony variant (AV-T), but the rate of AV-T to VIR-O switching was unchanged. In addition, the ΔrelA mutation resulted in a pronounced hypermotile phenotype on 0.35% agar plates. This hypermotility was dependent on the activation of a LysR regulator ABUW_1132, which was required for expression of AbaR, a LuxR family quorum-sensing regulator. In the ΔrelA mutant, ABUW_1132 was also required for the increased expression of an operon composed of the ABUW_3766-ABUW_3773 genes required for production of the surfactant-like lipopeptide acinetin 505. Additional phenotypes identified in the ΔrelA mutant included (i) cell elongation at high density, (ii) reduced formation of persister cells tolerant to colistin and rifampin, and (iii) decreased virulence in a Galleria mellonella model.IMPORTANCEAcinetobacter baumannii is a pathogen of worldwide importance. Due to the increasing prevalence of antibiotic resistance, these infections are becoming increasingly difficult to treat. New therapies are required to combat multidrug-resistant isolates. The role of RelA in A. baumannii is largely unknown. This study demonstrates that like in other bacteria, RelA controls a variety of functions, including virulence. Strategies to inhibit the activity of RelA and the resulting production of ppGpp could inhibit virulence and may represent a new therapeutic approach.

Allelic Exchange Mutagenesis in Proteus mirabilis.

This chapter outlines a method for making unmarked, in-frame deletion mutations in Proteus mirabilis by allelic replacement. This method utilizes an R6K-based suicide plasmid allowing for integration of the plasmid by homologous recombination via a cloned insert. The plasmid also contains the sacB gene to select for plasmid loss (excision) in the presence of sucrose to create a mutant allele. This method has been applied to the P. mirabilis strains PM7002 and BB2000 and should be generally applicable to other P. mirabilis strains. The same methods described in this chapter can be used to introduce marked or point mutations in a given gene.

Methods for Transposon Mutagenesis in Proteus mirabilis.

Several methods for transposon mutagenesis have been employed for use in P. mirabilis. The first method involves the use of mini-Tn5 derivatives, which are delivered by conjugation of a suicide plasmid containing this transposon, followed by transposition into the chromosome. A second method is the use of preformed transposon/transposase complexes (transposomes), which are introduced into P. mirabilis cells by electroporation. Each of these methods will be discussed along with the advantages and disadvantages of each.

Roles of two-component regulatory systems in antibiotic resistance.

Two-component regulatory systems (TCSs) are a major mechanism by which bacteria sense and respond to changes in their environment. TCSs typically consist of two proteins that bring about major regulation of the cell genome through coordinated action mediated by phosphorylation. Environmental conditions that activate TCSs are numerous and diverse and include exposure to antibiotics as well as conditions inside a host. The resulting regulatory action often involves activation of antibiotic defenses and changes to cell physiology that increase antibiotic resistance. Examples of resistance mechanisms enacted by TCSs contained in this review span those found in both Gram-negative and Gram-positive species and include cell surface modifications, changes in cell permeability, increased biofilm formation, and upregulation of antibiotic-degrading enzymes.

Targeting Multidrug-Resistant Acinetobacter spp.: Sulbactam and the Diazabicyclooctenone ß-Lactamase Inhibitor ETX2514 as a Novel Therapeutic Agent.

Multidrug-resistant (MDR) Acinetobacter spp. poses a significant therapeutic challenge in part due to the presence of chromosomally encoded ß-lactamases, including class C Acinetobacter-derived cephalosporinases (ADC) and class D oxacillinases (OXA), as well as plasmid-mediated class A ß-lactamases. Importantly, OXA-like ß-lactamases represent a gap in the spectrum of inhibition by recently approved ß-lactamase inhibitors such as avibactam and vaborbactam. ETX2514 is a novel, rationally designed, diazabicyclooctenone inhibitor that effectively targets class A, C, and D ß-lactamases. We show that addition of ETX2514 significantly increased the susceptibility of clinical Acinetobacterbaumannii isolates to sulbactam. AdeB and AdeJ were identified to be key efflux constituents for ETX2514 in A. baumannii The combination of sulbactam and ETX2514 was efficacious against A. baumannii carrying blaTEM-1, blaADC-82, blaOXA-23, and blaOXA-66 in a neutropenic murine thigh infection model. We also show that, in vitro, ETX2514 inhibited ADC-7 (k2/Ki 1.0 ± 0.1 × 106 M-1 s-1) and OXA-58 (k2/Ki 2.5 ± 0.3 × 105 M-1 s-1). Cocrystallization of ETX2514 with OXA-24/40 revealed hydrogen bonding interactions between ETX2514 and residues R261, S219, and S128 of OXA-24/40 in addition to a chloride ion occupied in the active site. Further, the C3 methyl group of ETX2514 shifts the position of M223. In conclusion, the sulbactam-ETX2514 combination possesses a broadened inhibitory range to include class D ß-lactamases as well as class A and C ß-lactamases and is a promising therapeutic candidate for infections caused by MDR Acinetobacter spp.IMPORTANCE The number and diversity of ß-lactamases are steadily increasing. The emergence of ß-lactamases that hydrolyze carbapenems poses a significant threat to our antibiotic armamentarium. The explosion of OXA enzymes that are carbapenem hydrolyzers is a major challenge (carbapenem-hydrolyzing class D [CHD]). An urgent need exists to discover ß-lactamase inhibitors with class D activity. The sulbactam-ETX2514 combination demonstrates the potential to become a treatment regimen of choice for Acinetobacter spp. producing class D ß-lactamases.