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.

Role of Capsule in Resistance to Disinfectants, Host Antimicrobials, and Desiccation in Acinetobacter baumannii.

Acinetobacter baumannii strain AB5075 forms two cell types distinguished by their opaque (VIR-O) or translucent (AV-T) colonies. VIR-O cells possess a thicker capsule and are more resistant to a variety of stressors than AV-T cells. However, the direct role of the capsule in these stressors was unknown. This study demonstrates that the capsule is required for resistance to disinfectants, lysozyme, and desiccation in Acinetobacter baumannii In addition, the capsule is required for survival in a mouse lung model of infection.

An ompR-envZ Two-Component System Ortholog Regulates Phase Variation, Osmotic Tolerance, Motility, and Virulence in Acinetobacter baumannii Strain AB5075.

Recently, a novel phase-variable colony opacity phenotype was discovered in Acinetobacter baumannii strain AB5075, where colonies interconvert between opaque and translucent variants. Opaque colonies become mottled or sectored after 24 h of growth due to translucent variants arising within the colony. This easily distinguishable opaque-colony phenotype was used to screen for random transposon insertions that increased the frequency of sectoring at a time point when wild-type colonies were uniformly opaque. A colony was identified that contained multiple papillae of translucent variants, and the insertion in this mutant mapped to an ortholog of the two-component system response regulator ompR Subsequent investigation of in-frame deletions of ompR and the sensor kinase envZ (located adjacent to ompR) showed that the switching frequency from opaque to translucent was increased 401- and 281-fold, respectively. The ompR mutant also exhibited sensitivity to sodium chloride in growth medium, whereas the envZ mutation did not elicit sensitivity to sodium chloride. Mutation of either gene reduced motility in A. baumannii strain AB5075, but a mutation in both ompR and envZ produced a more profound effect. The ompR and envZ genes were cotranscribed but were not subject to autoregulation by OmpR. Both ompR and envZ mutant opaque variants were attenuated in virulence in the Galleria mellonella infection model, whereas mutation of ompR had no effect on the virulence of the translucent variant. IMPORTANCEAcinetobacter baumannii is a well-known antibiotic-resistant pathogen; many clinical isolates can only be treated by a very small number of antibiotics (including colistin), while some exhibit panresistance. The current antimicrobial arsenal is nearing futility in the treatment of Acinetobacter infections, and new avenues of treatment are profoundly needed. Since phase variation controls the transition between opaque (virulent) and translucent (avirulent) states in A. baumannii, this may represent an "Achilles' heel" that can be targeted via the development of small molecules that lock cells in the translucent state and allow the host immune system to clear the infection. A better understanding of how phase variation is regulated may allow for the development of methods to target this process. The ompR-envZ two-component system ortholog negatively regulates phase variation in A. baumannii, and perturbation of this system leads to the attenuation of virulence in an invertebrate infection model.

Mutations Decreasing Intrinsic ß-Lactam Resistance Are Linked to Cell Division in the Nosocomial Pathogen Acinetobacter baumannii.

Transposon mutagenesis was used to identify novel determinants of intrinsic ß-lactam resistance in Acinetobacter baumannii An EZ-Tn5 transposon insertion in a gene corresponding to the A1S_0225 sequence resulted in a 4-fold decrease in resistance to ampicillin, cefotaxime, imipenem, and ceftriaxone but did not alter resistance to other classes of antibiotics. Based on this phenotype, the gene was designated blhA (ß-lactam hypersusceptibility). The blhA::EZ-Tn5 mutation conferred a similar phenotype in A. baumannii strain ATCC 17978. The wild-type blhA gene complemented the blhA::EZTn5 insertion and restored ß-lactam resistance levels back to wild-type levels. The blhA mutation also increased ß-lactam susceptibility in an adeB adeJ double mutant, indicating that the blhA mutation acted independently of these efflux systems to mediate susceptibility. In addition, mRNA levels for the blaOXA and blaADC ß-lactamase genes were not altered by the blhA mutation. The blhA mutation resulted in a prominent cell division and morphological defect, with cells exhibiting a highly elongated phenotype, combined with large bulges in some cells. The blhA gene is unique to Acinetobacter and likely represents a novel gene involved in cell division. Three additional mutations, in zipA, zapA, and ftsK, each of which encode predicted cell division proteins, also conferred increased ß-lactam susceptibility, indicating a common link between cell division and intrinsic ß-lactam resistance in A. baumannii.

The Rcs regulon in Proteus mirabilis: implications for motility, biofilm formation, and virulence.

The overall role of the Rcs phosphorelay in Proteus mirabilis is largely unknown. Previous work had demonstrated that the Rcs phosphorelay represses the flhDC operon and activates the minCDE cell division inhibition system. To identify additional cellular functions regulated by the Rcs phosphorelay, an analysis of RNA-seq data was undertaken. In this report, the results of the RNA-sequencing are discussed with an emphasis on the predicted roles of the Rcs phosphorelay in swarmer cell differentiation, motility, biofilm formation, and virulence. RcsB is shown to activate genes important for differentiation and fimbriae formation, while repressing the expression of genes important for motility and virulence. Additionally, to follow up on the RNA-Seq data, we demonstrate that an rcsB mutant is deficient in its ability to form biofilm and exhibits enhanced virulence in a Galleria mellonella waxworm model. Overall, these results indicate the Rcs regulon in P. mirabilis extends beyond flagellar genes to include those involved in biofilm formation and virulence. Furthermore, the information presented in this study may provide clues to additional roles of the Rcs phosphorelay in other members of the Enterobacteriaceae.

Phase-Variable Control of Multiple Phenotypes in Acinetobacter baumannii Strain AB5075.

UNLABELLED: Acinetobacter baumannii strain AB5075 produces colonies with two opacity phenotypes, designated opaque and translucent. These phenotypes were unstable and opaque and translucent colony variants were observed to interconvert at high frequency, suggesting that a phase-variable mechanism was responsible. The frequency of phase variation both within colonies and in broth cultures increased in a cell density-dependent manner and was mediated by the accumulation of an extracellular factor. This factor was distinct from the known A. baumannii signaling molecule 3-OH C12-homoserine lactone. Opaque and translucent colony variants exhibited a number of phenotypic differences, including cell morphology, surface motility, biofilm formation, antibiotic resistance, and virulence in a Galleria mellonella model. Additional clinical isolates exhibited a similar phase-variable control of colony opacity, suggesting that this may be a common feature of A. baumannii. IMPORTANCE: A novel phase-variable mechanism has been identified in Acinetobacter baumannii that results in an interconversion between opaque and translucent colony phenotypes. This phase variation also coordinately regulates motility, cell shape, biofilm formation, antibiotic resistance, and virulence. The frequency of phase variation is increased at high cell density via a diffusible extracellular signal. To our knowledge, this report presents the first example of phase variation in A. baumannii and also the first example of quorum sensing-mediated control of phase variation in a bacterium. The findings are important, as this phase-variable mechanism can be identified only via changes in colony opacity using oblique light; therefore, many researchers studying A. baumannii may unknowingly be working with different colony variants.

Regulation of the Min Cell Division Inhibition Complex by the Rcs Phosphorelay in Proteus mirabilis.

UNLABELLED: A key regulator of swarming in Proteus mirabilis is the Rcs phosphorelay, which represses flhDC, encoding the master flagellar regulator FlhD4C2. Mutants in rcsB, the response regulator in the Rcs phosphorelay, hyperswarm on solid agar and differentiate into swarmer cells in liquid, demonstrating that this system also influences the expression of genes central to differentiation. To gain a further understanding of RcsB-regulated genes involved in swarmer cell differentiation, transcriptome sequencing (RNA-Seq) was used to examine the RcsB regulon. Among the 133 genes identified, minC and minD, encoding cell division inhibitors, were identified as RcsB-activated genes. A third gene, minE, was shown to be part of an operon with minCD. To examine minCDE regulation, the min promoter was identified by 5' rapid amplification of cDNA ends (5'-RACE), and both transcriptional lacZ fusions and quantitative real-time reverse transcriptase (qRT) PCR were used to confirm that the minCDE operon was RcsB activated. Purified RcsB was capable of directly binding the minC promoter region. To determine the role of RcsB-mediated activation of minCDE in swarmer cell differentiation, a polar minC mutation was constructed. This mutant formed minicells during growth in liquid, produced shortened swarmer cells during differentiation, and exhibited decreased swarming motility. IMPORTANCE: This work describes the regulation and role of the MinCDE cell division system in P. mirabilis swarming and swarmer cell elongation. Prior to this study, the mechanisms that inhibit cell division and allow swarmer cell elongation were unknown. In addition, this work outlines for the first time the RcsB regulon in P. mirabilis. Taken together, the data presented in this study begin to address how P. mirabilis elongates upon contact with a solid surface.

Putrescine importer PlaP contributes to swarming motility and urothelial cell invasion in Proteus mirabilis.

Previously, we reported that the speA gene, encoding arginine decarboxylase, is required for swarming in the urinary tract pathogen Proteus mirabilis. In addition, this previous study suggested that putrescine may act as a cell-to-cell signaling molecule (Sturgill, G., and Rather, P. N. (2004) Mol. Microbiol. 51, 437-446). In this new study, PlaP, a putative putrescine importer, was characterized in P. mirabilis. In a wild-type background, a plaP null mutation resulted in a modest swarming defect and slightly decreased levels of intracellular putrescine. In a P. mirabilis speA mutant with greatly reduced levels of intracellular putrescine, plaP was required for the putrescine-dependent rescue of swarming motility. When a speA/plaP double mutant was grown in the presence of extracellular putrescine, the intracellular levels of putrescine were greatly reduced compared with the speA mutant alone, indicating that PlaP functioned as the primary putrescine importer. In urothelial cell invasion assays, a speA mutant exhibited a 50% reduction in invasion when compared with wild type, and this defect could be restored by putrescine in a PlaP-dependent manner. The putrescine analog Triamide-44 partially inhibited the uptake of putrescine by PlaP and decreased both putrescine stimulated swarming and urothelial cell invasion in a speA mutant.

Extensively drug-resistant pseudomonas aeruginosa isolates containing blaVIM-2 and elements of Salmonella genomic island 2: a new genetic resistance determinant in Northeast Ohio.

Carbapenems are a mainstay of treatment for infections caused by Pseudomonas aeruginosa. Carbapenem resistance mediated by metallo-ß-lactamases (MBLs) remains uncommon in the United States, despite the worldwide emergence of this group of enzymes. Between March 2012 and May 2013, we detected MBL-producing P. aeruginosa in a university-affiliated health care system in northeast Ohio. We examined the clinical characteristics and outcomes of patients, defined the resistance determinants and structure of the genetic element harboring the blaMBL gene through genome sequencing, and typed MBL-producing P. aeruginosa isolates using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR (rep-PCR), and multilocus sequence typing (MLST). Seven patients were affected that were hospitalized at three community hospitals, a long-term-care facility, and a tertiary care center; one of the patients died as a result of infection. Isolates belonged to sequence type 233 (ST233) and were extensively drug resistant (XDR), including resistance to all fluoroquinolones, aminoglycosides, and ß-lactams; two isolates were nonsusceptible to colistin. The blaMBL gene was identified as blaVIM-2 contained within a class 1 integron (In559), similar to the cassette array previously detected in isolates from Norway, Russia, Taiwan, and Chicago, IL. Genomic sequencing and assembly revealed that In559 was part of a novel 35-kb region that also included a Tn501-like transposon and Salmonella genomic island 2 (SGI2)-homologous sequences. This analysis of XDR strains producing VIM-2 from northeast Ohio revealed a novel recombination event between Salmonella and P. aeruginosa, heralding a new antibiotic resistance threat in this region's health care system.

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.

Regulation of the swarming inhibitor disA in Proteus mirabilis.

The disA gene encodes a putative amino acid decarboxylase that inhibits swarming in Proteus mirabilis. 5' rapid amplification of cDNA ends (RACE) and deletion analysis were used to identify the disA promoter. The use of a disA-lacZ fusion indicated that FlhD(4)C(2), the class I flagellar master regulator, did not have a role in disA regulation. The putative product of DisA, phenethylamine, was able to inhibit disA expression, indicating that a negative regulatory feedback loop was present. Transposon mutagenesis was used to identify regulators of disA and revealed that umoB (igaA) was a negative regulator of disA. Our data demonstrate that the regulation of disA by UmoB is mediated through the Rcs phosphorelay.

Streptomycin inhibits quorum sensing in Acinetobacter baumannii.

Streptomycin at subinhibitory concentrations was found to inhibit quorum sensing in Acinetobacter baumannii. Conditioned medium prepared by growth of A. baumannii in the presence of subinhibitory concentrations of streptomycin exhibited reduced activation of two quorum-sensing-regulated genes, abaI, encoding an autoinducer synthase, and A1S_0112. The reduced expression of AbaI resulted in greatly decreased levels of 3-OH-C(12)-HSL as confirmed by direct analysis using thin-layer chromatography. The effect on acyl-homoserine lactone (AHL) signal production was specific to streptomycin, as gentamicin and myomycin had no significant effect at subinhibitory levels.

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.

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.

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.

Role of the Umo proteins and the Rcs phosphorelay in the swarming motility of the wild type and an O-antigen (waaL) mutant of Proteus mirabilis.

Proteus mirabilis is a Gram-negative bacterium that exists as a short rod when grown in liquid medium, but during growth on surfaces it undergoes a distinct physical and biochemical change that culminates in the formation of a swarmer cell. How P. mirabilis senses a surface is not fully understood; however, the inhibition of flagellar rotation and accumulation of putrescine have been proposed to be sensory mechanisms. Our lab recently isolated a transposon insertion in waaL, encoding O-antigen ligase, that resulted in a loss of swarming but not swimming motility. The waaL mutant failed to activate flhDC, the class 1 activator of the flagellar gene cascade, when grown on solid surfaces. Swarming in the waaL mutant was restored by overexpression of flhDC in trans or by a mutation in the response regulator rcsB. To further investigate the role of the Rcs signal transduction pathway and its possible relationship with O-antigen surface sensing, mutations were made in the rcsC, rcsB, rcsF, umoB (igaA), and umoD genes in wild-type and waaL backgrounds. Comparison of the swarming phenotypes of the single and double mutants and of strains overexpressing combinations of the UmoB, UmoD, and RcsF proteins demonstrated the following: (i) there is a differential effect of RcsF and UmoB on swarming in wild-type and waaL backgrounds, (ii) RcsF inhibits UmoB activity but not UmoD activity in a wild-type background, and (iii) UmoD is able to modulate activity of the Rcs system.

Novel mechanism for fluoroquinolone resistance in Acinetobacter baumannii.

An EZ::TNTnp transposon insertion in an open reading frame of unknown function (ncr) in Acinetobacter baumannii resulted in an 8-fold increase in ciprofloxacin resistance (Cip(r)). Transposon insertions in an ncr mutant that reduced Cip(r) back to wild type mapped to three genes encoding subunits of the RecCBD exonuclease. The ncr mutation increased transcription of the recCBD genes, and overexpression of the recCBD genes in a wild-type background resulted in a 4-fold increase in Cip(r).

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.