Induced Heteroresistance in Carbapenem-Resistant Acinetobacter baumannii (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility.

Infections caused by Carbapenem-resistant Acinetobacter baumannii (CRAB) isolates, such as hospital-acquired pneumonia (HAP), bacteremia, and skin and soft tissue infections, among others, are particularly challenging to treat. Cefiderocol, a chlorocatechol-substituted siderophore antibiotic, was approved by the U.S. Food and Drug Administration (FDA) in 2019 and prescribed for the treatment of CRAB infections. Despite the initial positive treatment outcomes with this antimicrobial, recent studies reported a higher-than-average all-cause mortality rate in patients treated with cefiderocol compared to the best available therapy. The cause(s) behind these outcomes remains unconfirmed. A plausible hypothesis is heteroresistance, a phenotype characterized by the survival of a small proportion of cells in a population that is seemingly isogenic. Recent results have demonstrated that the addition of human fluids to CRAB cultures leads to cefiderocol heteroresistance. Here, we describe the molecular and phenotypic analyses of CRAB heteroresistant bacterial subpopulations to better understand the nature of the less-than-expected successful outcomes after cefiderocol treatment. Isolation of heteroresistant variants of the CRAB strain AMA40 was carried out in cultures supplemented with cefiderocol and human pleural fluid (HPF). Two AMA40 variants, AMA40 IHC1 and IHC2, were resistant to cefiderocol. To identify mutations and gene expression changes associated with cefiderocol heteroresistance, we subjected these variants to whole genome sequencing and global transcriptional analysis. We then assessed the impact of these mutations on the pharmacodynamic activity of cefiderocol via susceptibility testing, EDTA and boronic acid inhibition analysis, biofilm formation, and static time-kill assays. Heteroresistant variants AMA40 IHC1 and AMA40 IHC2 have 53 chromosomal mutations, of which 40 are common to both strains. None of the mutations occurred in genes associated with high affinity iron-uptake systems or ß-lactam resistance. However, transcriptional analyses demonstrated significant modifications in levels of expression of genes associated with iron-uptake systems or ß-lactam resistance. The blaNDM-1 and blaADC-2, as well as various iron-uptake system genes, were expressed at higher levels than the parental strain. On the other hand, the carO and ompA genes' expression was reduced. One of the mutations common to both heteroresistant strains was mapped within ppiA, a gene associated with iron homeostasis in other species. Static time-kill assays demonstrated that supplementing cation-adjusted Mueller-Hinton broth with human serum albumin (HAS), the main protein component of HPF, considerably reduced cefiderocol killing activity for all three strains tested. Notably, collateral resistance to amikacin was observed in both variants. We conclude that exposing CRAB to fluids with high HSA concentrations facilitates the rise of heteroresistance associated with point mutations and transcriptional upregulation of genes coding for ß-lactamases and biofilm formation. The findings from this study hold significant implications for understanding the emergence of CRAB resistance mechanisms against cefiderocol treatment. This understanding is vital for the development of treatment guidelines that can effectively address the challenges posed by CRAB infections.

Whole Genome Sequence Analysis of Burkholderia contaminans FFH2055 Strain Reveals the Presence of Putative ß-Lactamases.

Burkholderia contaminans is a member of the Burkholderia cepacia complex (Bcc), a pathogen with increasing prevalence among cystic fibrosis (CF) patients and the cause of numerous outbreaks due to the use of contaminated commercial products. The antibiotic resistance determinants, particularly ß-lactamases, have been poorly studied in this species. In this work, we explored the whole genome sequence (WGS) of a B. contaminans isolate (FFH 2055) and detected four putative ß-lactamase-encoding genes. In general, these genes have more than 93% identity with ß-lactamase genes found in other Bcc species. Two ß-lactamases, a class A (Pen-like, suggested name PenO) and a class D (OXA-like), were further analyzed and characterized. Amino acid sequence comparison showed that Pen-like has 82% and 67% identity with B. multivorans PenA and B. pseudomallei PenI, respectively, while OXA-like displayed strong homology with class D enzymes within the Bcc, but only 22-44% identity with available structures from the OXA family. PCR reactions designed to study the presence of these two genes revealed a heterogeneous distribution among clinical and industrial B. contaminans isolates. Lastly, blaPenO gene was cloned and expressed into E. coli to investigate the antibiotic resistance profile and confers an extended-spectrum ß-lactamase (ESBL) phenotype. These results provide insight into the presence of ß-lactamases in B. contaminans, suggesting they play a role in antibiotic resistance of these bacteria.

Amikacin: Uses, Resistance, and Prospects for Inhibition.

Aminoglycosides are a group of antibiotics used since the 1940s to primarily treat a broad spectrum of bacterial infections. The primary resistance mechanism against these antibiotics is enzymatic modification by aminoglycoside-modifying enzymes that are divided into acetyl-transferases, phosphotransferases, and nucleotidyltransferases. To overcome this problem, new semisynthetic aminoglycosides were developed in the 70s. The most widely used semisynthetic aminoglycoside is amikacin, which is refractory to most aminoglycoside modifying enzymes. Amikacin was synthesized by acylation with the l-(-)-γ-amino-α-hydroxybutyryl side chain at the C-1 amino group of the deoxystreptamine moiety of kanamycin A. The main amikacin resistance mechanism found in the clinics is acetylation by the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib], an enzyme coded for by a gene found in integrons, transposons, plasmids, and chromosomes of Gram-negative bacteria. Numerous efforts are focused on finding strategies to neutralize the action of AAC(6')-Ib and extend the useful life of amikacin. Small molecules as well as complexes ionophore-Zn+2 or Cu+2 were found to inhibit the acetylation reaction and induced phenotypic conversion to susceptibility in bacteria harboring the aac(6')-Ib gene. A new semisynthetic aminoglycoside, plazomicin, is in advance stage of development and will contribute to renewed interest in this kind of antibiotics.

Inhibition of aminoglycoside 6'-N-acetyltransferase type Ib-mediated amikacin resistance in Klebsiella pneumoniae by zinc and copper pyrithione.

The in vitro activity of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] was inhibited by CuCl2 with a 50% inhibitory concentration (IC50) of 2.8 µM. The growth of an amikacin-resistant Klebsiella pneumoniae strain isolated from a neonate with meningitis was inhibited when amikacin was supplemented by the addition of Zn(2+) or Cu(2+) in complex with the ionophore pyrithione. Coordination complexes between cations and ionophores could be developed for their use, in combination with aminoglycosides, to treat resistant infections.

Dynamics and quantitative contribution of the aminoglycoside 6'-N-acetyltransferase type Ib to amikacin resistance.

Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6')-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we used a fluorescent-based system to quantify the number of AAC(6')-Ib per cell in Escherichia coli, and we modulated this copy number with the CRISPR interference method. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6')-Ib until it reaches a plateau at a specific protein concentration. In vivo imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6')-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6')-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell.IMPORTANCEAntibiotic resistance is a growing threat to human health. Understanding antibiotic resistance mechanisms can serve as foundation for developing innovative treatment strategies to counter this threat. While numerous studies clarified the genetics and dissemination of resistance genes and explored biochemical and structural features of resistance enzymes, their molecular dynamics and individual contribution to resistance within the cellular context remain unknown. Here, we examined this relationship modulating expression levels of aminoglycoside 6'-N-acetyltransferase type Ib, an enzyme of clinical relevance. We show a linear correlation between copy number of the enzyme per cell and amikacin resistance levels up to a threshold where resistance plateaus. We propose that at concentrations below the threshold, the enzyme diffuses freely in the cytoplasm but aggregates at the cell poles at concentrations over the threshold. This research opens promising avenues for studying enzyme solubility's impact on resistance, creating opportunities for future approaches to counter resistance.

Structure-Activity Relationship of Pyrrolidine Pentamine Derivatives as Inhibitors of the Aminoglycoside 6'-N-Acetyltransferase Type Ib.

Resistance to amikacin and other major aminoglycosides is commonly due to enzymatic acetylation by the aminoglycoside 6'-N-acetyltransferase type I enzyme, of which type Ib [AAC(6')-Ib] is the most widespread among Gram-negative pathogens. Finding enzymatic inhibitors could be an effective way to overcome resistance and extend the useful life of amikacin. Small molecules possess multiple properties that make them attractive for drug development. Mixture-based combinatorial libraries and positional scanning strategy have led to the identification of a chemical scaffold, pyrrolidine pentamine, that, when substituted with the appropriate functionalities at five locations (R1-R5), inhibits AAC(6')-Ib-mediated inactivation of amikacin. Structure-activity relationship studies have shown that while truncations to the molecule result in loss of inhibitory activity, modifications of functionalities and stereochemistry have different effects on the inhibitory properties. In this study, we show that alterations at position R1 of the two most active compounds, 2700.001 and 2700.003, reduced inhibition levels, demonstrating the essential nature not only of the presence of an S-phenyl moiety at this location but also the distance to the scaffold. On the other hand, modifications on the R3, R4, and R5 positions had varied effects, demonstrating the potential for optimization. A correlation analysis between molecular docking values (ΔG) and the dose required for two-fold potentiation of the compounds described in this and the previous studies showed a significant correlation between ΔG values and inhibitory activity.

Structure-activity relationship of pyrrolidine pentamine derivatives as inhibitors of the aminoglycoside 6'- N -acetyltransferase type Ib.

Resistance to amikacin and other major aminoglycosides is commonly due to enzymatic acetylation by aminoglycoside 6'- N -acetyltransferase type I enzyme, of which type Ib [AAC(6')-Ib] is the most widespread among Gram-negative pathogens. Finding enzymatic inhibitors could be an effective way to overcome resistance and extend the useful life of amikacin. Small molecules possess multiple properties that make them attractive compounds to be developed as drugs. Mixture-based combinatorial libraries and positional scanning strategy led to the identification of a chemical scaffold, pyrrolidine pentamine, that, when substituted with the appropriate functionalities at five locations (R1 - R5), inhibits AAC(6')-Ib-mediated inactivation of amikacin. Structure-activity relationship (SAR) studies showed that while truncations to the molecule result in loss of inhibitory activity, modifications of functionalities and stereochemistry have different effects on the inhibitory properties. In this study, we show that alterations at position R1 of the two most active compounds, 2700.001 and 2700.003 , reduced inhibition levels, demonstrating the essential nature not only of the presence of an S -phenyl moiety at this location but also the distance to the scaffold. On the other hand, modifications on the R3, R4, and R5 positions have varied effects, demonstrating the potential for optimization. A correlation analysis between molecular docking values (ΔG) and the dose required for two-fold potentiation of compounds described in this and the previous studies showed a significant correlation between ΔG values and inhibitory activity. Highlights: Amikacin resistance in Gram-negatives is mostly caused by the AAC(6')-Ib enzymeAAC(6')-Ib has been identified in most Gram-negative pathogensInhibitors of AAC(6')-Ib could be used to treat resistant infectionsCombinatorial libraries and positional scanning identified an inhibitorThe lead compound can be optimized by structure activity relationship studies.

Intra-abdominal infections due to Comamonas kerstersii.

Herein, we report four cases of Comamonas kerstersii intra-abdominal infections representing the first report of human infections caused by this Comamonas species. In addition, our work demonstrates the association of C. kerstersii with peritonitis secondary to appendix rupture.

Inhibition of Enzymatic Acetylation-Mediated Resistance to Plazomicin by Silver Ions.

Plazomicin is a recent U.S. Food and Drug Administration (FDA)-approved semisynthetic aminoglycoside. Its structure consists of a sisomicin scaffold modified by adding a 2(S)-hydroxy aminobutyryl group at the N1 position and a hydroxyethyl substituent at the 6' position. These substitutions produced a molecule refractory to most aminoglycoside-modifying enzymes. The main enzyme within this group that recognizes plazomicin as substrate is the aminoglycoside 2'-N-acetyltransferase type Ia [AAC(2')-Ia], which reduces the antibiotic's potency. Designing formulations that combine an antimicrobial with an inhibitor of resistance is a recognized strategy to extend the useful life of existing antibiotics. We have recently found that several metal ions inhibit the enzymatic inactivation of numerous aminoglycosides mediated by the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib]. In particular, Ag+, which also enhances the effect of aminoglycosides by other mechanisms, is very effective in interfering with AAC(6')-Ib-mediated resistance to amikacin. Here we report that silver acetate is a potent inhibitor of AAC(2')-Ia-mediated acetylation of plazomicin in vitro, and it reduces resistance levels of Escherichia coli carrying aac(2')-Ia. The resistance reversion assays produced equivalent results when the structural gene was expressed under the control of the natural or the blaTEM-1 promoters. The antibiotic effect of plazomicin in combination with silver was bactericidal, and the mix did not show significant toxicity to human embryonic kidney 293 (HEK293) cells.

Dynamics and quantitative contribution of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] to amikacin resistance.

Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside acetyltransferase AAC(6')-Ib is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6')-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we used a fluorescent-based system to quantify the number of AAC(6')-Ib per cell in Escherichia coli, and we modulated this copy number with the CRISPR interference method. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6')-Ib until it reaches a plateau at a specific protein concentration. In vivo imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6')-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6')-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell.

First report of an extensively drug-resistant VIM-2 metallo-ß-lactamase-producing Brevundimonas diminuta clinical isolate.

In the literature, only three Brevundimonas diminuta environmental isolates carrying metallo-ß-lactamase genes were recently published. However, so far, no B. diminuta clinical isolates carrying these carbapenem resistance genes have been described. Here we report the first VIM-2 metallo-ß-lactamase-producing B. diminuta clinical isolate obtained from an immunocompromised patient.

Isolation of Kerstersia gyiorum from a patient with cholesteatomatous chronic otitis media.

We describe the first case of a Kerstersia gyiorum strain isolated from a patient with cholesteatomatous chronic otitis media. We emphasize the isolation of members of the family Alcaligenaceae in serious infections and unusual sites and the importance of polyphasic identification addressing the definitive identification.

First case of fulminant sepsis due to Wohlfahrtiimonas chitiniclastica.

We report the first case of fulminant sepsis due to Wohlfahrtiimonas chitiniclastica. This case is also the first one reported in South America. We emphasize the importance of recognizing bacteria that live in the larvae of a parasitic fly as the causative agent of severe infections in homeless patients.

Aminoglycoside modifying enzymes.

Aminoglycosides have been an essential component of the armamentarium in the treatment of life-threatening infections. Unfortunately, their efficacy has been reduced by the surge and dissemination of resistance. In some cases the levels of resistance reached the point that rendered them virtually useless. Among many known mechanisms of resistance to aminoglycosides, enzymatic modification is the most prevalent in the clinical setting. Aminoglycoside modifying enzymes catalyze the modification at different -OH or -NH2 groups of the 2-deoxystreptamine nucleus or the sugar moieties and can be nucleotidyltransferases, phosphotransferases, or acetyltransferases. The number of aminoglycoside modifying enzymes identified to date as well as the genetic environments where the coding genes are located is impressive and there is virtually no bacteria that is unable to support enzymatic resistance to aminoglycosides. Aside from the development of new aminoglycosides refractory to as many as possible modifying enzymes there are currently two main strategies being pursued to overcome the action of aminoglycoside modifying enzymes. Their successful development would extend the useful life of existing antibiotics that have proven effective in the treatment of infections. These strategies consist of the development of inhibitors of the enzymatic action or of the expression of the modifying enzymes.

Two Acinetobacter baumannii Isolates Obtained From a Fatal Necrotizing Fasciitis Infection Display Distinct Genomic and Phenotypic Characteristics in Comparison to Type Strains.

Acinetobacter baumannii has been recognized as a critical pathogen that causes severe infections worldwide not only because of the emergence of extensively drug-resistant (XDR) derivatives, but also because of its ability to persist in medical environments and colonize compromised patients. While there are numerous reports describing the mechanisms by which this pathogen acquires resistance genes, little is known regarding A. baumannii's virulence functions associated with rare manifestations of infection such as necrotizing fasciitis, making the determination and implementation of alternative therapeutic targets problematic. To address this knowledge gap, this report describes the analysis of the NFAb-1 and NFAb-2 XDR isolates, which were obtained at two time points during a fatal case of necrotizing fasciitis, at the genomic and functional levels. The comparative genomic analysis of these isolates with the ATCC 19606T and ATCC 17978 strains showed that the NFAb-1 and NFAb-2 isolates are genetically different from each other as well as different from the ATCC 19606T and ATCC 17978 clinical isolates. These genomic differences could be reflected in phenotypic differences observed in these NFAb isolates. Biofilm, cell viability and flow cytometry assays indicate that all tested strains caused significant decreases in A549 human alveolar epithelial cell viability with ATCC 17978, NFAb-1 and NFAb-2 producing significantly less biofilm and significantly more hemolysis and capacity for intracellular invasion than ATCC 19606T. NFAb-1 and NFAb-2 also demonstrated negligible surface motility but significant twitching motility compared to ATCC 19606T and ATCC 17978, likely due to the presence of pili exceeding 2 µm in length, which are significantly longer and different from those previously described in the ATCC 19606T and ATCC 17978 strains. Interestingly, infection with cells of the NFAb-1 isolate, which were obtained from a premortem blood sample, lead to significantly higher mortality rates than NFAb-2 bacteria, which were obtained from postmortem tissue samples, when tested using the Galleria mellonella in vivo infection model. These observations suggest potential changes in the virulence phenotype of the A. baumannii necrotizing fasciitis isolates over the course of infection by mechanisms and cell processes that remain to be identified.

First case of human infection due to Pseudomonas fulva, an environmental bacterium isolated from cerebrospinal fluid.

We report the first case of human infection due to Pseudomonas fulva. P. fulva caused acute meningitis following the placement of a drainage system in a 2-year-old female. Additionally, the isolate displayed a VIM-2 carbapenemase in a class 1 integron context.

Aminoglycoside 6'-N-acetyltransferase Type Ib [AAC(6')-Ib]-Mediated Aminoglycoside Resistance: Phenotypic Conversion to Susceptibility by Silver Ions.

Clinical resistance to amikacin and other aminoglycosides is usually due to the enzymatic acetylation of the antimicrobial molecule. A ubiquitous resistance enzyme among Gram-negatives is the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib], which catalyzes acetylation using acetyl-CoA as a donor substrate. Therapies that combine the antibiotic and an inhibitor of the inactivation reaction could be an alternative to treat infections caused by resistant bacteria. We previously observed that metal ions such as Zn2+ or Cu2+ in complex with ionophores interfere with the AAC(6')-Ib-mediated inactivation of aminoglycosides and reduced resistance to susceptibility levels. Ag1+ recently attracted attention as a potentiator of aminoglycosides' action by mechanisms still in discussion. We found that silver acetate is also a robust inhibitor of the enzymatic acetylation mediated by AAC(6')-Ib in vitro. This action seems to be independent of other mechanisms, like increased production of reactive oxygen species and enhanced membrane permeability, proposed to explain the potentiation of the antibiotic effect by silver ions. The addition of this compound to aac(6')-Ib harboring Acinetobacter baumannii and Escherichia coli cultures resulted in a dramatic reduction of the resistance levels. Time-kill assays showed that the combination of silver acetate and amikacin was bactericidal and exhibited low cytotoxicity to HEK293 cells.

Small Klebsiella pneumoniae Plasmids: Neglected Contributors to Antibiotic Resistance.

Klebsiella pneumoniae is the causative agent of community- and, more commonly, hospital-acquired infections. Infections caused by this bacterium have recently become more dangerous due to the acquisition of multiresistance to antibiotics and the rise of hypervirulent variants. Plasmids usually carry genes coding for resistance to antibiotics or virulence factors, and the recent sequence of complete K. pneumoniae genomes showed that most strains harbor many of them. Unlike large plasmids, small, usually high copy number plasmids, did not attract much attention. However, these plasmids may include genes coding for specialized functions, such as antibiotic resistance, that can be expressed at high levels due to gene dosage effect. These genes may be part of mobile elements that not only facilitate their dissemination but also participate in plasmid evolution. Furthermore, high copy number plasmids may also play a role in evolution by allowing coexistence of mutated and non-mutated versions of a gene, which helps to circumvent the constraints imposed by trade-offs after certain genes mutate. Most K. pneumoniae plasmids 25-kb or smaller replicate by the ColE1-type mechanism and many of them are mobilizable. The transposon Tn1331 and derivatives were found in a high percentage of these plasmids. Another transposon that was found in representatives of this group is the bla KPC-containing Tn4401. Common resistance determinants found in these plasmids were aac(6')-Ib and genes coding for ß-lactamases including carbapenemases.

Restoration of susceptibility to amikacin by 8-hydroxyquinoline analogs complexed to zinc.

Gram-negative pathogens resistant to amikacin and other aminoglycosides of clinical relevance usually harbor the 6'-N-acetyltransferase type Ib [AAC(6')-Ib], an enzyme that catalyzes inactivation of the antibiotic by acetylation using acetyl-CoA as donor substrate. Inhibition of the acetylating reaction could be a way to induce phenotypic conversion to susceptibility in these bacteria. We have previously observed that Zn2+ acts as an inhibitor of the enzymatic acetylation of aminoglycosides by AAC(6')-Ib, and in complex with ionophores it effectively reduced the levels of resistance in cellulo. We compared the activity of 8-hydroxyquinoline, three halogenated derivatives, and 5-[N-Methyl-N-Propargylaminomethyl]-8-Hydroxyquinoline in complex with Zn2+ to inhibit growth of amikacin-resistant Acinetobacter baumannii in the presence of the antibiotic. Two of the compounds, clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) and 5,7-diiodo-8-hydroxyquinoline, showed robust inhibition of growth of the two A. baumannii clinical isolates that produce AAC(6')-Ib. However, none of the combinations had any activity on another amikacin-resistant A. baumannii strain that possesses a different, still unknown mechanism of resistance. Time-kill assays showed that the combination of clioquinol or 5,7-diiodo-8-hydroxyquinoline with Zn2+ and amikacin was bactericidal. Addition of 8-hydroxyquinoline, clioquinol, or 5,7-diiodo-8-hydroxyquinoline, alone or in combination with Zn2+, and amikacin to HEK293 cells did not result in significant toxicity. These results indicate that ionophores in complex with Zn2+ could be developed into potent adjuvants to be used in combination with aminoglycosides to treat Gram-negative pathogens in which resistance is mediated by AAC(6')-Ib and most probably other related aminoglycoside modifying enzymes.

Identification of Potential Virulence Factors in the Model Strain Acinetobacter baumannii A118.

Acinetobacter baumannii A118, a strain isolated from the blood of an infected patient, is naturally competent and unlike most clinical strains, is susceptible to a variety of different antibiotics including those usually used for selection in genetic manipulations. These characteristics make strain A118 a convenient model for genetic studies of A. baumannii. To identify potential virulence factors, its complete genome was analyzed and compared to other A. baumannii genomes. A. baumannii A118 includes gene clusters coding for the acinetobactin and baumannoferrin iron acquisition systems. Iron-regulated expression of the BauA outer membrane receptor for ferric-acinetobactin complexes was confirmed as well as the utilization of acinetobactin. A. baumannii A118 also possesses the feoABC genes, which code for the main bacterial ferrous uptake system. The functionality of baumannoferrin was suggested by the ability of A. baumannii A118 culture supernatants to cross feed an indicator BauA-deficient strain plated on iron-limiting media. A. baumannii A118 behaved as non-motile but included the csuA/BABCDE chaperone-usher pilus assembly operon and produced biofilms on polystyrene and glass surfaces. While a known capsular polysaccharide (K) locus was identified, the outer core polysaccharide (OC) locus, which belongs to group B, showed differences with available sequences. Our results show that despite being susceptible to most antibiotics, strain A118 conserves known virulence-related traits enhancing its value as model to study A. baumannii pathogenicity.