Colistin Resistance In Acinetobacter Baumannii Research Articles

Resensitization to colistin results in rapid and stable recovery of adherence, serum resistance and ompW in Acinetobacter baumannii.

Colistin resistance in Acinetobacter baumannii is an emerging problem that limits antimicrobial therapy options. We isolated two pairs of colistin susceptible and colistin-resistant A. baumannii (K1007/K1006 and K408/K409) from two patients diagnosed with carbapenem-resistant A. baumannii infection. Colistin susceptible isolates were exposed to in vitro colistin induction for 50 generations. The selected cell populations were subjected to DNA and RNA sequencing and phenotypic assays. In the in vitro induction assay, K408 gained colistin resistance on the corresponding day of clinical resistance (K408-G25) and got resensitized to colistin in the consecutive generation (K408-G26). A significant upregulation of ompW, ata, adeFGH genes on K408-G25 was followed by a downregulation upon resensitization to colistin (G26). Despite the upregulation of the ompW gene in transcriptomic analysis, the ompW protein disappeared on K408-G25 and recovered in the resensitized generation (G26). In parallel, disrupted cell membrane integrity recovered in K408-G26. In the K408-G25, downregulation of pbpG and upregulation of pbp1a/pbp3 genes decreased serum-resistance which was reversed in the resensitized generation (G26). The K1007 did not gain colistin resistance amongst 50-generations, however, the generation corresponding to clinical resistance day (K1007-G9) had a similar trend with K408-G25. The clinical colistin-resistant K409 and K1006 had SNPs on pmrA and pmrB genes. In this study, we observed that A. baumannii regulates adhesion, efflux pumps and serum-resistance associated genes as an early response to colistin stress. Besides, the ompW protein disappears in the cell membrane of colistin resistant cells which recovers after resensitization to colistin. The lack of ompW protein in colistin-resistant cells should be taken into consideration for escape mutants in development of antivirulence vaccination or treatment options.

Characterization of a colistin resistant, hypervirulent hospital isolate of Acinetobacter courvalinii from Canada.

Non-baumannii Acinetobacter spp. are becoming more prevalent in clinical settings including those that present resistance to last-resort antibiotics such as colistin. AB222-IK40 is an Acinetobacter courvalinii strain isolated from the Ottawa Hospital Research Institute located in Ottawa, Canada. To our knowledge, it is the first report of clinical A. courvalinii in Canada. Based on the susceptibility profile, AB222-IK40 is resistant to colistin and non-susceptible to ertapenem. Whole-genome sequencing allowed for genomic investigation into colistin resistance mechanisms. No previously identified mechanism(s) were observed, but a mobile colistin resistance (mcr)-like gene and a UDP-glucose dehydrogenase gene were identified. Based on phylogenomic analyses, the mcr-like gene is an intrinsic phosphoethanolamine transferase. This gene family is implicated in one of the many mechanisms responsible for colistin resistance in Acinetobacter baumannii as well as Acinetobacter modestus. UDP-glucose dehydrogenase is involved in colistin resistance in Enterobacterales and has been shown to be involved in capsule formation in A. baumannii. Global lipidomics revealed greater abundance of phosphatidyl-myo-inositol and lyso-phosphatidyl ethanolamine moieties in the membrane of A. courvalinii than in A. baumannii. Lipidomic profiles showed differences that were probably responsible for the colistin resistance phenotype in AB222-IK40. This isolate was also hypervirulent based on survival assays in Galleria mellonella. As this is the first report of A. courvalinii from a hospital in Canada, this species may be an emerging clinical pathogen, and therefore, it is important to understand this mechanism of its colistin resistance and hypervirulence.

Molecular Characteristics of Colistin Resistance in Acinetobacter baumannii and the Activity of Antimicrobial Combination Therapy in a Tertiary Care Medical Center in Lebanon.

(1) Background: Infections with pan-drug-resistant (PDR) bacteria, such as A. baumannii, are becoming increasingly common, especially in healthcare facilities. In this study, we selected 15 colistin-resistant clinical A. baumannii isolates from a hospital in Beirut, Lebanon, to test combination therapies and determine their sequence types (STs) and the mechanism of colistin resistance using whole-genome sequencing (WGS). (2) Methods: Antimicrobial susceptibility testing via broth microdilution against 12 antimicrobials from different classes and growth rate assays were performed. A checkerboard assay was conducted on PDR isolates using six different antimicrobials, each in combination with colistin. Genomic DNA was extracted from all isolates and subjected to WGS. (3) Results: All isolates were resistant to all tested antimicrobials with the one exception that was susceptible to gentamicin. Combining colistin with either meropenem, ceftolozane-tazobactam, or teicoplanin showed synergistic activity. Sequencing data revealed that 67% of the isolates belonged to Pasteur ST2 and 33% to ST187. Furthermore, these isolates harbored a number of resistance genes, including blaOXA-23. Mutations in the pmrC gene were behind colistin resistance. (4) Conclusions: With the rise in antimicrobial resistance and the absence of novel antimicrobial production, alternative treatments must be found. The combination therapy results from this study suggest treatment options for PDR ST2 A. baumannii-infected patients.

Phage-mediated colistin resistance in Acinetobacter baumannii

AimsAntimicrobial resistance is a global threat to human health, and Acinetobacter baumannii is a paradigmatic example of how rapidly bacteria become resistant to clinically relevant antimicrobials. The emergence of multidrug-resistant A. baumannii strains has forced the revival of colistin as a last-resort drug, suddenly leading to the emergence of colistin resistance. We investigated the genetic and molecular basis of colistin resistance in A. baumannii, and the mechanisms implicated in its regulation and dissemination. MethodsComparative genomic analysis was combined with genetic, biochemical, and phenotypic assays to characterize Φ19606, an A. baumannii temperate bacteriophage that carries a colistin resistance gene. ResultsФ19606 was detected in 41% of 523 A. baumannii complete genomes and demonstrated to act as a mobile vehicle of the colistin resistance gene eptA1, encoding a functional lipid A phosphoethanolamine transferase. The eptA1 gene is coregulated with its chromosomal homolog pmrC via the PmrAB two-component system and confers colistin resistance when induced by low calcium and magnesium levels. Resistance selection assays showed that the eptA1-harbouring phage Ф19606 promotes the emergence of spontaneous colistin-resistant mutants. ConclusionsΦ19606 is an unprecedented example of a self-transmissible phage vector implicated in the dissemination of colistin resistance.

PmrAB controls virulence-associated traits and outer membrane vesicle biogenesis in Acinetobacter baumannii

The PmrAB two-component system modulates colistin resistance in Acinetobacter baumannii, but its association with the virulence traits of this bacterium remains uncharacterized. This study explored the role of A. baumannii PmrAB in surface motility, biofilm formation, and outer membrane vesicle (OMV) biogenesis using wild-type (WT) A. baumannii 17978 and ΔpmrA and ΔpmrB mutant strains. The two mutant strains exhibited significantly decreased surface motility compared with that of WT strain by the low expression of abaI, abaR, A1S_0113, A1S_0115, and A1S_0116. Biofilm mass also significantly decreased in the two mutant strains at 12 h of incubation, but restored at 24 h. Under static culture conditions for 12 h, the two mutant strains showed low pgaA expression. However, the other biofilm-associated genes, such as csuC, csuE, ompA, and bap, showed different expression between the two mutant strains. Although the size of OMVs was similar among the three strains, the number of OMVs secreted from the two mutant strains slightly decreased compared with that secreted from the WT strain. Protein concentrations in the OMVs of ΔpmrA mutant significantly decreased compared with those in the OMVs of WT and ΔpmrB strains. Overall, PmrAB modulates virulence traits and OMV biogenesis in A. baumannii.

Adaptation of Acinetobacter baumannii to Colistin Exposure: Laboratory Mimicking of a Clinical Case

The increase of colistin resistance in Acinetobacter baumannii is a great concern in various regions of the world such as Asia, Europe, and North and South America. Exposure to colistin is considered the most significant factor for emerging of colistin resistance; however, details of the in vivo response

Mutations at Novel Sites in pmrA/B and lpxA/D Genes and Absence of Reduced Fitness in Colistin-Resistant Acinetobacter baumannii from a Tertiary Care Hospital, India.

Background: Colistin resistance in Acinetobacter baumannii, the last resort drug for serious infections, is emerging worldwide. There has been paucity of data on this aspect from India, which is one of the largest producers of colistin. We studied colistin resistance in A. baumannii and characterized the isolates with respect to resistance mechanisms and virulence. Methods: A total of 365 A. baumannii isolates were studied. Antimicrobial susceptibility testing was performed as per standards. Colistin resistance mechanisms were studied by mutation detection in pmrA/B and lpxA/C/D genes, phenotypic loss of lipopolysaccharide, presence of mcr1-5 genes, and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) effects. Biofilm formation, desiccation survival, and growth kinetics were studied and statistically analyzed for colistin-resistant and colistin-susceptible isolates. Results: All the colistin-resistant isolates (9, 2.5%) showed multiple mutations at novel sites in pmrA/B and/or lpxA/D genes with reversion of resistance with CCCP. Majority of these isolates (6, 66.6%) were from patients without prior colistin therapy. All received prior carbapenems. The resistant isolates demonstrated no significant difference in biofilm formation and desiccation survival but were slow growers. Conclusion: Mutations in pmrA/B and/or lpxA/D genes were the main resistance mechanism in these colistin-resistant isolates that showed no reduction in fitness.

New Mutations Involved in Colistin Resistance in Acinetobacter baumannii.

Colistin is used as the "last resort" to treat infections caused by multidrug-resistant Acinetobacter baumannii, which is at the top of the World Health Organization's list of the most dangerous bacterial species that threaten human health. Unfortunately, colistin resistance has emerged in A. baumannii To broaden the study of the resistance mechanism of colistin in A. baumannii, we obtained colistin-resistant mutants via two methods: (i) screening and isolation from a mariner-based A. baumannii ATCC 19606 transposon mutant library; (ii) selection from challenge of ATCC 19606 with successively increasing concentrations of colistin. A total of 41 mutants with colistin MIC of 4 μg/ml to 64 μg/ml were obtained by transposon mutant library screening. Five highly resistant mutants with colistin MICs ranging from 256 μg/ml to 512 μg/ml were selected from successive colistin challenges. Genotypic complementation and remodeling of the transposon mutants revealed that the genes inactivated by the transposon insertion were not responsible for resistance. Whole-genome sequence analysis of the colistin-resistant strains revealed that the main causes of the resistance to colistin were mutations in the pmrA-pmrB genes, including pmrAP102R, pmrBP233S, and pmrBT235N and the novel alleles pmrAI13M and pmrBQ270P Interestingly, we found that miaAI221V mutation of A. baumannii strain ATCC 19606 (pmrAP102R) resulted in 4-fold increases in the colistin MIC, which rose from 32 μg/ml to 128 μg/ml. But miaAI221V itself had little effect on the colistin susceptibility of ATCC 19606. These data broaden knowledge of the scope of chromosomally encoded mechanisms of resistance to colistin.IMPORTANCEAcinetobacter baumannii is an important Gram-negative opportunistic pathogen commonly infecting critically ill patients. It possesses a remarkable ability to survive in the hospital environment and acquires resistance determinants corresponding to a wide range of antibacterial agents. Given that the current treatment options for multidrug resistant A. baumannii are extremely limited, colistin administration has become the treatment of last resort. However, colistin-resistant A. baumannii strains have recently been reported. The mechanism of resistance to colistin in A. baumannii has rarely been reported. Here, we found two novel mutations in pmrA (I13M) and pmrB (Q270P) that caused colistin resistance. It is also first reported here that the presence of miaA with a I221V mutation enhanced the colistin resistance of pmrAP102R.

Screen of Unfocused Libraries Identified Compounds with Direct or Synergistic Antibacterial Activity.

Antibiotic resistance is an increasingly important global public health issue, as major opportunistic pathogens are evolving toward multidrug- and pan-drug resistance phenotypes. New antibiotics are thus needed to maintain our ability to treat bacterial infections. According to the WHO, carbapenem-resistant Acinetobacter, Enterobactericaeae, and Pseudomonas are the most critical targets for the development of new antibacterial drugs. An automated phenotypic screen was implemented to screen 634 synthetic compounds obtained in-house for both their direct-acting and synergistic activity. Fourteen percent and 10% of the compounds showed growth inhibition against tested Gram-positive and Gram-negative bacteria, respectively. The most active direct-acting compounds showed a broad-spectrum antibacterial activity, including on some multidrug-resistant clinical isolates. In addition, 47 compounds were identified for their ability to potentiate the activity of other antibiotics. Compounds of three different scaffolds (2-quinolones, phenols, and pyrazoles) showed a strong potentiation of colistin, some being able to revert colistin resistance in Acinetobacter baumannii.

Colistin resistance in Acinetobacter baumannii isolated from critically ill patients: clinical characteristics, antimicrobial susceptibility and outcome.

Acinetobacter baumannii (AB) is increasingly becoming a clinically relevant organism due to the rising number of associated nosocomial infections. The therapeutic options are extremely minimal because of its ability to develop resistance to all available antimicrobials, including colistin (CST). Data on the clinical and microbiological characteristics of colistin-resistant A. baumannii infections remain scarce to date. In this prospective study, clinical isolates of colistin resistance among Acinetobacter strain was evaluated from the database of Microbiology Laboratory of King Khalid University Hospital, Saudi Arabia. In a total of 142 patients with 136 Acinetobacter isolates, Acintobacter baumannii was the predominant serotype 73% of the isolates and Acinetobacter lwoffii constituted 27% of the isolate . There was 8.5% colistin resistant isolates with colistin E-test MIC >4. The clinical characteristics were determined for colistin resistant Acinetobacter baumannii. All patients were critically ill and 64% of them were hositalized in the Intensive Care Unit (ICU). All patients have been previously given antibiotics. Other associated clinical characteristics included; morbid obesity and sleeve gastrectomy (21 %), mechanical ventilation and central venous catheter (50%). High mortality rate was found(28%). There is an increase of colistin resistance among clinical isolates of Acinetobacter baumannii causing serious infections especially in critically ill patients.

Promoters of Colistin Resistance in Acinetobacter baumannii Infections.

Objectives: We aimed to describe the mechanisms of colistin resistance in Acinetobacter baumannii. Materials and Methods: Twenty-nine patients diagnosed with colistin-resistant A. baumannii infection were included to the study. The mutations in pmrCAB, lpxA, lpxC, and lpxD genes, expression of pmrCAB, carbapenemases, and mcr-1 positivity were studied. Results: Twenty-seven (93%) of the patients received IV colistin therapy during their stay, and the case fatality rate was 45%. All mutations in pmrC and pmrB were found to be accompanied with a mutation in lpxD. The most common mutations were I42V and L150F in pmrC (65%), E117K in lpxD (65%), and A138T in pmrB (58.6%). The colistin minimum inhibitory concentrations (MICs) of the isolates having any of these four mutations were higher than the isolates with no mutations (p < 0.001). The two most common mutations in pmrC (I42V and L150F) were found to be associated with higher expressions of pmrA and pmrC and higher colistin MIC values (p = 0.010 and 0.031). All isolates were blaOXA-23 positive. Conclusion: Coexistence of the lpxD mutation along with mutations in pmrCAB indicates synergistic function of these genes in development of colistin resistance in A. baumannii.

Rapid detection of colistin resistance in Acinetobacter baumannii using MALDI-TOF-based lipidomics on intact bacteria

With the dissemination of extremely drug resistant bacteria, colistin is now considered as the last-resort therapy for the treatment of infection caused by Gram-negative bacilli (including carbapenemase producers). Unfortunately, the increase use of colistin has resulted in the emergence of resistance as well. In A. baumannii, colistin resistance is mostly caused by the addition of phosphoethanolamine to the lipid A through the action of a phosphoethanolamine transferase chromosomally-encoded by the pmrC gene, which is regulated by the two-component system PmrA/PmrB. In A. baumannii clinical isolate the main resistance mechanism to colistin involves mutations in pmrA, pmrB or pmrC genes leading to the overexpression of pmrC. Although, rapid detection of resistance is one of the key issues to improve the treatment of infected patient, detection of colistin resistance in A. baumannii still relies on MIC determination through microdilution, which is time-consuming (16–24 h). Here, we evaluated the performance of a recently described MALDI-TOF-based assay, the MALDIxin test, which allows the rapid detection of colistin resistance-related modifications to lipid A (i.e phosphoethanolamine addition). This test accurately detected all colistin-resistant A. baumannii isolates in less than 15 minutes, directly on intact bacteria with a very limited sample preparation prior MALDI-TOF analysis.

Association of virulence gene expression with colistin-resistance in Acinetobacter baumannii: analysis of genotype, antimicrobial susceptibility, and biofilm formation

BackgroundAcinetobacter baumannii causes difficult-to-treat nosocomial infections, which often lead to morbidity due to the development of antimicrobial drug resistance and expression of virulence genes. Data regarding the association of resistance to colistin, a last treatment option, and the virulence gene expression of A. baumannii is scarce.MethodsWe evaluated the MLVA genotype, antimicrobial resistance, and biofilm formation of 100 A. baumannii isolates from burn patients, and further compared the in vitro and in vivo expression of four virulence genes among five colistin-resistant A. baumannii (Cst-R-AB) isolates. Five Cst-R-AB isolates were tested; one from the present study, and four isolated previously.ResultsOur results showed that reduced expression of recA, along with increased in vivo expression of lpsB, dnaK, and blsA; are associated with colistin resistance among Cst-R-AB isolates. Differences in virulence gene expressions among Cst-R-AB isolates, may in part explain common discrepant in vitro vs. in vivo susceptibility data during treatment of infections caused by Cst-R-AB.ConclusionsOur findings highlight the intricate relationship between colistin-resistance and virulence among A. baumannii isolates, and underscore the importance of examining the interactions between virulence and antimicrobial resistance toward efforts to control the spread of multidrug-resistant A. baumannii (MDR-AB) isolates, and also to reduce disease severity in burn patients with MDR-AB infection.

Transition of colistin dependence into colistin resistance in Acinetobacter baumannii

We recently demonstrated a high rate of colistin dependence in Acinetobacter baumannii isolates exposed to colistin in vitro. In the present study, we obtained a colistin-resistant (H08-391R) and colistin-dependent mutant (H08-391D) from a colistin-susceptible parental strain (H08-391). We found that the colistin-dependent mutant converted into a stable colistin-resistant mutant (H08-391D-R) in vitro after four serial passages without colistin. H08-391D and H08-391D-R were both found to harbor defective lipid A, as indicated by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis. Additionally, both contained an ISAba1 insertion in lpxC, which encodes a lipid A biosynthetic enzyme. Further, membrane potential measurements using the fluorescent dye 3,3′-diethyloxacarbocyanine iodide (DiOC2[3]) showed that the membrane potential of H08-391D and H08-391D-R was significantly decreased as compared to that of the parental strain, H08-391. Moreover, these mutant strains exhibited increased susceptibilities to antibiotics other than colistin, which may be attributed to their outer membrane fragility. Such phenomena were identified in other A. baumannii strains (H06-855 and its derivatives). Taken together, our study reveals that the colistin-dependent phenotype is a transient phenotype that allows A. baumannii to survive under colistin pressure, and can transition to the extremely resistant phenotype, even in an antibiotic-free environment.

Small molecule adjuvants that suppress both chromosomal and mcr-1 encoded colistin-resistance and amplify colistin efficacy in polymyxin-susceptible bacteria

Bacterial resistance to polymyxin antibiotics has taken on a new and more menacing form. Common are genomically-encoded resistance mechanisms to polymyxins, specifically colistin (polymyxin E), however, the plasmid-borne mobile colistin resistance-1 (mcr-1) gene has recently been identified and poses a new threat to global public health. Within six months of initial identification in Chinese swine in November 2015, the first human clinical isolation in the US was reported (Apr. 2016). Herein we report successful reversion of mcr-1-driven colistin resistance in Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli with adjuvants we previously reported as modulators of chromosomally-encoded colistin resistance. Further screening of our in-house library of nitrogen-dense heterocycles has identified additional chemical scaffolds that actively attenuate colistin resistance. Ultimately, we present a diverse cohort of adjuvants that both sensitize colistin-resistant and colistin-susceptible bacteria to this antibiotic, thus providing a potential avenue to both reduce colistin dosage and toxicity, and overcome colistin resistance.

Synergy between Colistin and the Signal Peptidase Inhibitor MD3 Is Dependent on the Mechanism of Colistin Resistance in Acinetobacter baumannii.

Synergy between colistin and the signal peptidase inhibitor MD3 was tested against isogenic mutants and clinical pairs of Acinetobacter baumannii isolates. Checkerboard assays and growth curves showed synergy against both colistin-susceptible strains (fractional inhibitory concentration index [FICindex] = 0.13 to 0.24) and colistin-resistant strains with mutations in pmrB and phosphoethanolamine modification of lipid A (FICindex = 0.14 to 0.25) but not against colistin-resistant Δlpx strains with loss of lipopolysaccharide (FICindex = 0.75 to 1). A colistin/MD3 combination would need to be targeted to strains with specific colistin resistance mechanisms.

Impaired growth under iron-limiting conditions associated with the acquisition of colistin resistance in Acinetobacter baumannii

Acquisition of colistin resistance in Acinetobacter baumannii has been associated with reduced bacterial fitness and virulence, although the mechanisms underlying this fitness loss have not been well characterised. In this study, the role played by environmental iron levels on the growth and survival of colistin-resistant strains of A. baumannii was assessed. Growth assays with the colistin-susceptible ATCC 19606 strain and its colistin-resistant derivative RC64 [colistin minimum inhibitory concentration (MIC) of 64 mg/L] demonstrated that the strains grew similarly in rich laboratory medium (Mueller–Hinton broth), whereas RC64 demonstrated impaired growth compared with ATCC 19606 in human serum (>100-fold at 24 h). Compared with RC64, ATCC 19606 grew in the presence of higher concentrations of the iron-specific chelator 2,2′-bipyridine and grew more readily under iron-limiting conditions in solid and liquid media. In addition, iron supplementation of human serum increased the growth of RC64 compared with unsupplemented human serum to a greater extent than ATCC 19606. The ability of 11 colistin-resistant clinical isolates with mutations in the pmrB gene to grow in iron-replete and iron-limiting conditions was assessed, demonstrating that eight of the strains showed reduced growth under iron limitation. Individual mutations in the pmrB gene did not directly correlate with a decreased capacity for growth under iron limitation, suggesting that mutations in pmrB may not directly produce this phenotype. Together these results indicate that acquisition of colistin resistance in A. baumannii can be associated with a decreased ability to grow in low-iron environments.

Correlation between overexpression and amino acid substitution of the PmrAB locus and colistin resistance in Acinetobacter baumannii

Relationships between the PmrAB two-component system and colistin resistance were investigated in Acinetobacter baumannii. The sequences of pmrA, pmrB and pmrC in 26 colistin-susceptible (ColS) and 7 colistin-resistant (ColR) A. baumannii isolates were determined. In addition, 30 ColR mutants (colistin minimum inhibitory concentration >64 mg/L) were selected in vitro from 10 ColS strains and the pmrA, pmrB and pmrC sequences of the in-vitro-selected ColR mutants were also determined. Expression of pmrA and pmrB was compared between the ColR mutants and their parent ColS strains using a quantitative real-time polymerase chain reaction method. Elevated expression of pmrA and pmrB genes was evident both in wild-type and in in-vitro-selected ColR strains. However, no amino acid differences in the pmrA, pmrB and pmrC genes were found between wild-type ColR and ColS isolates. Although six kinds of amino acid alterations in pmrB were identified in in-vitro-selected ColR mutants, no changes were found in some of the mutants. These findings indicate that increased expression of the PmrAB system is essential for colistin resistance in A. baumannii but that amino acid alterations might be only partially responsible for resistance.

The cost of resistance to colistin in Acinetobacter baumannii: a proteomic perspective

Colistin resistance in Acinetobacter baumannii, a pathogen of clinical concern, was induced in the susceptible strain ATCC 19606 by growth under increasing pressure of the antibiotic, the only drug universally active against multi-resistant clinical strains. In 2-D difference gel electrophoresis (DIGE) experiments, 35 proteins with differences in expression between both phenotypes were identified, most of them appearing as down regulated in the colistin-resistant strain. These include outer membrane (OM) proteins, chaperones, protein biosynthesis factors, and metabolic enzymes, all suggesting substantial loss of biological fitness in the resistant phenotype, as substantiated by complementary experiments in the absence of colistin. Results shed light on the scarcity of widespread clinical outbreaks for resistant phenotypes.