Growth Retardation,Reduced Invasiveness,and Impaired Colistin-Mediated Cell Death Associated with Colistin Resistance Development in Acinetobacter baumannii

Two colistin-susceptible/colistin-resistant (Col^s/Col^r) pairs of Acinetobacter baumannii strains assigned to international clone 2, which is prevalent worldwide, were sequentially recovered from two patients after prolonged colistin administration. Compared with the respective Col^s isolates (Ab248 and Ab299, both having a colistin MIC of 0.5 μg/ml), both Col^r isolates (Ab249 and Ab347, with colistin MICs of 128 and 32 μg/ml, respectively) significantly overexpressed pmrCAB genes, had single-amino-acid shifts in the PmrB protein, and exhibited significantly slower growth. The Col^r isolate Ab347, tested by proteomic analysis in comparison with its Col^s counterpart Ab299, underexpressed the proteins CsuA/B and C from the csu operon (which is necessary for biofilm formation). This isolate also underexpressed aconitase B and different enzymes involved in the oxidative stress response (KatE catalase, superoxide dismutase, and alkyl hydroperoxide reductase), suggesting a reduced response to reactive oxygen species (ROS) and, consequently, impaired colistin-mediated cell death through hydroxyl radical production. Col^s isolates that were indistinguishable by macrorestriction analysis from Ab299 caused six sequential bloodstream infections, and isolates indistinguishable from Ab248 caused severe soft tissue infection, while Col^r isolates indistinguishable from Ab347 and Ab249 were mainly colonizers. In particular, a Col^s isolate identical to Ab299 was still invading the bloodstream 90 days after the colonization of this patient by Col^r isolates. These observations indicate considerably lower invasiveness of A. baumannii clinical isolates following the development of colistin resistance.     

Resistance to Colistin Associated with a Single Amino Acid Change in Protein PmrB among Klebsiella pneumoniae Isolates of Worldwide Origin

A series of colistin-resistant Klebsiella pneumoniae isolates recovered from different countries was investigated in order to evaluate the involvement of the PmrA/PmrB two-component system in this resistance. Six isolates possessed a mutated PmrB protein, which is encoded by the pmrB gene, part of the pmrCAB operon involved in lipopolysaccharide modification. The same amino acid substitution (Thr157Pro) in PmrB was identified in the six isolates. The six isolates belonged to four distinct clonal groups, recovered in South Africa (sequence type 14 [ST14]), Turkey (ST101), and Colombia (ST258 and ST15). Three out of the four clones produced a carbapenemase, OXA-181, OXA-48, or KPC-3, while a single isolate did not produce any carbapenemase. Expression assays revealed an overexpression of the pmrA (70-fold), pmrB (70-fold), pmrC (170-fold), and pmrK (40-fold) genes in the pmrB-mutated isolate compared to expression of the pmrB wild-type isogenic K. pneumoniae isolate, confirming that the PmrB substitution was responsible for increased expression levels of those genes. Complementation assays leading to the expression of a wild-type PmrB protein restored the susceptibility to colistin in all isolates, confirming that the substitution in PmrB was responsible for the resistance phenotype. This study identified a key amino acid located in the PmrB protein as being responsible for the overexpression of pmrCAB and pmrHFIJKLM operons, leading to resistance to colistin.     

Elucidation of molecular mechanism for colistin resistance among Gram-negative isolates from tertiary care hospitals

Antimicrobial resistance is a growing concern of global public health. The emergence of colistin-resistance among carbapenem-resistant (CPR) Gram-negative bacteria causing fear of pan-resistance, treatment failure, and high mortality across the globe.AimTo determine the genotypic colistin-resistance mechanisms among colistin-resistant (CR)Gram-negative clinical isolates along with genomic insight into hypermucoviscous(hv)-CR-Klebsiella pneumoniae.MethodsPhenotypic colistin-resistance via broth-microdilution method. PCR-based detection of plasmid-mediated colistin resistance genes(mcr-1,2,3). Characterization of selected hvCR-K. pneumoniae via Whole-genome sequencing.ResultsPhenotypic colistin-resistance was 28% among CPR-Gram-negative isolates of which 90% of CR-isolates displayed MDR profile with overall low plasmid-mediated colistin resistance (mcr-2 = 9.4%;mcr-3 = 6%). Although K. pneumoniae isolates showed the highest phenotypic colistin-resistance (51%) however, relatively low plasmid-mediated gene-carriage (mcr-2 = 11.5%;mcr-3 = 3.4%) pointed toward other mechanisms of colistin-resistance. mcr-negative CR-K. pneumoniae displaying hv-phenotype were subjected to WGS. In-silico analysis detected 7-novel mutations in lipid-A modification genes includes eptA(I38V; V50L; A135P), opgE(M53L; T486A; G236S), and arnD(S164P) in addition to several non-synonymous mutations in lipid-A modification genes conferring resistance to colistin. Insertion of 6.6-kb region harboring putative-PEA-encoding gene(yjgX) was detected for the first time in K. pneumoniae (hvCRKP4771). In-silico analysis further confirmed the acquisition of not only MDR determinants but several hypervirulent-determinants displaying a convergent phenotype.Conclusionoverall high prevalence of phenotypic colistin resistance but low mcr-gene carriage suggested complex chromosomal mediated resistance mechanism especially in K. pneumoniae isolates. The presence of novel mutations in lipid-A modification genes and the acquisition of putative-PEA-encoding gene by hvCR-K. pneumoniae points toward the role of chromosomal determinants conferring resistance to colistin in the absence of mcr-genes.     

Synergistic Activity of Colistin plus Rifampin against Colistin-Resistant KPC-Producing Klebsiella pneumoniae

Infections caused by carbapenem-resistant KPC-producing Klebsiella pneumoniae are responsible for high rates of mortality and represent a major therapeutic challenge, especially when the isolates are also resistant to colistin. We used the checkerboard method to evaluate the synergistic activity of 10 antibiotic combinations against 13 colistin-resistant KPC-producing K. pneumoniae isolates (colistin MIC range of 8 to 128 mg/liter). Colistin plus rifampin was the only combination that demonstrated consistent synergistic bacteriostatic activity against 13/13 strains tested, reducing the colistin MIC below the susceptibility breakpoint (MIC ≤ 2 mg/liter) in 7/13 strains at rifampin concentrations ranging from 4 to 16 mg/liter. Bactericidal synergistic activity was also documented for 8/13 tested strains. Other antimicrobial combinations with carbapenems, gentamicin, and tigecycline showed variously synergistic results. Colistin plus rifampin also exhibited bacteriostatic synergistic activity against 4/4 colistin-susceptible KPC-producing K. pneumoniae isolates (colistin MIC range of 0.5 to 2 mg/liter) and 4/4 ertapenem-resistant extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae isolates (ertapenem MIC range of 16 to 32 mg/liter). Collectively, our data suggest that colistin plus rifampin is the most consistently synergistic combination against KPC-producing K. pneumoniae isolates, including colistin-resistant strains. Colistin-rifampin combinations may have a role in the treatment of multidrug-resistant K. pneumoniae and may possibly slow the selection of heteroresistant subpopulations during colistin therapy.     

Mutations and expression of PmrAB and PhoPQ related with colistin resistance in Pseudomonas aeruginosa clinical isolates

To comprehend the resistance of colistin resistance, we investigated the relationships between amino acid alterations and expression of PmrAB and PhoPQ and colistin resistance in 16 colistin-nonsusceptible clinical Pseudomonas aeruginosa isolates. In addition, we obtained induced colistin-resistant mutants and their colistin-susceptible revertants. Expression levels of the pmrA, phoP, parR, cprR, and pmrH genes were determined for them. Nine amino acid substitutions unique to 10 colistin-nonsusceptible P. aeruginosa (CNPA) isolates were identified: 7 in PmrB and 1 each in PmrA and PhoQ. However, 6 CNPA isolates did not show amino acid substitutions compared with colistin-susceptible P. aeruginosa isolates. Among 16 CNPA isolates, 7 and 8 isolates displayed activated expression of pmrA and phoP, respectively. Activated expression of pmrA and/or phoP was identified in 13 isolates of CNPA isolates, but some had no noticeable PmrAB and PhoPQ amino acid substitutions. In addition, in vitro selected colistin-resistant mutants (P5R and P155R) showed higher expression level in pmrA, phoP, and pmrH than their parent strains (P5 and P155) and colistin-susceptible, revertant strains (P5R-rev and P155R-rev). However, expression of the parR and cprR genes was not consistent. Our data may indicate that amino acid substitutions of PmrAB or PhoPQ do not have an immediate connection with decreased susceptibility of colistin in P. aeruginosa isolates, although activated expression of pmrAB and/or phoPQ resulting in overexpression of pmrH may be required for colistin resistance. Expression of pmrAB or phoPQ related with colistin nonsusceptibility may not explained by a single mechanism, which may suggest that colistin resistance appears easily by diverse pathways in clinical settings as well as in laboratory.     

Emergence and dissemination of colistin-resistant Klebsiella pneumoniae isolates expressing OXA-48 plus CTX-M-15 in patients not previously treated with colistin in a Spanish university hospital

Dissemination of multidrug-resistant Klebsiella pneumoniae in the hospital environment represents a primary target of resistance containment and stewardship programs. At present, polymyxins, mostly in combination, exemplify a last-resort alternative. Colistin-resistant K. pneumoniae isolates harboring OXA-48 plus CTX-M-15 (n?=?21) with the simultaneous colistin-susceptible counterparts (n?=?9) were recovered from 14 hospitalized patients (January 2014–January 2015) admitted in different wards. In most cases, patients had not previously received colistin. Genetic relatedness experiments demonstrated that 93% (28/30) of isolates belonged to the ST11 high-risk clone. Heteroresistance and the fitness cost of colistin resistance were addressed in susceptible and resistant isolates as well as in in vitro–obtained stable mutants, and results appeared to be strain dependent. Whole genome sequencing demonstrated molecular changes in pmrA, pmrB, and mgrB genes. Plasmid-mediated colistin resistance genes were not found. Colistin resistance in multidrug-resistant K. pneumoniae isolates should be continuously monitored to detect its potential emergence, even in patients not previously exposed to colistin.     

Loss of Hypermucoviscosity and Increased Fitness Cost in Colistin-Resistant Klebsiella pneumoniae Sequence Type 23 Strains

In this study, we investigated the effects of colistin resistance on virulence and fitness in hypermucoviscous (HV) Klebsiella pneumoniae sequence type 23 (ST23) strains. Colistin-resistant mutants were developed from three colistin-susceptible HV K. pneumoniae ST23 strains. The lipid A structures of strains were analyzed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. Changes in HV were investigated using the string test, and extracellular polysaccharide production was quantified. The expression levels of the phoQ, pmrD, pmrB, pbgP, magA, and p-rmpA2 genes, serum resistance, and biofilm-forming activity were determined. The fitness of colistin-resistant mutants compared to that of the parental strains was examined by determining the competitive index (CI). The colistin-resistant mutants exhibited reduced HV, which was accompanied by decreased formation of capsular polysaccharides (CPS) and reduced expression of genes (magA and p-rmpA2). While there was enhanced expression of pmrD and pbgP in all colistin-resistant derivatives, there were differences in the expression levels of phoQ and pmrB between strains. MALDI-TOF analysis detected the addition of aminoarabinose or palmitate to the lipid A moiety of lipopolysaccharide in the colistin-resistant derivatives. In addition, survival rates in the presence of normal human serum were decreased in the mutant strains, and CI values (0.01 to 0.19) indicated significant fitness defects in the colistin-resistant derivatives compared to the respective parental strains. In hypervirulent HV K. pneumoniae strains, the acquisition of colistin resistance was accompanied by reduced CPS production, impaired virulence, and a significant fitness cost.     

Preservation of Acquired Colistin Resistance in Gram-Negative Bacteria

Colistin-resistant mutants were obtained from 17 colistin-susceptible strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. The stability of colistin resistance in these mutants was investigated. Three of four colistin-resistant P. aeruginosa mutants recovered colistin susceptibility in colistin-free medium; however, colistin-susceptible revertants were obtained from only one strain each of A. baumannii and E. coli. No susceptible revertants were obtained from K. pneumoniae mutants.     

Heteroresistance to Colistin in Klebsiella pneumoniae Associated with Alterations in the PhoPQ Regulatory System

A multidrug-resistant Klebsiella pneumoniae isolate exhibiting heteroresistance to colistin was investigated. The colistin-resistant subpopulation harbored a single amino acid change (Asp191Tyr) in protein PhoP, which is part of the PhoPQ two-component system that activates pmrHFIJKLM expression responsible for l-aminoarabinose synthesis and polymyxin resistance. Complementation assays with a wild-type phoP gene restored full susceptibility to colistin. Then, analysis of the colistin-susceptible subpopulation showed a partial deletion (25 bp) in the phoP gene compared to that in the colistin-resistant subpopulation. That deletion disrupted the reading frame of phoP, leading to a longer and inactive protein (255 versus 223 amino acids long). This is the first report showing the involvement of mutation(s) in PhoP in colistin resistance. Furthermore, this is the first study to decipher the mechanisms leading to colistin heteroresistance in K. pneumoniae.     

Evaluation of Double- and Triple-Antibiotic Combinations for VIM- and NDM-Producing Klebsiella pneumoniae by In Vitro Time-Kill Experiments

Combination therapy is recommended for infections with carbapenemase-producing Klebsiella pneumoniae. However, limited data exist on which antibiotic combinations are the most effective. The aim of this study was to find effective antibiotic combinations against metallo-beta-lactamase-producing K. pneumoniae (MBL-KP). Two VIM- and two NDM-producing K. pneumoniae strains, all susceptible to colistin, were exposed to antibiotics at clinically relevant static concentrations during 24-h time-kill experiments. Double- and triple-antibiotic combinations of aztreonam, ciprofloxacin, colistin, daptomycin, fosfomycin, meropenem, rifampin, telavancin, tigecycline, and vancomycin were used. Synergy was defined as a ≥2 log₁₀ decrease in CFU/ml between the combination and its most active drug after 24 h, and bactericidal effect was defined as a ≥3 log₁₀ decrease in CFU/ml after 24 h compared with the starting inoculum. Synergistic or bactericidal activity was demonstrated for aztreonam, fosfomycin, meropenem, and rifampin in double-antibiotic combinations with colistin and also for aztreonam, fosfomycin, and rifampin in triple-antibiotic combinations with meropenem and colistin. Overall, the combination of rifampin-meropenem-colistin was the most effective regimen, demonstrating synergistic and bactericidal effects against all four strains. Meropenem-colistin, meropenem-fosfomycin, and tigecycline-colistin combinations were not bactericidal against the strains used. The findings of this and other studies indicate that there is great potential of antibiotic combinations against carbapenemase-producing K. pneumoniae. However, our results deviate to some extent from those of previous studies, which might be because most studies to date have included KPC-producing rather than MBL-producing strains. More studies addressing MBL-KP are needed.     

Performance of CHROMID® Colistin R agar,a new chromogenic medium for screening of colistin-resistant Enterobacterales

Recent emergence of transferable plasmid-borne colistin resistance (mcr genes) raised fear for pan-resistance. We evaluated the performance of a new chromogenic medium [CHROMID® Colistin R agar (COLR)] for the screening of colistin-resistant Enterobacterales. Specificity was evaluated using 89 rectal swabs and 89 stools prospectively collected. COLR sensitivity was evaluated by seeding 59 negative clinical samples artificially contaminated (10⁵?CFU/mL) with 59 colistin-resistant Enterobacterales, including 20 mcr-1–positive strains. Twelve samples with an Enterobacterales with nonintrinsic resistance to colistin were recovered during the specificity study, including one mcr-1–positive Escherichia coli, representing a 6.7% prevalence of colistin resistance in fecal carriage. Overall, specificity was 100.0% [95% CI: 97.8–100.0] and sensitivity yielded 88.1% [95% CI: 77.5–94.1]. False negatives corresponded to 3 Enterobacter spp. (MIC>64?mg/L), 2 Salmonella spp. (MIC?=?16?mg/L), 1 E. coli (MIC?=?4?mg/L), and 1?K. pneumoniae (MIC?=?8?mg/L). COLR appears to be a sensitive and specific chromogenic agar for screening colistin-resistant Enterobacterales, including those carrying mcr-1 gene.     

Resistance to Colistin in Acinetobacter baumannii Associated with Mutations in the PmrAB Two-Component System

The mechanism of colistin resistance (Col^r) in Acinetobacter baumannii was studied by selecting in vitro Col^r derivatives of the multidrug-resistant A. baumannii isolate AB0057 and the drug-susceptible strain ATCC 17978, using escalating concentrations of colistin in liquid culture. DNA sequencing identified mutations in genes encoding the two-component system proteins PmrA and/or PmrB in each strain and in a Col^r clinical isolate. A colistin-susceptible revertant of one Col^r mutant strain, obtained following serial passage in the absence of colistin selection, carried a partial deletion of pmrB. Growth of AB0057 and ATCC 17978 at pH 5.5 increased the colistin MIC and conferred protection from killing by colistin in a 1-hour survival assay. Growth in ferric chloride [Fe(III)] conferred a small protective effect. Expression of pmrA was increased in Col^r mutants, but not at a low pH, suggesting that additional regulatory factors remain to be discovered.Among gram-negative pathogens that are reported as “multidrug resistant” (MDR), Acinetobacter baumannii is rapidly becoming a focus of significant attention (1, 7, 25, 32, 38, 39, 46, 51). In intensive care units, up to 30% of A. baumannii clinical isolates are resistant to at least three classes of antibiotics, often including fluoroquinolones and carbapenems (25).The emergence of MDR gram-negative pathogens, including A. baumannii, has prompted increased reliance on the cationic peptide antibiotic colistin (12). Regrettably, increasing colistin use has led to the discovery of resistant strains (10, 11, 22, 26). For example, in a recent study, 12% of carbapenemase-producing Enterobacteriaceae were found to be colistin resistant (Col^r) (6). Although still uncommon, A. baumannii isolates resistant to all available antimicrobial agents have been reported (26, 45) and are of enormous concern, given their potential to spread in the critical care environment.Colistin and other polymyxins are cyclic cationic peptides produced by the soil bacterium Bacillus polymyxa that act by disrupting the negatively charged outer membranes of gram-negative bacteria (37, 50). The following three distinct mechanisms that give rise to colistin resistance are known: (i) specific modification of the lipid A component of the outer membrane lipopolysaccharide, resulting in a reduction of the net negative charge of the outer membrane; (ii) proteolytic cleavage of the drug; and (iii) activation of a broad-spectrum efflux pump (13, 14, 49). The mechanism of colistin resistance in Acinetobacter spp. is not yet known. Heteroresistance to colistin in A. baumannii has been described (17, 24), but it is uncertain whether the basis for this resistance is the presence of a genetically distinct population of cells or whether variation in the regulatory program among genetically identical cells may be sufficient for the expression of resistance.In Salmonella enterica, the two-component signaling systems PmrAB and PhoPQ are involved in sensing environmental pH, Fe³⁺, and Mg²⁺ levels, leading to altered expression of a set of genes involved in lipid A modification (14, 43, 53). A small adapter protein, PmrD, serves as an interface between the two-component systems by stabilizing the activated form of PmrA in S. enterica (19), but other mechanisms of coordinated regulation are described for other species (52). Mutations causing constitutive activation of PmrA and PmrB are associated with colistin resistance (31, 33). Interestingly, the phoPQ and pmrD genes do not appear to be present in Acinetobacter spp., based on computational analysis of the genome sequences (2).PmrA-regulated resistance to colistin in S. enterica and P. aeruginosa results from modification of lipid A with 4-deoxy-aminoarabinose (Ara4N) or phosphoethanolamine via activation of ugd, the pmrF (or pbgP) operon, and pmrC, which encode UDP-glucose dehydrogenase (the first step in Ara4N biosynthesis), Ara4N biosynthetic enzymes, and lipid A phosphoethanolamine transferase, respectively (8, 15, 21, 41, 48). The Ara4N biosynthesis and attachment genes are not present in A. baumannii or Neisseria meningitidis (36, 47). N. meningitidis is intrinsically resistant to polymyxins, demonstrating that Ara4N modification of lipid A is not required for resistance. Mutations in the pmrC ortholog lptA, encoding the lipid A phosphoethanolamine transferase, reduce colistin resistance in N. meningitidis, suggesting that this modification alone may be sufficient for conferring colistin resistance (49). Here we show that the PmrAB system is involved in regulating colistin resistance in A. baumannii by identification of mutations in resistant isolates that exhibit constitutive expression of pmrA.     

Electron microscopic structures, serum resistance, and plasmid restructuring of New Delhi metallo-β-lactamase-1 (NDM-1)-producing ST42 Klebsiella pneumoniae emerging in Japan

Enterobacteriaceae, carrying the New Delhi metallo-β-lactamase-1 (NDM-1) gene (bla _NDM-1), have emerged and posed a threat since 2006. In Japan, bla _NDM-1-carrying Escherichia coli was first described in 2010. In this study, we characterized NDM-1-positive Klebsiella pneumoniae strain 419 in Japan, which was isolated from the urine of a 90-year-old Japanese patient who had never been to the Indian subcontinent. K. pneumoniae 419 belonged to ST42. It possessed a surface capsule (with untypeable capsular PCR types) and was resistant to serum killing. K. pneumoniae 419 cells were occasionally flagellated or piliated and autoaggregated. K. pneumoniae 419 was resistant to β-lactams (including carbapenems), aminoglycosides, and fluoroquinolones, and was susceptible to imipenem (or biapenem), aztreonam, polymixin B, and colistin. It possessed at least eight plasmids; of those, a 74-kb plasmid (pKPJ1) of the replicon FIIA carried bla _NDM-1 and was conjugally transferred to E. coli strains, with a 71-kb transferable azithromycin-resistant (mphA ⁺) plasmid of the replicon F (pKPJ2), as a large (145-kb) plasmid (pKPJF100) through a transposition event. In addition to bla _NDM-1, pKPJ1 carried arr-2, pKPJ2 carried mphA, and pKPJF100 carried both. They were negative for the 16S rRNA methylase gene, e.g., which is frequently associated with bla _NDM-1. The data demonstrate that K. pneumoniae 419 possessed virulence- and fitness-associated surface structures, was resistant to serum killing, and possessed a unique (or rare) genetic background in terms of ST type and bla _NDM-1-carrying plasmid.