SUMMARY Polymyxins are well-established antibiotics that have recently regained significant interest as a consequence of the increasing incidence of infections due to multidrug-resistant Gram-negative bacteria. Colistin and polymyxin B are being seriously reconsidered as last-resort antibiotics in many areas where multidrug resistance is observed in clinical medicine. In parallel, the heavy use of polymyxins in veterinary medicine is currently being reconsidered due to increased reports of polymyxin-resistant bacteria. Susceptibility testing is challenging with polymyxins, and currently available techniques are presented here. Genotypic and phenotypic methods that provide relevant information for diagnostic laboratories are presented. This review also presents recent works in relation to recently identified mechanisms of polymyxin resistance, including chromosomally encoded resistance traits as well as the recently identified plasmid-encoded polymyxin resistance determinant MCR-1. Epidemiological features summarizing the current knowledge in that field are presented.

The inactivation or down-regulation of the mgrB gene was shown to be a source of colistin resistance in K. pneumoniae. Interestingly, identical genetic events were identified among clonally unrelated isolates.

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.

cA 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. Klebsiella pneumoniae is a Gram-negative bacterium often associated with nosocomial infections, including urinary tract infection, pneumonia, and bloodstream infection (1). While multidrug resistance is increasingly reported in that species due to acquisition of numerous resistance traits, including extended-spectrum ␤-lactamase and carbapenemase genes, colistin is increasingly used for treating infections due to multidrug-resistant isolates. Colistin and polymyxin B correspond to important therapeutic options for treating infections caused by multidrug-resistant K. pneumoniae, particularly in countries with a high prevalence of carbapenemase producers (2). Those drugs are bactericidal for Gram-negative bacteria, interacting with the lipid A moiety of lipopolysaccharide (LPS) and subsequently causing disorganization of the outer membrane (3).In K. pneumoniae, colistin resistance is being increasingly reported, and some cases of heteroresistant K. pneumoniae isolates have also been observed (4, 5). Colistin heteroresistance is defined as "the emergence of resistance to colistin by a subpopulation from an otherwise susceptible (MIC of Յ2 mg/liter) population" (6) that may be related to exposure to a suboptimal polymyxin concentration (7). A comparison of the in vitro susceptibility testing methods for colistin showed that the Etest and agar dilution methods are reliable for detecting resistant subpopulations in contrast to some automatic methods; nonetheless, the broth microdilution method remains the reference test for determination of colistin MICs (8).Several studies have shown that mutations in the PmrAB or PhoPQ regulatory systems may confer colistin resistance in K. pneumoniae (9-12). In addition, inactivation of the mgrB gene, which encodes the MgrB protein known to negatively regulate the PhoPQ signaling system, may also be the source of acquired resistance to colistin (13-16).Acquired resistance to polymyxins is mediated by the addition of 4-deoxyaminoarabinose (LAra4N) and/or phosphoethanolamine (pEtN) to lipid A. LAra4N synthesis requires the products of ...

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