Absence of mgrB Alleviates Negative Growth Effects of Colistin Resistance in Enterobacter cloacae.
ABSTRACT: Colistin is an important last-line antibiotic to treat highly resistant Enterobacter infections. Resistance to colistin has emerged among clinical isolates but has been associated with a significant growth defect. Here, we describe a clinical Enterobacter isolate with a deletion of mgrB, a regulator of colistin resistance, leading to high-level resistance in the absence of a growth defect. The identification of a path to resistance unrestrained by growth defects suggests colistin resistance could become more common in Enterobacter.
Project description:Multidrug-resistant strains belonging to the Enterobacter cloacae complex (ECC) group, and especially those belonging to clusters C-III, C-IV, and C-VIII, have increasingly emerged as a leading cause of health care-associated infections, with colistin used as one of the last lines of treatment. However, colistin-resistant ECC strains have emerged. The aim of this study was to prove that MgrB, the negative regulator of the PhoP/PhoQ two-component regulatory system, is involved in colistin resistance in ECC of cluster C-VIII, formerly referred to as Enterobacter hormaechei subsp. steigerwaltii An in vitro mutant (Eh22-Mut) was selected from a clinical isolate of Eh22. The sequencing analysis of its mgrB gene showed the presence of one nucleotide deletion leading to the formation of a truncated protein of six instead of 47 amino acids. The wild-type mgrB gene from Eh22 and that of a clinical strain of Klebsiella pneumoniae used as controls were cloned, and the corresponding recombinant plasmids were used for complementation assays. The results showed a fully restored susceptibility to colistin and confirmed for the first time that mgrB gene expression plays a key role in acquired resistance to colistin in ECC strains.
Project description:Colistin is one of the last-resort therapeutic agents to combat multidrug-resistant Gram-negative bacteria (GNB) including Klebsiella pneumoniae. Although it happens rarely, resistance to colistin has been reported for several GNB. A total of 20 colistin resistant (col-R) and three colistin susceptible (col-S) clinical isolates of K. pneumoniae were studied to explore the underlying mechanisms of colistin resistance. The presence of plasmid encoded resistance genes, mcr-1, mcr-2, mcr-3, and mcr-4 genes were examined by PCR. The nucleotide sequences of pmrA, pmrB, phoP, phoQ, and mgrB genes were determined. To evaluate the association between colistin resistance and upregulation of pmrHFIJKLM and pmrCAB operons, transcriptional level of the pmrK and pmrC genes encoding for lipopolysaccharide target modifying enzymes was quantified by RT-qPCR analysis. None of the plasmid encoded resistance genes were detected in the studied isolates. Inactivation of MgrB due to nonsense mutations and insertion of IS elements was observed in 15 col-R isolates (75%). IS elements (IS5-like and IS1-like families) most commonly targeted the coding region and in one case the promoter region of the mgrB. Complementation with wild-type MgrB restored colistin susceptibility in isolates with altered mgrB. All col-R isolates lacked any genetic alterations in the pmrA, phoP, and phoQ genes and substitutions identified in the pmrB were not found to be involved in resistance conferring determined by complementation assay. Colistin resistance linked with upregulation of pmrHFIJKLM and pmrCAB operons with the pmrK and pmrC being overexpressed in 20 and 11 col-R isolates, respectively. Our results demonstrated that MgrB alterations are the major mechanisms contributing to colistin resistance in the tested K. pneumoniae isolates from Iran.
Project description:Colistin is one of the few agents that retain activity against extensively drug-resistant strains of Klebsiella pneumoniae producing KPC-type carbapenemases (KPC-KP). However, resistance to colistin is increasingly reported among KPC-KP. Comparative genomic analysis of a pair of sequential KPC-KP isolates from the same patient including a colistin-susceptible isolate (KKBO-1) and a colistin-resistant isolate (KKBO-4) selected after colistin exposure revealed that insertional inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, is a genetic mechanism for acquired colistin resistance. The role of mgrB inactivation in acquired colistin resistance was confirmed by complementation experiments with wild-type mgrB, which restored colistin susceptibility in KKBO-4, and by construction of an mgrB deletion mutant from KKBO-1, which exhibited a colistin-resistant phenotype. Insertional mgrB inactivation was also detected in 60% of colistin-resistant mutants selected from KKBO-1 in vitro, following plating on colistin-containing medium, confirming the role (although not unique) of this mechanism in the emergence of acquired colistin resistance. In colistin-resistant mutants carrying insertional inactivation or deletion of the mgrB gene, upregulated transcription of phoP, phoQ, and pmrK (which is part of the pmrHFIJKLM operon) was detected. These findings confirmed the MgrB regulatory role in K. pneumoniae and were in agreement with the known association between upregulation of the PhoQ/PhoP system and activation of the pmrHFIJKLM operon, which eventually leads to resistance to polymyxins by modification of the lipopolysaccharide target.
Project description:Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP) are challenging multidrug-resistant pathogens due to their extensively drug-resistant phenotypes and potential for epidemic dissemination in health care settings. Colistin is a key component of the combination antimicrobial regimens used for treatment of severe KPC-KP infections. We previously reported that insertional inactivation of the mgrB gene, encoding a negative-feedback regulator of the PhoQ-PhoP signaling system, can be responsible for colistin resistance in KPC-KP, due to the resulting upregulation of the Pmr lipopolysaccharide modification system. In this work we investigated the status of the mgrB gene in a collection of 66 colistin-resistant nonreplicate clinical strains of KPC-KP isolated from different hospitals in Italy and Greece. Overall, 35 strains (53%) exhibited alterations of the mgrB gene, including insertions of different types of mobile elements (IS5-like, IS1F-like, or ISKpn14), nonsilent point mutations, and small intragenic deletions. Four additional strains had a larger deletion of the mgrB locus, while the remaining 27 strains (41%) did not show mgrB alterations. Transcriptional upregulation of the phoQ and pmrK genes (part of the phoPQ and pmrHFIJKLM operon, respectively) was observed in all strains with mgrB alterations. Complementation experiments with a wild-type mgrB gene restored colistin susceptibility and basal expression levels of phoQ and pmrK genes in strains carrying different types of mgrB alterations. The present results suggest that mgrB alteration can be a common mechanism of colistin resistance among KPC-KP in the clinical setting.
Project description:Antibiotic resistance is a major public health threat, further complicated by unexplained treatment failures caused by bacteria that appear antibiotic susceptible. We describe an Enterobacter cloacae isolate harbouring a minor subpopulation that is highly resistant to the last-line antibiotic colistin. This subpopulation was distinct from persisters, became predominant in colistin, returned to baseline after colistin removal and was dependent on the histidine kinase PhoQ. During murine infection, but in the absence of colistin, innate immune defences led to an increased frequency of the resistant subpopulation, leading to inefficacy of subsequent colistin therapy. An isolate with a lower-frequency colistin-resistant subpopulation similarly caused treatment failure but was misclassified as susceptible by current diagnostics once cultured outside the host. These data demonstrate the ability of low-frequency bacterial subpopulations to contribute to clinically relevant antibiotic resistance, elucidating an enigmatic cause of antibiotic treatment failure and highlighting the critical need for more sensitive diagnostics.
Project description:Background:In Klebsiella pneumoniae, mgrB and components of pmrHFIJKLM operon play a major role in colistin resistance. Methods:We analyzed 23 nonduplicating colistin-resistant K. pneumoniae isolates, collected during the years 2011-2015, for the possible mechanism underlying their nonsusceptibility to colistin. Isolates were tested for their minimum inhibitory concentrations and antibiotic resistance determinants and genotyped by multilocus sequence typing (MLST). The MLST genes, antibiotic-resistant genes, and the genes of two component system (TCS), including mgrB, PhoQ/PhoP, pmrA/B, and CrrAB, were investigated by PCR amplification and Sanger sequencing. Results:All isolates were distributed in eight sequence types (STs) and showed mutations either in mgrB or PhoP genes. ISKpn14 was found in 10, ISKpn28 in four, and IS903 in three isolates. One isolate showed deletion of a single nucleotide in mgrB open reading frame causing premature stop codon. L26Q substitution in PhoP was found in five isolates. Conclusion:The mutations in mgrB were mostly mediated by insertion elements (IS). ISKpn14 is the major IS while ISKpn28 is reported for the first time in mediating mgrB disruption. IS903, an IS5 family member, involved in mgrB disruption in three ST-152 NDM-1-positive isolates, was previously responsible for omp-36 disruption in our carbapenem-resistant K. pneumoniae and appears to contribute to transform the isolates into a pan-drug ones. Also, the abundance of insertion sites in mgrB indicates the plasticity of this gene. In our isolates, IS-mediated colistin resistance appears to be a later phenomenon than mutation in PhoP gene.
Project description:Here, we describe the first identification of colistin-heteroresistant Enterobacter cloacae in the United States. Treatment of this isolate with colistin increased the frequency of the resistant subpopulation and induced cross-resistance to the host antimicrobial lysozyme. This is the first description of heteroresistance conferring cross-resistance to a host antimicrobial and suggests that clinical treatment with colistin may inadvertently select for bacteria that are resistant to components of the host innate immune system.
Project description:Resistance to carbapenems in Enterobacteriaceae is a clinical problem of growing significance. Difficulty in treating multidrug-resistant Gram-negative organisms with conventional antibiotics has led to a renewed and increasing use of polymyxin compounds, such as colistin. Here, we report the isolation of carbapenem- and colistin-resistant Enterobacter cloacae from a polymicrobial lower extremity wound in an ambulatory patient. Whole-genome sequencing demonstrated the presence of chromosomal blaIMI-1 and blaAmpC, as well as numerous efflux pump genes.
Project description:Transmission of colistin-resistant <i>Enterobacterales</i> from companion animals to humans poses a clinical risk as colistin is a last-line antimicrobial agent for treatment of multidrug-resistant Gram-negative bacteria including <i>Enterobacterales</i>. In this study, we investigated the colistin susceptibility of 285 <i>Enterobacterales</i> (including 140 <i>Escherichia coli</i>, 86 <i>Klebsiella</i> spp., and 59 <i>Enterobacter</i> spp.) isolated from companion animals in Japan. We further characterized colistin-resistant isolates by multilocus sequence typing (MLST), phylogenetic analysis of <i>hsp60</i> sequences, and population analysis profiling, to evaluate the potential clinical risk of companion animal-derived colistin-resistant <i>Enterobacterales</i> to humans in line with the One Health approach. All <i>E. coli</i> isolates were susceptible to colistin, and only one <i>Klebsiella</i> spp. isolate (1.2%, 1/86 isolates) was colistin resistant. <i>Enterobacter</i> spp. isolates were frequently colistin resistant (20.3%, 12/59 isolates). In colistin-resistant <i>Enterobacter</i> spp., all except one isolate exhibited colistin heteroresistance by population analysis profiling. These colistin-heteroresistant isolates belonged to clusters I, II, IV, VIII, and XII based on <i>hsp60</i> phylogeny. MLST analysis revealed that 12 colistin-resistant <i>Enterobacter</i> spp. belonged to the <i>Enterobacter cloacae</i> complex; five <i>Enterobacter kobei</i> (four ST591 and one ST1577), three <i>Enterobacter asburiae</i> (one ST562 and two ST1578), two <i>Enterobacter roggenkampii</i> (ST606 and ST1576), and <i>Enterobacter hormaechei</i> (ST1579) and <i>E. cloacae</i> (ST765) (each one strain). Forty-two percent of the colistin-resistant <i>E. cloacae</i> complex isolates (predominantly ST562 and ST591) belonged to lineages with human clinical isolates. Four <i>E. kobei</i> ST591 isolates were resistant to third-generation cephalosporines, aminoglycosides, and fluroquinolones but remained susceptible to carbapenems. In conclusion, our study is the first to our knowledge to report the frequent isolation of the colistin-resistant <i>E. cloacae</i> complex from companion animals. Furthermore, a subset of isolates belonged to human-associated lineages with resistance to multiple classes of antibiotics. These data warrant monitoring carriage of the colistin-resistant <i>E. cloacae</i> complex in companion animals as part of a domestic infection control procedure in line with the One Health approach.
Project description:We describe the first report of a clinical colistin-resistant ST84 Enterobacter cloacae isolate coharboring mcr-4.3 (previously named mcr-4.2) and blaNDM-1 from a patient in China. The blaNDM-1-harboring IncX3 plasmid and the novel mcr-4.3-harboring ColE plasmid were completely sequenced. Although this isolate showed a high level of resistance to colistin, mcr-4.3 plasmid transformation, gene subcloning, susceptibility testing, and lipid A matrix-assisted laser desorption ionization mass spectrometry analysis indicated that mcr-4.3 itself does not confer resistance to colistin.