mcr-1 Gene Expression Modulates the Inflammatory Response of Human Macrophages to Escherichia coli.

MCR-1 is a plasmid-encoded phosphoethanolamine transferase able to modify the lipid A structure. It confers resistance to colistin and was isolated from human, animal, and environmental strains of Enterobacteriaceae, raising serious global health concerns. In this paper, we used recombinant mcr-1-expressing Escherichia coli to study the impact of MCR-1 products on E. coli-induced activation of inflammatory pathways in activated THP-1 cells, which was used as a model of human macrophages. We found that infection with recombinant mcr-1-expressing E. coli significantly modulated p38-MAPK and Jun N-terminal protein kinase (JNK) activation and pNF-κB nuclear translocation as well as the expression of genes for the relevant proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and IL-1ß compared with mcr-1-negative strains. Caspase-1 activity and IL-1ß secretion were significantly less activated by mcr-1-positive E. coli strains than the mcr-1-negative parental strain. Similar results were obtained with clinical isolates of mcr-1-positive E. coli, suggesting that, in addition to colistin resistance, the expression of mcr-1 allows the escape of early host innate defenses and may promote bacterial survival.

Results of the Italian infection-Carbapenem Resistance Evaluation Surveillance Trial (iCREST-IT): activity of ceftazidime/avibactam against Enterobacterales isolated from urine.

OBJECTIVES: To assess the in vitro antibacterial activity of ceftazidime/avibactam against a recent Italian collection of carbapenem-resistant Enterobacterales (CRE) isolated from urine specimens. METHODS: Consecutive Gram-negative isolates from urine specimens, collected from inpatients in five Italian hospitals during the period October 2016 to February 2017, were screened for CRE phenotype using chromogenic selective medium and identified using MALDI-TOF MS. Antimicrobial susceptibility testing was performed by reference broth microdilution (BMD) and, for ceftazidime/avibactam, also by Etest® CZA. Results were interpreted according to the EUCAST breakpoints. All confirmed CRE were subjected to real-time PCR targeting blaKPC-type, blaVIM-type, blaNDM-type and blaOXA-48-type carbapenemase genes. Non-MBL-producing isolates resistant to ceftazidime/avibactam were subjected to WGS and their resistome and clonality were analysed. RESULTS: Overall, 318 non-replicate presumptive CRE were collected following screening of 9405 isolates of Enterobacterales (3.4%) on chromogenic selective medium. Molecular analysis revealed that 216 isolates were positive for a carbapenemase gene (of which 92.1%, 2.8%, 1.4% and 1.4% were positive for blaKPC-type, blaOXA-48-type, blaNDM-type and blaVIM-type, respectively). Against the confirmed carbapenemase-producing Enterobacterales (CPE), ceftazidime/avibactam was the most active compound, followed by colistin (susceptibility rates 91.6% and 69.4%, respectively). Compared with BMD, Etest® for ceftazidime/avibactam yielded consistent results (100% category agreement). All class B ß-lactamase producers were resistant to ceftazidime/avibactam, while OXA-48 and KPC producers were susceptible, with the exception of seven KPC-producing isolates (4.2%). The latter exhibited an MIC of 16 to >32 mg/L, belonged to ST512, produced KPC-3 and showed alterations in the OmpK35 and Ompk36 porins. CONCLUSIONS: Ceftazidime/avibactam showed potent in vitro activity against a recent Italian collection of CPE from urine.

Characterization of Extensively Drug-Resistant or Pandrug-Resistant Sequence Type 147 and 101 OXA-48-Producing Klebsiella pneumoniae Causing Bloodstream Infections in Patients in an Intensive Care Unit.

Carbapenem-resistant Klebsiella pneumoniae causes important health care-associated infections worldwide. An outbreak of sequence type 11 (ST11) OXA-48-producing K. pneumoniae (OXA-48-Kp) isolates occurred in Tzaneio Hospital in 2012 and was contained until 2014, when OXA-48-Kp reemerged. The present study involved 19 bloodstream infection (BSI) OXA-48-Kp isolates recovered from 19 intensive care unit (ICU) patients hospitalized between August 2014 and July 2016. MICs were determined by broth microdilution. Beta-lactamase genes were detected by PCR. All isolates were typed by pulsed-field gel electrophoresis/multilocus sequence typing (PFGE/MLST), and 10 representative isolates were typed by next-generation sequencing (NGS). Of the 19 study patients, 9 had previous hospitalizations, and 10 carried OXA-48-Kp prior to BSI isolation; median time from ICU admission to BSI was 29 days. Four OXA-48-Kp isolates belonged to PFGE profile A (ST147) and were pandrug resistant (PDR), while 15 isolates exhibited PFGE profile B (ST101) and were extensively drug resistant. Genes detected via NGS resistome analysis accounted for most of the resistance phenotypes, except for tigecycline and fosfomycin. Insertional inactivation of mgrB (distinct per clone) conferred colistin resistance in all 19 isolates. NGS single nucleotide polymorphism (SNP) analysis validated the clonal relatedness of the ST147 and ST101 strains and revealed the possible presence of two index ST147 strains and the microevolution of ST101 strains. Distinct, but highly related, IncL OXA-48-encoding plasmid lineages were identified; plasmids of the ST147 strains were identical with the plasmid of ST11 OXA-48-Kp which caused the 2012 outbreak. In conclusion, biclonal circulation of OXA-48-Kp and, alarmingly, emergence of a PDR clone are reported. These observations, along with the challenging phenotypic detection of OXA-48 producers and the high reported transmissibility of blaOXA-48, necessitate intensive efforts to prevent their further spread.

mcr-1.2, a New mcr Variant Carried on a Transferable Plasmid from a Colistin-Resistant KPC Carbapenemase-Producing Klebsiella pneumoniae Strain of Sequence Type 512.

A novel mcr variant, named mcr-1.2, encoding a Gln3-to-Leu functional variant of MCR-1, was detected in a KPC-3-producing ST512 Klebsiella pneumoniae isolate collected in Italy from a surveillance rectal swab from a leukemic child. The mcr-1.2 gene was carried on a transferable IncX4 plasmid whose structure was very similar to that of mcr-1-bearing plasmids previously found in Escherichia coli and K. pneumoniae strains from geographically distant sites (Estonia, China, and South Africa).

Colistin Resistance Caused by Inactivation of the MgrB Regulator Is Not Associated with Decreased Virulence of Sequence Type 258 KPC Carbapenemase-Producing Klebsiella pneumoniae.

Using aGalleria mellonellaanimal model, we compared the virulence of two sequence type 258 (ST258) KPC-producingKlebsiella pneumoniaestrains, which were representative of the two clades of this clonal lineage, with that of isogenic colistin-resistantmgrBmutants. With both strains, themgrBmutants did not exhibit modification in virulence. In theG. mellonellamodel, the clade 1 strain (capsular typecps-1[wzi29, producing KPC-2]) was significantly more virulent than the clade 2 strain (capsular typecps-2[wzi154, producing KPC-3]).

Polymyxin resistance caused by mgrB inactivation is not associated with significant biological cost in Klebsiella pneumoniae.

The inactivation of the mgrB gene, which encodes a negative-feedback regulator of the PhoPQ signaling system, was recently shown to be a common mutational mechanism responsible for acquired polymyxin resistance among carbapenemase-producing Klebsiella pneumoniae strains from clinical sources. In this work, we show that mgrB mutants can easily be selected in vitro from different K. pneumoniae lineages, and mgrB inactivation is not associated with a significant biological cost.

MgrB inactivation is a common mechanism of colistin resistance in KPC-producing Klebsiella pneumoniae of clinical origin.

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.

In vivo evolution to colistin resistance by PmrB sensor kinase mutation in KPC-producing Klebsiella pneumoniae is associated with low-dosage colistin treatment.

Colistin is a key drug for the treatment of infections caused by extensively drug-resistant strains of Enterobacteriaceae producing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially among Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription of pmrA and of pmrK, which is part of the pmrHFIJKLM operon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-type pmrB in KPB-2 restored colistin susceptibility and reduced the transcription of pmrA and pmrK to basal levels, while expression of PmrB(L82R) in KPB-1 did not alter colistin susceptibility or upregulate pmrA and pmrK expression, confirming the dominance of wild-type PmrB versus the PmrB(L82R) mutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment.

In vivo emergence of colistin resistance in Klebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrB regulator.

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.

Synergistic Activity of Colistin in Combination With Resveratrol Against Colistin-Resistant Gram-Negative Pathogens.

Objectives: In this study, we investigated the antimicrobial activity of resveratrol in combination with colistin, a last-resort agent for the treatment of severe infections caused by multidrug resistant Gram-negative pathogens. Methods: The synergistic activity and the bactericidal activity of colistin in combination with resveratrol was investigated by checkerboard assays and time-kill assays, respectively. A total of 21 strains were investigated, including 16 strains of different species (Klebsiella pneumoniae, n = 6, Escherichia coli, n = 6; Citrobacter braakii, n = 1; Stenotrophomonas malthophilia, n = 1; Enterobacter cloaceae, n = 1; Acinetobacter baumannii, n = 1) with acquired colistin resistance, three colistin-susceptible K. pneumoniae precursors, and two strains of intrinsically colistin-resistant species (Serratia marcescens, n = 1; Proteus mirabilis, n = 1). Mechanisms of acquired colistin resistance included chromosomal mutations (i.e., mgrB, pmrAB) and plasmid genes (mcr-1, mcr-1.2). Results: Resveratrol did not show any significant intrinsic antimicrobial activity. Overall, a relevant synergistic antimicrobial activity of resveratrol in combination with colistin was observed with all tested strains, except for the three colistin-susceptible K. pneumoniae strains, and for two mcr-1-positive E. coli strains. In time-kill assays, performed with 15 selected strains, the combination of colistin 2 mg/L plus resveratrol 128 mg/L was bactericidal with 11 strains, and bacteriostatic for the remaining ones. Conclusions: Resveratrol was found to potentiate colistin activity against a wide panel of colistin-resistant strains, regardless of species and resistance mechanisms, which would deserve further investigation for potential clinical applications.

In vitro activity of N-acetylcysteine against Stenotrophomonas maltophilia and Burkholderia cepacia complex grown in planktonic phase and biofilm.

Stenotrophomonas maltophilia and Burkholderia cepacia complex (Bcc) have been increasingly recognized as relevant pathogens in hospitalized, immunocompromised and cystic fibrosis (CF) patients. As a result of complex mechanisms, including biofilm formation and multidrug resistance phenotype, S. maltophilia and Bcc respiratory infections are often refractory to therapy, and have been associated with a worse outcome in CF patients. Here we demonstrate for the first time that N-acetylcysteine (NAC), a mucolytic agent with antioxidant and anti-inflammatory properties, may exhibit antimicrobial and antibiofilm activity against these pathogens. The antimicrobial and antibiofilm activity of high NAC concentrations, potentially achievable by topical administration, was tested against a collection of S. maltophilia (n = 19) and Bcc (n = 19) strains, including strains from CF patients with acquired resistance traits. Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal Concentrations (MBCs) ranged from 16 to 32 mg/ml and from 32 to >32 mg/ml, respectively. Sub-MIC concentrations (i.e., 0.25 × MIC) slowed down the growth kinetics of most strains. In time-kill assays, 2-day-old biofilms were more affected than planktonic cultures, suggesting a specific antibiofilm activity of NAC against these pathogens. Indeed, a dose- and time-dependent antibiofilm activity of NAC against most of the S. maltophilia and Bcc strains tested was observed, with a sizable antibiofilm activity observed also at 0.5 and 1 × MIC NAC concentrations. Furthermore, at those concentrations, NAC was also shown to significantly inhibit biofilm formation with the great majority of tested strains.

An allelic variant of the PmrB sensor kinase responsible for colistin resistance in an Escherichia coli strain of clinical origin.

We investigated the colistin resistance mechanism in an Escherichia coli strain (LC711/14) isolated in Italy in 2014, from an urinary tract infection, which was previously shown to express a colistin resistance mechanism different from mcr-1. LC711/14 was found to carry a novel mutation in the pmrB gene, resulting in a leucine to proline amino acid substitution at position 10 of the PmrB sensor kinase component of the PmrAB signal transduction system. The role of this substitution in colistin resistance was documented by expression of the wild-type and mutated alleles in a pmrB deletion derivative of the E. coli reference strain MG1655, in which expression of the mutated allele conferred colistin resistance and upregulation of the endogenous pmrHFIJKLM lipid A modification system. Complementation of LC711/14 with the wild-type pmrB allele restored colistin susceptibility and decreased expression of pmrHFIJKLM, confirming the role of this PmrB mutation. Substitution of leucine at position 10 of PmrB with other amino acids (glycine and glutamine) resulted in loss of function, underscoring a key role of this residue which is located in the cytoplasmic secretion domain of the protein. This work demonstrated that mutation in this domain of the PmrB sensor kinase can be responsible for acquired colistin resistance in E. coli strains of clinical origin.

CXC Chemokines Exhibit Bactericidal Activity against Multidrug-Resistant Gram-Negative Pathogens.

The continued rise and spread of antimicrobial resistance among bacterial pathogens pose a serious challenge to global health. Countering antimicrobial-resistant pathogens requires a multifaceted effort that includes the discovery of novel therapeutic approaches. Here, we establish the capacity of the human CXC chemokines CXCL9 and CXCL10 to kill multidrug-resistant Gram-negative bacteria, including New Delhi metallo-beta-lactamase-1-producing Klebsiella pneumoniae and colistin-resistant members of the family Enterobacteriaceae that harbor the mobile colistin resistance protein MCR-1 and thus possess phosphoethanolamine-modified lipid A. Colistin-resistant K. pneumoniae isolates affected by genetic mutation of the PmrA/PmrB two-component system, a chromosomally encoded regulator of lipopolysaccharide modification, and containing 4-amino-4-deoxy-l-arabinose-modified lipid A were also found to be susceptible to chemokine-mediated antimicrobial activity. However, loss of PhoP/PhoQ autoregulatory control, caused by disruption of the gene encoding the negative regulator MgrB, limited the bactericidal effects of CXCL9 and CXCL10 in a variable, strain-specific manner. Cumulatively, these findings provide mechanistic insight into chemokine-mediated antimicrobial activity, highlight disparities amongst determinants of colistin resistance, and suggest that chemokine-mediated bactericidal effects merit additional investigation as a therapeutic avenue for treating infections caused by multidrug-resistant pathogens.IMPORTANCE As bacterial pathogens become resistant to multiple antibiotics, the infections they cause become increasingly difficult to treat. Carbapenem antibiotics provide an essential clinical barrier against multidrug-resistant bacteria; however, the dissemination of bacterial enzymes capable of inactivating carbapenems threatens the utility of these important antibiotics. Compounding this concern is the global spread of bacteria invulnerable to colistin, a polymyxin antibiotic considered to be a last line of defense against carbapenem-resistant pathogens. As the effectiveness of existing antibiotics erodes, it is critical to develop innovative antimicrobial therapies. To this end, we demonstrate that the chemokines CXCL9 and CXCL10 kill the most concerning carbapenem- and colistin-resistant pathogens. Our findings provide a unique and timely foundation for therapeutic strategies capable of countering antibiotic-resistant "superbugs."

Inhibitory activity of avibactam against selected ß-lactamases expressed in an isogenic Escherichia coli strain.

Avibactam restored the in-vitro antibacterial activity of ceftazidime, ceftaroline, and aztreonam against isogenic Escherichia coli expressing class A, class C, and class D ß-lactamases. The enzymes included TEM and CTX-M extended spectrum ß-lactamases, ACT, CMY and FOX AmpC-type enzymes, and carbapenemases including rarer KPC variants and OXA-139.