Colistin MIC Research Articles

Diversity of amino acid substitutions in PmrCAB associated with colistin resistance in clinical isolates of Acinetobacter baumannii

This study aimed to investigate the mechanisms of colistin resistance in 64 Acinetobacter baumannii isolates obtained from patients with ventilator-associated pneumonia hospitalised in Greece, Italy and Spain. In total, 31 A. baumannii isolates were colistin-resistant. Several novel amino acid substitutions in PmrCAB were found in 27 colistin-resistant A. baumannii. Most substitutions were detected in PmrB, indicating the importance of the histidine kinase for colistin resistance. In two colistin-resistant isolates, 93 amino acid changes were observed in PmrCAB compared with A. baumannii ACICU, and homologous recombination across different clonal lineages was suggested. Analysis of gene expression revealed increased pmrC expression in isolates harbouring pmrCAB mutations. Complementation of A. baumannii ATCC 19606 and ATCC 17978 with a pmrAB variant revealed increased pmrC expression but unchanged colistin MICs, indicating additional unknown factors associated with colistin resistance. Moreover, a combination of PmrB and PmrC alterations was associated with very high colistin MICs, suggesting accumulation of mutations as the mechanism for high-level resistance. The pmrC homologue eptA was detected in 29 colistin-susceptible and 26 colistin-resistant isolates. ISAba1 was found upstream of eptA in eight colistin-susceptible and one colistin-resistant isolate and eptA was disrupted by ISAba125 in two colistin-resistant isolates. Whilst in most isolates an association of eptA with colistin resistance was excluded, in one isolate an amino acid substitution in EptA (R127L) combined with a point mutation in ISAba1 upstream of eptA contributed to elevated colistin MICs. This study helps to gain an insight into the diversity and complexity of colistin resistance in A. baumannii.

Restoring colistin sensitivity in colistin-resistant E. coli: Combinatorial use of MarR inhibitor with efflux pump inhibitor

Antibiotics like colistin are the last resort to deal with infections by carbapenem-resistant Enterobacteriaceae (CREB). Resistance to colistin severely restricts therapeutic options. To tackle this dire situation, urgent measures to restore colistin sensitivity are needed. In this study, whole-genome sequencing of colistin-resistant E. coli strain was performed and the genome analysis revealed that the strain belonged to the sequence type ST405. Multiple mutations were observed in genes implicated in colistin resistance, especially those related to the L-Ara-4-N pathway but mgrB was unmutated and mcr1-9 genes were missing. MarR inhibitor salicylate was used to re-sensitize this strain to colistin, which increased the negative charge on the cell surface especially in colistin resistant E. coli (U3790 strain) and thereby facilitated a decrease in colistin MIC by 8 fold. It is indeed well known that MarR inhibition by salicylate triggers the expression of AcrAB efflux pumps through MarA. So, in order to fully restore colistin sensitivity, a potent efflux pump inhibitor (BC1), identified earlier by this group was employed. The combination of colistin with both salicylate and BC1 caused a remarkable 6 log reduction in cell counts of U3790 in time-kill assay. Infection of muscle tissue of zebrafish with U3790 followed by various treatments showed that the combination of colistin + salicylate + BC1 was highly effective in reducing bioburden in infected muscle tissue by 4 log fold. Thus, our study shows that a combination of MarR inhibitor to enhance colistin binding and efflux pump inhibitor to reduce colistin extrusion was highly effective in restoring colistin sensitivity in colistin-resistant clinical isolate of E. coli in vitro and in vivo.

Draft genome sequence of an mcr-1/IncI2-carrying multidrug-resistant Escherichia coli B1:ST101 isolated from meat and meat products in Egypt.

The aim of this study was to investigate the occurrence of plasmid-encoded colistin resistance among Gram-negative bacteria isolated from meat and meat products in Egypt and to report the draft genome sequence of anmcr-1/IncI2-carrying multidrug-resistant (MDR) Escherichia coli B1:ST101 isolate. A total of 128 colistin-resistant strains were isolated from various meat and meat product samples in different cities in Egypt. Multiplex PCR screening for plasmid-mediated colistin resistance genes was performed. Whole-genome sequencing was performed using an Illumina NextSeq platform and the genome was assembled using CLC Genomics Workbench 7.5.1. A singlemcr-1-positive MDR E. coli strain was isolated from beef sausages. The genome size of the E. coli strain was calculated at 5 044 715bp, with a total of 226 contigs and a G+C content of 50.5%. The strain belonged to ST101 (phylogroup B1). The mcr-1 gene was located on an IncI2-type self-conjugative plasmid of 64.6kb in size. The strain showed a MDR phenotype, with a colistin MIC of 4mg/L. A large number of acquired antimicrobial resistance genes was identified, including genes encoding resistance to colistin (mcr-1), β-lactams (blaTEM-1), phenicols (floR), trimethoprim (dfrA12), aminoglycosides [aac(3)-IIa, aph(3")-Ib and aadA2], macrolides (mphA and mdfA), tetracyclines (tetA), sulfonamides (sul1 and sul2) and quinolones (qnrS1). Here we report the first draft genome sequence of anmcr-1/IncI2-carrying MDR E. coli B1:ST101 isolated from beef sausage in Egypt. This study highlights the potential role played by food products in the spread of colistin resistance to humans.

Co-occurrence of Variants of mcr-3 and mcr-8 Genes in a Klebsiella pneumoniae Isolate From Laos.

Colistin is considered as a last resort antibiotic. The re-use of this antibiotic highlighted the emergence of colistin resistance mediated by chromosomal and plasmidic resistance mechanisms. Five colistin-resistant Klebsiella pneumoniae strains from Laos and Thailand were analyzed by Next Generation Sequencing (NGS) approaches to determine their colistin resistance mechanisms. Antimicrobial susceptibility testing, conjugation and transformation were performed on these strains. Moreover, whole genome sequencing (WGS) combining Illumina (MiSeq) and Oxford Nanopore technologies (MinION) was realized to obtain closed genomes and plasmids. Resistome analyses as well as location of mcr genes and its genetic environments were done in silico. All five strains had colistin MIC of 32 mg/L and were positive for mcr-3 variants including additionally positive for a mcr-8 variant gene. The novel variants were named mcr-3.21, mcr-3.26, mcr-3.28, and mcr-8.3 genes. The mcr-3 variants genes were located on plasmids IncP1, IncFII, and IncI1 type, while mcr-8.3 gene was found on an IncFII type plasmid. The genetic environment of mcr-3.21 and mcr-3.26 genes were composed of a composite transposon ISKpn40- mcr-3-dgkA- ISKpn40. Concerning mcr-8.3 gene, a similar genetic environment of mcr-8.1 gene surrounded by ISIX2 and IS903B was observed. To the best of our knowledge, this is the first description of the novel variants mcr-3.21, mcr-3.26, mcr-3.28 and mcr-8.3 genes as well as the first study on co-occurrence of mcr-3 and mcr-8 genes. Spread and evolution of mcr genes should be monitored.

Emergence of a ST307 clone carrying a novel insertion element MITEKpn1 in the mgrB gene among carbapenem-resistant Klebsiella pneumoniae from Moscow, Russia

Carbapenem-resistant Klebsiella pneumoniae (CRKP) represents a major nosocomial pathogen with only a few antimicrobial agents, including colistin, remaining active. However, the emergence of colistin-resistant (Col-R) isolates is compromising the activity of colistin. In this study, a collection of 159 CRKP recovered from three hospitals in Moscow (Russian Federation) was examined. The isolates demonstrated resistance to cephalosporins (100%), ciprofloxacin (92.5%), fosfomycin (90.1%), netilmicin (81.1%), gentamicin (84.3%) and amikacin (49.7%). The rate of colistin resistance (MIC > 2 mg/L by broth microdilution) was 44.7%; moreover, 6.7% of isolates were tigecycline-resistant. Among 18 sequence types (STs) discovered, isolates of five lineages including ST307 (n=46; 28.9%), ST395 (n=40; 25.2%), ST377 (n=17; 10.7%), ST48 (n=17; 10.7%) and ST23 (n=16; 10.1%) dominated. Carriage of a blaOXA-48-like carbapenemase gene was detected in 146 CRKP (91.8%); 11 (6.9%) and 2 (1.3%) isolates harboured blaNDM-1 and blaKPC-3, respectively. Among 71 Col-R isolates, colistin MICs ranged from 4 mg/L to >1024 mg/L (MIC50/90, 2/512 mg/L). All Col-R isolates were mcr-1-negative. In 19 (26.8%) Col-R isolates, inactivation of mgrB by insertion sequences IS1A, IS1R, ISKpn14 and ISKpn26 and a novel miniature inverted-repeat transposable element (MITE) Kpn1 was observed. Carriage of MITEKpn1 was restricted to six ST307 isolates and affected mgrB at nucleotide position 75. mgrB deletion was observed in four (5.6%) Col-R isolates. Moreover, PmrA and/or PmrB were altered in three (4.5%) Col-R isolates with wild-type mgrB. Thus, blaOXA-48-like-carrying Col-R ST307 K. pneumoniae is emerging as a dominant clone in Moscow.

2277. Comparison of 30-day Crude Mortality Rates in Patients with Bloodstream Infections (BSIs) Caused by Colistin Susceptible-(ColS-CRKp) vs. Colistin and Carbapenem-Resistant Klebsiella pneumoniae(ColR-CRKp)

BackgroundColistin is a last resort antibiotic against infections with CRKp. Its increased use has led to resistance to this antibiotic.MethodsThis was a single-center, retrospective, cohort study including all CRKp BSIs treated between January 1, 2014 and July 31, 2018. Antibiotic therapy was appropriate if initiated within 5 days from the onset of BSI including at least an active drug with an adequate dosage. Exclusion criteria were missing key data, death < 24 h after inclusion, subsequent episodes in the same patients, pregnancy, polymicrobial BSI, < 18 years of age, no ID consultation, no carbapenemase gene detection by multiplex PCR for blaKPC, blaNDM, blaVIM, blaIMPand blaOXA48. EUCAST breakpoints were used for antibiotic susceptibilities. Colistin MIC was determined by using broth microdilution. FDA criteria were applied for tigecycline susceptibilityResultsAmong 174 CRKp BSIs, 129 met all inclusion criteria. Susceptibility to antibiotics was as follows: colistin (51.9%), tigecycline (67.4%), gentamicine (38.0%), amikacin (43.0%), meropenem (32.6%), ciprofloxacin (18.6%), TMP-SMX (24.0%), ceftazidime (7.8%), cefepime (7.8%).66.1% and 19.4% of the patients received inappropriate therapy in ColR-CRKp and ColS-CRKp groups, respectively (P ≤ 0.001). We incorporated interaction between colistin susceptibility and inappropriate therapy into multivariate logistic regression analysis (MLRA) that was constructed for identification of independent risk factors for 30-day mortality. The variables having P ≤ 0.1 in crude analysis were included in MLRA. After checking correlation between variables by Pearson correlation analysis and multicollinearity analysis, the final model was builded. The results are shown in Table 2.ConclusionColistin resistance and inappropriate therapy were not associated with decreased mortality individually, however their interaction significantly increased 30-day mortality rate (OR: 4.04; 95% CI: 1.62–10.02; P = 0.003).85.5% of ColR-CRKp isolates produced an OXA-48 enzyme which can be inhibited by ceftazidime–avibactam, but not available in our setting, treatment with this agent may have altered the mortality rates. Thus, high rate colistin resistance among CRKp isolates remains as a significant cause of mortality in our setting. Disclosures All authors: No reported disclosures.

135. Clinical and Microbiologic Analysis of Risk Factors for Mortality in Patients with Carbapenem-Resistant Acinetobacter baumannii Bacteremia

BackgroundCarbapenem-resistant Acinetobacter baumannii (CRAB) infection is an emerging clinical issue and shows high mortality rates. There are a few studies that have evaluated the microbiologic risk factors for mortality in CRAB bacteremia. Aim of this study is to identify the clinical and microbiologic risk factors for mortality in CRAB bacteremia.MethodsAdult patients with monomicrobial CRAB bacteremia at a 2,700-bed tertiary hospital between December 2012 and December 2018 were retrospectively enrolled in the study. Risk factors for 30-day mortality were evaluated through a detailed clinical and microbiological analysis of study patients. All isolates collected on the first day of bacteremia were subjected to colistin susceptibility testing by broth microdilution and genotyping by multilocus sequence typing (MLST).ResultsA total of 164 patients were enrolled and 90 (55%) died within 30 days. Of the 164 patients, 111 (68%) were male and median age was 66.5 years. The most common MLST genotype was ST191 (80 isolates, 49%), followed by ST451 (14%) and ST784 (13%), and 12 (7%) isolates were resistant to colistin (MIC ≥4 mg/L). Deceased patients were more likely to have hematologic malignancy, neutropenia, pneumonia, and primary bacteremia; less likely to have solid tumor, catheter-related infection, and biliary tract infection; more likely to have a high Pitt bacteremia score; and less likely to receive appropriate antibiotic treatment, colistin, and combination therapy with colistin and tigecycline, compared with surviving patients (Table 1). Genotype, colistin MIC, and colistin resistance were not associated with mortality (Figure 1 and 2). In multivariable analysis, neutropenia (aOR, 3.25; 95% CI, 1.18–8.95), catheter-related infection (aOR, 0.33; 95% CI, 0.11–0.99), biliary tract infection (aOR, 0.20; 95% CI, 0.04–0.99), a high Pitt bacteremia score (aOR,1.42; 95% CI, 1.20–1.67), and combination therapy with colistin and tigecycline (aOR, 0.36; 95% CI, 0.14–0.92) were independent risk factors for mortality (Table 2).ConclusionClinical factors such as the site of infection, severity of bacteremia, and specific combination therapy rather than microbiologic factors contributed to mortality in CRAB bacteremia. Appropriate combination therapy may help improving outcomes in CRAB bacteremia. Disclosures All authors: No reported disclosures.

494. Fitness Cost of mcr-1-Mediated Colistin Resistance in Carbapenemase-Producing Klebsiella pneumoniae

BackgroundThe emergence of mobile colistin resistance gene mcr-1, a plasmid-borne polymyxin resistance mechanism, in carbapenem-resistant Klebsiella pneumoniae is an alarming concern. However, previous studies showed that the acquisition of mcr-1 was associated with a significant biological fitness cost in K. pneumoniae. We aimed to study the impact of mcr-1 on the biological fitness in clinical carbapenemase-producing K. pneumoniae strains.MethodsClinical carbapenemase-producing K. pneumoniae strains were collected consecutively at the Taipei Veterans General Hospital between November 2017 and December 2018. The strain positive for mcr-1 was subjected to whole-genome sequencing to delineate its genomic features. Escherichia coli J53 strain was used as the recipient strain in plasmid conjugation assay and the transconjugants were selected with sodium azide and colistin. Plasmid stability was tested by serial passaging in antibiotic-free LB broth for 28 days. The growth rate was compared between the parental mcr-1-bearing strain and the plasmid-cured strain.ResultsOne ST11 strain isolated from a fatal case with bacteremia (KP2509) was found to harbor blaKPC-2, blaOXA-48, and mcr-1. This strain was resistant to colistin (MIC=8 mg/L) and imipenem (MIC≥16 mg/L). Whole-genome sequencing of KP2509 showed that mcr-1, blaKPC-2 and blaOXA-48 were located on an IncHI-FIB type plasmid of 319 Kb, an IncFII type plasmid of 96 Kb, and an IncL type plasmid of 64 Kb, respectively. Conjugation efficiency of mcr-1-bearing plasmid was 2.24 × 10–4, and the colistin MIC of E. coli J53 transconjugant increased from 0.5 to 8 mg/L. The mcr-1-bearing plasmid in KP2509 showed high plasmid stability, and only ~1% were lost after 27-day passages. The resulting plasmid-cured strain (PC-KP2509) was susceptible to colistin (MIC=0.5 mg/L) and had a similar growth rate to that of parental mcr-1-bearing strain KP2509.ConclusionWe identified an ST11 K. pneumoniae strain carrying blaKPC-2, blaOXA-48, and mcr-1 genes causing a fatal bacteremia. The large mcr-1-bearing plasmid confers a moderate level of colistin resistance but without significant biological fitness cost in carbapenemase-producing K. pneumoniae, which could result in a serious threat clinically.Disclosures All authors: No reported disclosures.

A novel plasmid-encoded mcr-4.3 gene in a colistin-resistant Acinetobacter baumannii clinical strain.

To identify the molecular mechanism of colistin resistance in an MDR Acinetobacter baumannii clinical strain isolated in 2008 from a meningitis case in Brazil. Long- and short-read WGS was used to identify colistin resistance genes in A. baumannii strain 597A with a colistin MIC of 64 mg/L. MS was used to analyse lipid A content. mcr was cloned into pET-26b (+) and transformed into Escherichia coli BL21(λDE3)pLysS for analysis. A novel plasmid (pAb-MCR4.3) harbouring mcr-4.3 within a Tn3-like transposon was identified. The A. baumannii 597A lipid A MS spectra showed a main molecular ion peak at m/z=2034, which indicated the addition of phosphoethanolamine to the lipid A structure. E. coli BL21 transformed with pET-26b-mcr-4.3 gained colistin resistance with a colistin MIC of 8 mg/L. Colistin resistance in A. baumannii 597A was correlated with the presence of a novel plasmid-encoded mcr-4.3 gene.

In vitro activity of methylene blue and eosin methylene blue agar on colistin-resistant A. baumannii: an experimental study.

Introduction. Colistin is a last-resort antibiotic used against carbapenem-resistant Acinetobacter baumannii (AB); however, colistin resistance has been reported recently. Methylene blue (MB) is used in microbiology for staining, and in medicine as an antidote drug.Aim. We aimed to investigate the antimicrobial effects of MB and eosin methylene blue (EMB) agar against colistin-resistant AB strains.Methodology. Firstly, a standard strain and AB clinical isolate were included in the study. After determining MICs, two strains were transformed into colistin-resistant forms, using Li's method. At each step, new MICs were determined and subcultures were inoculated onto EMB and sheep blood agar (SBA). Colistin MICs of the subcultures were also determined using Mueller-Hinton agar (MHA) containing MB. Secondly, colistin-resistant clones from 31 multidrug-resistant AB clinical isolates were screened to investigate their susceptibilities to EMB agar.Results. In the first round, MICs of both strains had risen to 64 μg ml-1. Subpopulations with high colistin resistance were inhibited by MB and EMB agar, but could grow well on SBA. In MHA plates containing MB, the MICs decreased to 0.5 μg ml-1 for colistin-susceptible or moderately resistant clones. Additionally, clones with high colistin resistance showed atypical colony morphology on SBA. In the second round, 35 % of the clinical isolates, which had gained resistance to colistin, were inhibited by EMB agar.Conclusion. MB may have inhibitory effects against colistin-resistant AB. Secondly, using only EMB agar for subculturing may cause missing of colistin-resistant strains.

Implementation of the Rapid Polymyxin™ NP test directly to positive blood cultures bottles

The present study assessed the performance of Rapid Polymyxin™ NP test to detect colistin resistance directly from 132 blood cultures found positive for Enterobacterales by FilmArray. Additionally, colistin MICs of isolated microorganisms were determined by the commercial broth microdilution method ComASP™ Colistin, used as the gold standard comparator. The Rapid Polymyxin™ NP test correctly detected all colistin-resistant isolates. However, the test has misidentified as resistant 4 colistin-susceptible isolates (1 Escherichia coli and 3 Klebsiella pneumoniae). Molecular characterization of colistin-resistant isolates showed that K. pneumoniae, which belonged to ST15 and ST147, carried alterations in the mgrB. Moreover, the first Greek mcr-1–positive colistin-resistant E. coli was detected. The rapidity (2–3 h) of the results, combined with its excellent negative predictive value (100%), allows implementation of the test for routine testing of blood cultures mainly in the clinical settings that are endemic for carbapenem-resistant bacteria, avoiding misuse of colistin and preventing the spread of colistin-resistant bacteria.

Discrepancies in susceptibility testing to colistin in Acinetobacter baumannii: The influence of slow growth and heteroresistance

The increasing use of polymyxins as last-resort drugs for managing infections by Acinetobacter baumannii has led to the emergence of resistance. This study aimed to determine the resistance mechanisms in Acinetobacter baumannii isolates with colistin MIC ≥ 4 mg/L and to relate the mechanisms of resistance with the difficulties in detecting them. Absolute agreement among the different methodologies (Phoenix automatized system, broth and agar dilution, and a rapid colorimetric test) in the 140 colistin-susceptible isolates was observed; whereas in the 25 resistant isolates, the performance varied according to the colistin MIC value. Most of the discrepancies (irrespective of the methodology that was used) were observed in isolates with an MIC value close to the breakpoint. The number of errors in each method in the resistant isolates was as follows: rapid test, four of 25 (16%); agar dilution, eight of 25 (32%); Phoenix system, 13 of 25 (52%) and its manual reading at 24 h, eight of 25 (32%). Categorical errors were detected in 13 isolates: slow growth was the main reason in five isolates, whereas in the remaining eight isolates, slow growth was detected together with a low proportion of colistin-resistant subpopulations and the colistin MIC value was close to the breakpoint value. To understand the probable reason for the observed MIC values, sequencing of genes associated with colistin resistance was performed. Mutations at lpxA, lpxC, and pmrB genes were detected and it was observed that isolates carrying mutations in lpxC presented slow growth at killing curves.

Antimicrobial Resistance Pattern of Extensively Drug Resistant Carbapenemase Producing Enterobacteriaceae

Carbapenems, often agents of last resort for multidrug resistant bacterial infections are now threatened by widespread dissemination of carbapenem-resistant Enterobacteriaceae (CRE). Production of carbapenemases remain the most clinically important mechanism of carbapenem resistance in Enterobacteriaceae. The objective of this study was to determine the antibiogram pattern of carbapenemase producing Enterobacteriaceae. A cross sectional study was conducted at department of Microbiology and Immunology, BSMMU from September 2018 to August 2019. A total of 145 CRE isolates from different clinical samples were studied.Antimicrobial susceptibility was examinedby disk diffusion method and MIC of colistin by broth microdilution method. Resistant carbapenemase genes NDM and OXA-48 were identified by polymerase chain reaction. Out of 145 CRE isolates, 104 were NDM, 73 were OXA-48and 34 isolates were both NDM and OXA-48 co-producers. All the NDM and OXA-48 carbapenemase producing isolates were 100% resistant to meropenem, imipenem, ertapenem, ceftriaxone, ceftazidime, cefotaxime, cefuroxime, amoxicillin + clavulanic acid and piperacillin + tazobactam. Resistance rates of reserved antimicrobials to treat CRE isolates were also alarming. Thirty seven percent, 9.6% and 5.5 % of OXA-48 carbapenemase producers and 26.0%, 10.6% and 2.9% of NDM carbapenemase producers were resistant to colistin, polymyxin B and tigecycline respectively.Among the carbapenemase producing isolates, 16.6% (24) were multidrug resistant (MDR), 82.1% (119) were extensively drug resistant (XDR) and 1.3% (2) isolates were pan drug resistantwhich highlights the emerging therapeutic challenge for these superbugs.&#x0D; Bangladesh J Med Microbiol 2019; 13 (2): 7-10

Colistin-containing cement spacer for treatment of experimental carbapenemase-producing Klebsiella pneumoniae prosthetic joint infection

Carbapenemase-producing Enterobacteriaceae (CPE) are emerging multidrug-resistant bacteria responsible for invasive infections, including prosthetic joint infections (PJIs). Local administration of colistin may provide bactericidal concentrations in situ. This study evaluated the efficacy of a colistin-impregnated cement spacer, alone and in combination with systemic antibiotics, in a rabbit model of CPE-PJI. Elution of 3 MIU of colistimethate sodium (CMS) in 40 g of poly(methyl methacrylate) cement was studied in vitro. In vivo, 5 × 108 CFU of KPC-producing Klebsiella pneumoniae (colistin and meropenem MICs of 1 mg/L and 4 mg/L, respectively) were injected close to a prosthetic knee. Surgical debridement and prosthesis removal were performed 7 days later, and rabbits were assigned to six treatment groups (11-13 rabbits each): drug-free spacer; colistin-loaded spacer; colistin intramuscular (i.m.); colistin i.m. + colistin spacer; colistin i.m. + meropenem subcutaneous (s.c.); and colistin i.m. + meropenem s.c. + colistin spacer. Systemic treatment was administered at doses targeting pharmacokinetics in humans, and rabbits were euthanised 7 days later to evaluate bacterial counts in infected bones. In vitro, CMS elution was low (<0.1% at 24 h) but reached a local concentration of ≥20 mg/L (>20 × MIC). In vivo, combinations of local and systemic colistin, with or without meropenem, were the only regimens superior to the control group (P ≤ 0.05) in terms of viable bacterial counts and the proportion of rabbits with sterile bone, with no emergence of colistin-resistant strains. Colistin-loaded cement spacer in combination with systemic antibiotics were the most effective regimens in this CPE-PJI model.

Nationwide surveillance of antimicrobial resistance among clinically important Gram-negative bacteria, with an emphasis on carbapenems and colistin: Results from the Surveillance of Multicenter Antimicrobial Resistance in Taiwan (SMART) in 2018

Multicentre surveillance of antimicrobial susceptibility of clinically important Gram-negative bacteria (GNB) from 16 Taiwanese hospitals was performed. Escherichia coli (n = 398), Klebsiella pneumoniae (n = 346), Pseudomonas aeruginosa (n = 252) and Acinetobacter baumannii complex (ABC) (n = 188) bloodstream isolates, non-typhoidal Salmonella (n = 230) and Shigella flexneri (n = 18) from various sources were collected. Antimicrobial MICs were determined using broth microdilution. Genes encoding K. pneumoniae carbapenemases (KPCs), New Delhi metallo-β-lactamases (NDMs), Verona integron-encoded metallo-β-lactamase (VIM), OXA-48-like carbapenemase (OXA-48) as well as mcr-1-5 genes were detected by molecular methods. Rates of carbapenem non-susceptibility were 2.8%, 9.0%, 0.4%, 0%, 10.3% and 48.8% for E. coli, K. pneumoniae, Salmonella, Shigella, P. aeruginosa and ABC, respectively. For carbapenemases, one (0.3%) E. coli harboured blaNDM-1. Fifteen (4.3%), two (0.6%) and two (0.6%) K. pneumoniae contained blaKPC, blaOXA-48 and blaVIM, respectively. Two (0.5%) E. coli and fourteen (4.0%) K. pneumoniae were non-wild-type according to the colistin MIC. Among Enterobacteriaceae with a colistin MIC ≥ 2 mg/L, mcr-1 was detected in one E. coli, two K. pneumoniae and three Salmonella spp. All three mcr-1-positive Salmonella isolates were collected from community-acquired infections; none of the six mcr-1-positive Enterobacteriaceae were carbapenem-resistant. Carbapenem resistance has increased among clinically important GNB, especially among hospital-acquired infections. blaKPC, especially the blaKPC-2 variant, was detected in approximately one-half of the carbapenem-resistant K. pneumoniae isolates in this study. Although resistance rates to colistin remained low among Enterobacteriaceae, the finding of mcr-1 from different species raises concern of potential dissemination.

Population structure and antimicrobial resistance traits of avian-origin mcr-1-positive Escherichia coli in Eastern China, 2015 to 2017.

Recent emergence of mcr-1-positive Escherichia coli (MCRPEC) is causing serious concern around the world. Due to poultry-origin E.coli holding zoonotic potential, the improved understandings of MCRPEC population structure and antimicrobial resistance are critical to public health purposes. This study provided novel insights into the molecular epidemiology of avian-origin MCRPEC. For the mcr genes prevalence study, we analysed 1,360 E.coli recovered from avian colibacillosis in eastern China from 2015 to 2017. The mcr-1 was present in 172 (12.6%) E.coli isolates. For all of MCRPEC isolates, MICs of colistin were ≥4mg/L. Avian-origin MCRPEC was widely distributed throughout phylogroups A, B1, B2, D, and F. Moreover, those isolates were assigned to 52 unique STs, such as ST48, ST117, ST131, and ST648, suggesting substantial horizontal dissemination of mcr-1 gene through avian-origin E.coli populations. The susceptibility of MCRPEC isolates was tested with 26 antimicrobial agents from 16 antimicrobial categories. There were high resistance rates of MCRPEC isolates against the clinically used antibiotics. All MCRPEC isolates in this study presented the multidrug-resistant (MDR) trait were even considered as extensively drug-resistant (XDR) strains. Resistance genotypes and plasmid replicon profiling showed that a majority of MCRPEC isolates contained plasmid-mediated resistance genes and exhibited the co-existence of mcr-1 with ESBLs and pAmpCs genes. Furthermore, the overlapped distribution of ST types and resistance gene contents was detected among MCRPEC isolates from humans and poultry. Besides mcr-1, our findings highlighted a significant prevalence of plasmid-mediated resistance genes among avian-origin MCRPEC isolates.

A Novel Phenotypic Method To Screen for Plasmid-Mediated Colistin Resistance among Enterobacteriales.

Plasmid-mediated colistin resistance (PMCR), a consequence of the mcr genes, is a significant public health concern given its potential to easily spread among clinical pathogens. Recently, it was discovered that MCR enzymes require zinc for activity. Thus, we modified the colistin broth-disk elution (CBDE) test to screen for plasmid-mediated colistin resistance (PMCR) genes based on any reduction of colistin MIC in the presence of EDTA. Eighty-five isolates of the order Enterobacteriales (12 mcr positive) were tested by CBDE ± EDTA. The sensitivity and specificity of the EDTA-CBDE method to detect PMCR compared to the molecular genotype results were 100% and 95.8%, respectively. Isolates positive by the EDTA-CBDE test should be further evaluated to confirm the presence of mcr genes.

Modification of outer membrane permeability and alteration of LPS in veterinary enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a worrying cause of diarrhoea in calves and the drug multiresistance phenotype concerning various antibiotic families are of concern. Resistance mechanisms associated with envelope changes (porin expression, efflux pump overexpression, lipolysaccahride (LPS) modification) were studied in 14 ETEC isolates selected for their resistance. We performed determinations of (i) antimicrobials Minimal Inhibitory Concentrations with or without the efflux pump inhibitor phenylalanine arginine β-naphthylamide; (ii) colistin and polymyxin MICs with and without EDTA, (iii) intracellular accumulation of chloramphenicol in presence of an energy uncoupler of pump energy, (iv) and immunodetection of porins and evaluation of porin trimers thermostability.Results indicated that 9 strains presented significant efflux mechanisms overexpression, among them 8 were resistant to colistin and polymyxin B due to a modification of LPS structure as evidenced by EDTA effect and silver staining electrophoresis. The high resistant strains to colistin and polymyxin exhibited identical LPS patterns. Studies of E. coli porins indicated that the majority of strains didn't show modification in their amount, however analysis of porin thermostability showed that porin trimers of some resistant strains were relatively heat-labile, suggesting a misassembly of the functional trimer. The multidrug resistance (MDR) phenotypes detected in these selected ETEC corresponded to association of LPS modifications, abordive assembly of porin trimers and active efflux which drastically alter the antibiotic activity currently used to combat enteric infections caused by this pathogen.

Comparative evaluation of the UMIC Colistine kit to assess MIC of colistin of gram-negative rods

BackgroundThe recent description of the first plasmid-mediated colistin-resistant gene mcr-1, conferring transferable and low-level resistance to colistin, raised concern about the need to implement a rapid and reliable screening method to detect colistin-resistant clinical isolates. The only valid method to assess the MIC of colistin is the broth microdilution according to the joint CLSI-EUCAST Polymyxin Breakpoints Working Group. UMIC Colistine is a ready-to-use broth microdilution kit developed to easily assess colistin MIC by proposing unitary polystyrene strips containing 11 concentrations of dehydrated colistin. Here, we evaluated the UMIC Colistine kit on 235 Gram-negative rods (176 Enterobacterales, including 70 harboring a mcr gene, and 59 non-fermentative), through comparison to the reference broth microdilution method prepared in accordance with EN ISO 20776-1:2006 standard. Reproducibility of the UMIC Colistine was assayed with the three recommended quality control strains E. coli ATCC 25922, E. coli NCTC 13846 (mcr-1 positive), and P. aeruginosa ATCC 27853, as for stability testing.ResultsCategorical agreement was 100% with 63.4% (n = 149) of colistin-resistant strains, and 36.6% (n = 86) of colistin-susceptible strains with both methods (S ≤ 2 μg/mL and R > 2 μg/mL). No major error or very major error was reported. Essential agreement was 94.0% (n = 221), and 100% for detection of colistin-resistant strains as compared to the reference method. Pearson’s correlation between UMIC Colistine and the reference method was 0.98. Reproducibility of the UMIC Colistine system was 97.8% with MICs of the quality control strains within the target ranges. However, some isolates had lower MIC with UMIC Colistine, but that did not change their categorization as colistin-susceptible, and this phenomenon should be further explored.ConclusionsThe UMIC Colistine kit is an easy to perform unitary device that showed excellent results when compared to the reference method. The UMIC Colistine system is a rapid and reliable broth microdilution method that is suitable to assess the colistin MIC of clinical isolates in clinical microbiology laboratories.

Investigation of Novel pmrB and eptA Mutations in Isogenic Acinetobacter baumannii Isolates Associated with Colistin Resistance and Increased Virulence In Vivo.

Colistin resistance in Acinetobacter baumannii is of great concern and is a threat to human health. In this study, we investigate the mechanisms of colistin resistance in four isogenic pairs of A. baumannii isolates displaying an increase in colistin MICs. A mutation in pmrB was detected in each colistin-resistant isolate, three of which were novel (A28V, I232T, and ΔL9-G12). Increased expression of pmrC was shown by semi-quantitative reverse transcription-PCR (qRT-PCR) for three colistin-resistant isolates, and the addition of phosphoethanolamine (PEtN) to lipid A by PmrC was revealed by mass spectrometry. Interestingly, PEtN addition was also observed in some colistin-susceptible isolates, indicating that this resistance mechanism might be strain specific and that other factors could contribute to colistin resistance. Furthermore, the introduction of pmrAB carrying the short amino acid deletion ΔL9-G12 into a pmrAB knockout strain resulted in increased pmrC expression and lipid A modification, but colistin MICs remained unchanged, further supporting the strain specificity of this colistin resistance mechanism. Of note, a mutation in the pmrC homologue eptA and a point mutation in ISAba1 upstream of eptA were associated with colistin resistance and increased eptA expression, which is a hitherto undescribed resistance mechanism. Moreover, no cost of fitness was observed for colistin-resistant isolates, while the virulence of these isolates was increased in a Galleria mellonella infection model. Although the mutations in pmrB were associated with colistin resistance, PEtN addition appears not to be the sole factor leading to colistin resistance, indicating that the mechanism of colistin resistance is far more complex than previously suspected and is potentially strain specific.