A novel homozygous synonymous splicing variant in SELENOI gene causes spastic paraplegia 81.

BACKGROUND: Hereditary spastic paraplegia 81 is a recently identified, rare autosomal recessive disease, caused by biallelic pathogenic variants in the SELENOI gene, with only two families reported to date. The features documented in the two previous affected families include sensorineural deafness, blindness, cleft palate, delayed motor development, regression of motor skills, impaired intellectual development, poor speech and language acquisition, spasticity, hyperreflexia, white matter abnormalities and cerebral and cerebellar atrophy. METHODS: In the present study, we performed exome sequencing analysis in a single family with two affected siblings to identify the genetic cause of complicated hereditary spastic paraplegia. The results were further confirmed by Sanger sequencing, cDNA analysis and 3D protein modelling. RESULTS: Exome sequencing identified a homozygous, synonymous variant in the SELENOI gene (NM_033505.4:c.126G>A:p.(Lys42Lys)) in both of the siblings. Sanger sequencing confirmed the heterozygous status in both parents consistent with the autosomal recessive inheritance. This variant has been found to disrupt normal splicing and lead to skipping of exon 2, causing in-frame deletion of SELENOI N-terminal 23 amino acids [NM_033505.4:c.57_126del:p.(Tyr20_Lys42del)] and further leading to structural changes in the protein. CONCLUSIONS: We report a novel homozygous synonymous variant in the SELENOI gene causing abnormal splicing in two patients affected with hereditary spastic paraplegia 81. This report further expands the phenotypic and genotypic spectrum of hereditary spastic paraplegia 81.

Emergence of colistin-resistant Acinetobacter modestus harbouring the intrinsic phosphoethanolamine transferase EptA.

OBJECTIVES: Colistin-resistant Gram-negative pathogens have become a serious worldwide medical problem. This study was designed to reveal the effects of an intrinsic phosphoethanolamine transferase from Acinetobacter modestus on Enterobacterales. METHODS: A strain of colistin-resistant A. modestus was isolated from a sample of nasal secretions taken in 2019 from a hospitalised pet cat in Japan. The whole genome was sequenced by next generation sequencing, and transformants of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae harbouring the phosphoethanolamine transferase-encoding gene from A. modestus were constructed. Lipid A modification in E. coli transformants was analysed using electrospray ionization mass spectrometry. RESULTS: Sequencing of the entire genome revealed that the isolate harboured a phosphoethanolamine transferase-encoding gene, eptA_AM, on its chromosome. Transformants of E. coli, K. pneumoniae, and E. cloacae harbouring both the promoter and eptA_AM gene from A. modestus had 32-fold, 8-fold, and 4-fold higher minimum inhibitory concentrations (MICs) for colistin, respectively, than transformants harbouring a control vector. The genetic environment surrounding eptA_AM in A. modestus was similar to that surrounding eptA_AM in Acinetobacter junii and Acinetobacter venetianus. Electrospray ionization mass spectrometry analysis revealed that EptA_AM modified lipid A in Enterobacterales. CONCLUSION: This is the first report to describe the isolation of an A. modestus strain in Japan and show that its intrinsic phosphoethanolamine transferase, EptA_AM, contributes to colistin resistance in Enterobacterales and A. modestus.

Emergence of a highly colistin-resistant Aeromonas jandaei clinical isolate harbouring four genes encoding phosphoethanolamine transferases in Nepal.

OBJECTIVES: This study aimed to describe a clinical isolate of Aeromonas jandaei (A. jandaei) in Nepal that harboured four types of genes encoding phosphoethanolamine transferases. METHODS: An isolate of colistin-resistant A. jandaei was obtained from a blood sample of an inpatient in a hospital in Nepal, and its complete genome sequence was determined. Escherichia coli (E. coli) and Aeromonas hydrophila (A. hydrophila) transformants expressing genes encoding novel phosphoethanolamine transferase variants were constructed and colistin-susceptibility profiles were determined. RESULTS: The isolate harboured four genes encoding phosphoethanolamine transferases on the chromosome, which were designated eptAv3.2, eptAv3.3, eptAv3.4 and eptAv7.2. The amino acid sequences of EptAv3.2, 3.3 and 3.4 were > 80% identical to MCR-3.1, and that of EptAv7.2 was > 79% identical to MCR-7.1. E. coli expressing eptAv3.2, 3.3 and 3.4 showed reduced susceptibility to colistin, whereas E. coli expressing eptAv7.2 did not. In contrast, A. hydrophila expressing eptAv7.2 showed reduced susceptibility to colistin, whereas A. hydrophila expressing eptAv3.2, 3.3 and 3.4 did not; eptAv3.3 and 3.4 formed a tandem structure. The genomic environments surrounding eptAv3.2, 3.3 and 3.4 were similar to Aeromonas veronii obtained from the effluent of a treatment plant in Japan in 2018. The genomic environment surrounding eptAv7.2 was similar to that of A. jandaei obtained from a chicken in the USA in 2019. CONCLUSIONS: The highly colistin-resistant A. jandaei clinical isolate harboured four chromosomal genes encoding phosphoethanolamine transferases, suggesting that Aeromonas spp. harbouring eptAv genes with strong similarities to mcr-3 and mcr-7 are emerging in medical settings as well as environments.

Biophysical Impact of Lipid A Modification Caused by Mobile Colistin Resistance Gene on Bacterial Outer Membranes.

Expression of mobile colistin resistance gene mcr-1 results in the addition of phosphoethanolamine (pEtN) to the lipid A headgroup in the bacterial outer membrane (OM) of Gram-negative bacteria, increasing the resistance to the last-line polymyxins. However, the potential biological consequences of such modification remain unclear. Using coarse-grained molecular simulations with quantitative lipidomics models, we discovered pEtN modification of the lipid A headgroup caused substantial changes to the morphology and physicochemical properties of the OM. Single-lipid level structural and energetic analyses revealed that this modification resulted in lipid A-pEtN adopting an abnormally twisted and slanted conformation with a closer packing state because of strengthened inter-lipid attraction. The consequent accumulation of lipid A-pEtN produced a negative curvature of the OM and altered the membrane's tension, fluidity, and rigidity. Our results provide a key mechanistic connection between mcr-1 expression and biophysical changes in the bacterial OM.

Reduction trend of mcr-1 circulation in Emilia-Romagna Region, Italy.

This study aims to describe trends of mcr-positive Enterobacterales in humans based on laboratory surveillance with a defined catchment population. The data source is the Micro-RER surveillance system, established in Emilia-Romagna region (Italy), to monitor the trend of mcr resistance. Enterobacterales isolates from human clinical samples with minimum inhibitory concentration (MIC) ≥ 2 mg/L for colistin were sent to the study reference laboratory for the detection of mcr genes. Isolates prospectively collected in the period 2018-2020 were considered for the assessment of population rates and trends; further analyses were carried out for the evaluation of clonality and horizontal mcr gene transfer. Previous isolates from local laboratory collection were also described. In the period 2018-2020, 1164 isolates were sent to the reference laboratory, and 51 (4.4%) were confirmed as mcr-positive: 50 mcr-1 (42 Escherichia coli, 6 Klebsiella pneumoniae, 2 Salmonella enterica) and 1 mcr-4 (Enterobacter cloacae). The number of mcr-positive isolates dropped from 24 in the first half of 2018 to 3 in the whole of 2020 (trend p value < 0.001). Genomic analyses showed the predominant role of the horizontal transfer of mcr genes through plasmids or dissemination of transposable elements compared to clonal dissemination of mcr-positive microorganisms. The study results demonstrate a substantial decrease in the circulation of mcr-1 plasmid genes in Emilia-Romagna Region.

Molecular genetic characteristics of mcr-9-harbouring Salmonella enterica serotype Typhimurium isolated from raw milk.

Among the 10 reported mcr genes, mcr-9 was first identified in Salmonella enterica serotype Typhimurium, which is a leading cause of foodborne illness worldwide. However, information about the prevalence and genetic features of mcr-9 is still lacking, especially among food samples. This study reports the presence of mcr-9 in raw milk samples from China; the prevalence rate was low (0.83%, 1/120). mcr-9 was located on a transferable plasmid, and was stable in wild-type S. enterica. However, it had a biological fitness cost when transferred to an Escherichia coli recipient. Whole-genome sequencing revealed that mcr-9 was located on the IncHI2A-type plasmid, and was surrounded by IS903B and IS26 in its flanking regions. The mcr-9-carrying S. enterica 19SE belonged to ST26 and had a multi-drug-resistant phenotype. It was confirmed that mcr-9 did not mediate colistin resistance in this study, indicating that its transfer may not facilitate the dissemination of colistin resistance.

Global impact of mcr-1-positive Enterobacteriaceae bacteria on "one health".

Polymyxins, especially polymyxin B and polymyxin E (colistin), are considered to be the last line of defence against infections caused by multi-drug-resistant (MDR) gram-negative bacteria such as carbapenem-resistant Enterobacteriaceae (CRE). However, the recent emergence and dissemination of the plasmid-mediated colistin resistance gene mcr-1 and its variants pose a serious challenge to public health and the livestock industry. This review describes the prevalence and dissemination of mcr-1-positive isolates from different sources, including animals (food animals, pet animals and wildlife), humans (healthy populations and patients) and the environment (farms, urban and rural communities and natural environments) based on existing epidemiological studies of mcr-1 and MCR-1-producing Enterobacteriaceae bacteria around the world. The major mechanisms of mcr-1 transmission across humans, animals and the environment are discussed.

Lipid A Phosphoethanolamine Transferase: Regulation, Structure and Immune Response.

A wide variety of antibiotics are targeted to the bacterial membrane due to its unique arrangement and composition relative to the host mammalian membranes. By modification of their membranes, some gram-negative pathogens resist the action of antibiotics. Lipid A phosphoethanolamine transferase (EptA) is an intramembrane enzyme that modifies the lipid A portion of lipopolysaccharide/lipooligosaccharide by the addition of phosphoethanolamine. This modification reduces the overall net-negative charge of the outer membrane of some gram-negative bacteria, conferring resistance to polymyxin. This resistance mechanism has resulted in a global public health issue due to the increased use of polymyxin as last-resort antibiotic treatments against multi-drug-resistant pathogens. Studies show that, without EptA, pathogenic bacteria become more sensitive to polymyxin and to clearance by the host immune system, suggesting the importance of this target enzyme for the development of novel therapeutic agents. In this review, EptA will be discussed comprehensively. Specifically, this review will cover the regulation of eptA expression by the two component systems PmrA/PmrB and PhoP/PhoQ, the site of modification on lipid A, the structure and catalytic mechanism of EptA in comparison to MCR-1 and Escherichia coli alkaline phosphatase, and the host immune system's response to lipid A modification by EptA. The overarching aim of this review is to provide a comprehensive overview of polymyxin resistance mediated by EptA.

Co-existence of two novel phosphoethanolamine transferase gene variants in Aeromonas jandaei from retail fish.

Two novel phosphoethanolamine transferase genes, eptAv7 and eptAv3, were identified in the chromosome of an Aeromonas jandaei isolate from retail fish. The variants showed 79.9% and 80.0% amino acid identity to MCR-7.1 and MCR-3.1, respectively, and increased colistin resistance 128- to 256-fold in Aeromonas salmonicida. The two variants with no mobile genetic element in the flanking regions were also observed in other Aeromonas species. This finding supports the view that Aeromonas is a reservoir for MCR-3 and MCR-7 mobile colistin resistance.

Reduced Fitness Costs of mcr-1.2 Compared to Mutated pmrB in Isogenic Colistin-Resistant KPC-3-Producing Klebsiella pneumoniae.

In the present study, we provide the results of a detailed genomic analysis and the growth characteristics of a colistin-resistant KPC-3-producing Klebsiella pneumoniae sequence type 512 (ST512) isolate (the colR-KPC3-KP isolate) with a mutated pmrB and isogenic isolates of colR-KPC3-KP with mcr-1.2 isolated from an immunocompromised patient. From 2014 to 2017, four colR-KPC3-KP isolates were detected in rectal swab samples collected from a pediatric hematology patient at the Azienda Ospedaliero-Universitaria Pisana in Pisa, Italy. Whole-genome sequencing was performed by MiSeq sequencing (Illumina). Growth experiments were performed using different concentrations of colistin. The growth lag phases both of an isolate harboring a deletion in pmrB and of clonal variants with mcr-1.2 were assessed by the use of real-time light-scattering measurements. In the first isolate (isolate 1000-pmrBΔ, recovered in September 2014), a 17-nucleotide deletion in pmrB was detected. In subsequent isolates, the mcr-1.2 gene associated with the plasmid pIncX4-AOUP was found, while pmrB was intact. Additionally, plasmid pIncQ-AOUP, harboring aminoglycoside resistance genes, was detected. The growth curves of the first three isolates were identical without colistin exposure; however, at higher concentrations of colistin, the growth curves of the isolate with a deletion in pmrB showed longer lag phases. We observed the replacement of mutated colR-KPC3-KP pmrB by isogenic isolates with multiple resistance plasmids, including mcr-1.2-carrying pIncX4, probably due to coselection under gentamicin treatment in a patient with prolonged colR-KPC3-KP carriage. The carriage of these isolates persisted in follow-up cultures. Coselection and the advantages in growth characteristics suggest that the plasmid-mediated resistance conferred by mcr has fewer fitness costs in colR-KPC3-KP than mutations in chromosomal pmrB, contributing to the success of this highly resistant hospital-adapted epidemiological lineage.IMPORTANCE Our study shows a successful prolonged human colonization by a colistin-resistant Klebsiella pneumoniae isolate harboring mcr-1.2 An intense antibiotic therapy contributed to the maintenance of this microorganism through the acquisition of new resistance genes. The isolates carrying mcr-1.2 showed fewer fitness costs than isogenic isolates with a pmrB mutation in the chromosome. Coselection and reduced fitness costs may explain the replacement of isolates with the pmrB mutation by other isolates and the ability of the microorganism to persist despite antibiotic treatment.

Polymyxin resistance in Klebsiella pneumoniae: multifaceted mechanisms utilized in the presence and absence of the plasmid-encoded phosphoethanolamine transferase gene mcr-1.

OBJECTIVES: Until plasmid-mediated mcr-1 was discovered, it was believed that polymyxin resistance in Gram-negative bacteria was mainly mediated by the chromosomally-encoded EptA and ArnT, which modify lipid A with phosphoethanolamine (pEtN) and 4-amino-4-deoxy-l-arabinose (l-Ara4N), respectively. This study aimed to construct a markerless mcr-1 deletion mutant in Klebsiella pneumoniae, validate a reliable reference gene for reverse transcription quantitative PCR (RT-qPCR) and investigate the interactions among mcr-1, arnT and eptA, in response to polymyxin treatments using pharmacokinetics/pharmacodynamics (PK/PD). METHODS: An isogenic markerless mcr-1 deletion mutant (II-503Δmcr-1) was generated from a clinical K. pneumoniae II-503 isolate. The efficacy of different polymyxin B dosage regimens was examined using an in vitro one-compartment PK/PD model and polymyxin resistance was assessed using population analysis profiles. The expression of mcr-1, eptA and arnT was examined using RT-qPCR with a reference gene pepQ, and lipid A was profiled using LC-MS. In vivo polymyxin B efficacy was investigated in a mouse thigh infection model. RESULTS: In K. pneumoniae II-503, mcr-1 was constitutively expressed, irrespective of polymyxin exposure. Against II-503Δmcr-1, an initial bactericidal effect was observed within 4 h with polymyxin B at average steady-state concentrations of 1 and 3 mg/L, mimicking patient PK. However, substantial regrowth and concomitantly increased expression of eptA and arnT were detected. Predominant l-Ara4N-modified lipid A species were detected in II-503Δmcr-1 following polymyxin B treatment. CONCLUSIONS: This is the first study demonstrating a unique markerless deletion of mcr-1 in a clinical polymyxin-resistant K. pneumoniae. The current polymyxin B dosage regimens are suboptimal against K. pneumoniae, regardless of mcr, and can lead to the emergence of resistance.

mcr-9, an Inducible Gene Encoding an Acquired Phosphoethanolamine Transferase in Escherichia coli, and Its Origin.

The plasmid-located mcr-9 gene, encoding a putative phosphoethanolamine transferase, was identified in a colistin-resistant human fecal Escherichia coli strain belonging to a very rare phylogroup, the D-ST69-O15:H6 clone. This MCR-9 protein shares 33% to 65% identity with the other plasmid-encoded MCR-type enzymes identified (MCR-1 to -8) that have been found as sources of acquired resistance to polymyxins in Enterobacteriaceae Analysis of the lipopolysaccharide of the MCR-9-producing isolate revealed a function similar to that of MCR-1 by adding a phosphoethanolamine group to lipid A and subsequently modifying the structure of the lipopolysaccharide. However, a minor impact on susceptibility to polymyxins was noticed once the mcr-9 gene was cloned and produced in an E. coli K-12-derived strain. Nevertheless, we showed here that subinhibitory concentrations of colistin induced the expression of the mcr-9 gene, leading to increased MIC levels. This inducible expression was mediated by a two-component regulatory system encoded by the qseC and qseB genes located downstream of mcr-9 Genetic analysis showed that the mcr-9 gene was carried by an IncHI2 plasmid. In silico analysis revealed that the plasmid-encoded MCR-9 shared significant amino acid identity (ca. 80%) with the chromosomally encoded MCR-like proteins from Buttiauxella spp. In particular, Buttiauxella gaviniae was found to harbor a gene encoding MCR-BG, sharing 84% identity with MCR-9. That gene was neither expressed nor inducible in its original host, which was fully susceptible to polymyxins. This work showed that mcr genes may circulate silently and remain undetected unless induced by colistin.

microRNA-16-5p promotes 3T3-L1 adipocyte differentiation through regulating EPT1.

Adipogenesis is an organized process of cellular differentiation by which pre-adipocytes differentiate towards mature adipocytes. miR-16-5p has been reported to be involved in cell proliferation, apoptosis, differentiation and angiogenesis. However little is known about miR-16-5p functional role in 3T3-L1 adipocyte differentiation. In this study, we found that miRNA-16-5p was significantly upregulated during 3T3-L1 preadipocytes differentiation towards mature adipocytes. Over-expression of miRNA-16-5p promoted mature adipocytes specific genes expression and fat droplet accumulation in vitro and in vivo. Meanwhile we have identified EPT1 as the target gene of miRNA-16-5p. Taken together, our data provided evidence to support that miRNA-16-5p promotes adipocyte differentiation by suppressing EPT1.

Colistin-Resistant mcr-Positive Enterobacteriaceae in Fresh Vegetables, an Increasing Infectious Threat in China.

The presence of mobilized colistin resistance (mcr) genes is a global concern. However, data concerning mcr in fresh vegetables, a reservoir for antibiotic resistance genes, are still rare. In this study, mcr genes were analysed in 528 vegetable samples from 53 supermarkets or farmer's markets in 23 cities of 9 provinces in China, and the mcr-positive Enterobacteriaceae were characterized. Nineteen (3.6%) samples carried one or more mcr-positive isolates, and the highest three detection rates were found in carrot, pak choi and green pepper. Twenty-four mcr-1-positive isolates (23 Escherichia coli and one Enterobacter cloacae) were obtained, and E. coli isolates showed high genetic diversity. Different multilocus sequence type (MLST) isolates were also observed within the same sample. All 24 isolates showed multidrug resistance, and 14 carried blaCTX-M genes. Most isolates harbored similarly conjugative IncX4-type (∼33 kb) or IncI2-type (∼60 kb) mcr-1-bearing plasmids. The sequenced prevalent IncX4 plasmid and IncI2 plasmid from tomato were similar to the relevant plasmids from animals and clinical isolates in various countries. mcr-1-bearing IncHI2/ST3 plasmid highly similar to that carrying 14 resistance genes from E. coli of chicken was also observed. In conclusion, a high prevalence of mcr-1 in fresh vegetables was found in China, and the dissemination of mcr-1 was mediated by similar IncX4 or IncI2 plasmids. The plasmids from vegetables showed high similarity to plasmids from clinical isolates, indicating MCR-1-producers in ready-to-eat vegetables may pose a huge threat to public health and measures need to be taken to ensure food safety.

Detection of Enterobacterales resistant to polymyxins using Rapid Polymyxins NP test.

Two hundred isolates of Enterobacterales were tested by Rapid Polymyxins NP for the detection of polymyxin resistance and compared to the reference test broth microdilution (BMD). The sensitivity and specificity of the NP test were 98% and the results are faster than the BMD, decreasing from approximately 24 to 2 h.

Characterization of mcr-5-Harboring Salmonella enterica subsp. enterica Serovar Typhimurium Isolates from Animal and Food Origin in Germany.

We characterized eight mcr-5-positive Salmonella enterica subsp. enterica serovar Typhimurium sequence type 34 (ST34) isolates obtained from pigs and meat in Germany. Five plasmid types were identified harboring mcr-5 on Tn6452 or putative mobile insertion cassettes. The mobility of mcr-5 was confirmed by integration of Tn6452 into the bacterial chromosomes of two strains and the detection of conjugative mcr-5 plasmids. The association with mobile genetic elements might further enhance mcr-5 distribution.

PmrC (EptA) and CptA Negatively Affect Outer Membrane Vesicle Production in Citrobacter rodentium.

Outer membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria by a bulging of the outer membrane (OM) and subsequent release into the environment. By serving as vehicles for various cargos, including proteins, nucleic acids and small metabolites, OMVs are central to interbacterial interactions and both symbiotic and pathogenic host bacterial interactions. However, despite their importance, the mechanism of OMV formation remains unclear. Recent evidence indicates that covalent modifications of lipopolysaccharides (LPS) influence OMV biogenesis. Several enteric bacteria modify LPS with phosphoethanolamine (pEtN) using the iron-regulated PmrC (EptA) and CptA pEtN transferases. In wild-type Citrobacter rodentium, the presence of increasing subtoxic concentrations of iron was found to stimulate OMV production 4- to 9-fold above baseline. C. rodentium uses the two-component system PmrAB to sense and adapt to environmental iron. Compared to the wild type, the C. rodentium ΔpmrAB strain exhibited heightened OMV production at similar iron concentrations. PmrAB regulates transcription of pmrC (also known as eptA) and cptA OMV production in strains lacking either pmrC (eptA) or cptA was similarly increased in comparison to that of the wild type. Importantly, plasmid complementation of C. rodentium strains with either pmrC (eptA) or cptA resulted in a drastic inhibition of OMV production. Finally, we showed that ß-lactamase and CroP, two enzymes found in the C. rodentium periplasm and outer membrane (OM), respectively, are associated with OMVs. These data suggest a novel mechanism by which C. rodentium and possibly other Gram-negative bacteria can negatively affect OMV production through the PmrAB-regulated genes pmrC (eptA) and cptAIMPORTANCE Although OMVs secreted by Gram-negative bacteria fulfill multiple functions, the molecular mechanism of OMV biogenesis remains ill defined. Our group has previously shown that PmrC (also known as EptA) and CptA maintain OM integrity and provide resistance to iron toxicity and antibiotics in the murine pathogen Citrobacter rodentium In several enteric bacteria, these proteins modify the lipid A and core regions of lipopolysaccharide with phosphoethanolamine moieties. Here, we show that these proteins also repress OMV production in response to environmental iron in C. rodentium These data support the emerging understanding that lipopolysaccharide modifications are important regulators of OMV biogenesis in Gram-negative bacteria.

Investigation of carbapenemase and mcr-1 genes in carbapenem-resistant Klebsiella pneumoniae isolates.

INTRODUCTION: Carbapenem-resistant Klebsiella pneumoniae are a major problem. We aimed to investigate carbapenemase-encoding genes and transferable mcr-1 genes among 57 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates from hospitalized patients. METHODOLOGY: Antibiotic susceptibility tests were performed by Phoenix (BD). Results for ertapenem and colistin were confirmed by gradient diffusion and microdilution methods. Carbapenemase and mcr-1 genes were investigated by Polymerase Chain Reaction (PCR). RESULTS: Thirty-two (56.14%) isolates were from intensive care units (ICU). Antibiotic resistance rates by Phoenix: 52.63% for amikacin; 73.69% trimethoprim sulfamethoxazole; 91.23% cefepime; 82.46% tigecycline; 59.65% colistin. Carbapenemases positivity: 82.45% (47) for blaOXA-48, 40.35% (23) blaOXA-55, 3.50% (2) blaOXA-51, 1.75% (1) blaOXA-23, 1.75% (1) blaOXA-24, 1.75% (1) blaIMP. blaOXA-58, blaKPC, blaNDM-1, and blaVIM were not detected. Twenty (35.08%) isolates had both blaOXA-48 and blaOXA-55. Three isolates were mcr-1 (+) and blaOXA-48 (+). One mcr-1 (+) isolates was blaOXA-51 (+). One colistin sensitive isolate determined by Phoenix, was found colistin resistant by microdilution. CONCLUSION: OXA-48 and OXA-55 co-harboring isolates and mcr-1 gene (+) isolates were spreading. Automated colistin susceptibility results should be confirmed by microdilution method. Resistance mechanisms in Enterobacteriaceae should be determined and the isolates should be monitored by molecular epidemiological methods. Effective infection control measures will contribute to reduce risk of antibiotic resistant bacterial infections and dissemination of antibiotic resistance.