Mechanisms Of Colistin Resistance Research Articles

A Review on Colistin Resistance: An Antibiotic of Last Resort

Antibiotic resistance has emerged as a significant global public health issue, driven by the rapid adaptation of microorganisms to commonly prescribed antibiotics. Colistin, previously regarded as a last-resort antibiotic for treating infections caused by Gram-negative bacteria, is increasingly becoming resistant due to chromosomal mutations and the acquisition of resistance genes carried by plasmids, particularly the mcr genes. The mobile colistin resistance gene (mcr-1) was first discovered in E. coli from China in 2016. Since that time, studies have reported different variants of mcr genes ranging from mcr-1 to mcr-10, mainly in Enterobacteriaceae from various parts of the world, which is a major concern for public health. The co-presence of colistin-resistant genes with other antibiotic resistance determinants further complicates treatment strategies and underscores the urgent need for enhanced surveillance and antimicrobial stewardship efforts. Therefore, understanding the mechanisms driving colistin resistance and monitoring its global prevalence are essential steps in addressing the growing threat of antimicrobial resistance and preserving the efficacy of existing antibiotics. This review underscores the critical role of colistin as a last-choice antibiotic, elucidates the mechanisms of colistin resistance and the dissemination of resistant genes, explores the global prevalence of mcr genes, and evaluates the current detection methods for colistin-resistant bacteria. The objective is to shed light on these key aspects with strategies for combating the growing threat of resistance to antibiotics.

Influence of PhoPQ and PmrAB two component system alternations on colistin resistance from non-mcr colistin resistant clinical E. Coli strains

BackgroundThe current understanding of acquired chromosomal colistin resistance mechanisms in Enterobacterales primarily involves the disruption of the upstream PmrAB and PhoPQ two-component system (TCS) control caused by mutations in the regulatory genes. Interestingly, previous studies have yielded conflicting results regarding the interaction of regulatory genes related to colistin resistance in Escherichia coli, specifically those surrounding PhoPQ and PmrAB TCS.ResultsIn our study, we focused on two clinical non-mcr colistin-resistant strains of E. coli, TSAREC02 and TSAREC03, to gain a better understanding of their resistance mechanisms. Upon analysis, we discovered that TSAREC02 had a deletion (Δ27–45) in MgrB, as well as substitutions (G206R, Y222H) in PmrB. On the other hand, TSAREC03 exhibited a long deletion (Δ84–224) in PhoP, along with substitutions (M1I, L14P, P178S, T235N) in PmrB. We employed recombinant DNA techniques to explore the interaction between the PhoPQ and PmrAB two-component systems (TCSs) and examine the impact of the mutated phoPQ and pmrB genes on the minimum inhibitory concentrations (MICs) of colistin. We observed significant changes in the expression of the pmrD gene, which encodes a connector protein regulated by the PhoPQ TCS, in the TSAREC02 wild-type (WT)-mgrB replacement mutant and the TSAREC03 WT-phoP replacement mutant, compared to their respective parental strains. However, the expressions of pmrB/pmrA, which reflect PmrAB TCS activity, and the colistin MICs remained unchanged. In contrast, the colistin MICs and pmrB/pmrA expression levels were significantly reduced in the pmrB deletion mutants from both TSAREC02 and TSAREC03, compared to their parental strains. Moreover, we were able to restore colistin resistance and the expressions of pmrB/pmrA by transforming a plasmid containing the parental mutated pmrB back into the TSAREC02 and TSAREC03 mutants, respectively.ConclusionWhile additional data from clinical E. coli isolates are necessary to validate whether our findings could be broadly applied to the E. coli population, our study illuminates distinct regulatory pathway interactions involving colistin resistance in E. coli compared to other species of Enterobacterales. The added information provided by our study contribute to a deeper understanding of the complex pathway interactions within Enterobacterales.

Carbapenem-resistant Klebsiella pneumoniae in the Balkans: Clonal distribution and associated resistance determinants.

Carbapenems are considered to be among the last line antibiotics against extended-spectrum β-lactamase producing Enterobacterales. Carbapenem-resistant Klebsiella pneumoniae (CRKP) has been frequently reported and its spread in Europe is indisputable and poses an enormous threat to hospitalized patients which is of growing concern. This review aims to record prevalence of CRKP in the Balkan region and to review the current knowledge about this life-threatening pathogen. In this review, we summarize data about clinical isolates of carbapenem-resistant K. pneumoniae from Greece, Croatia, Romania, Bulgaria, Serbia, Slovenia, Montenegro, Bosnia-Herzegovina and Albania from published reports between 2000 and 2023. Among Balkan countries, Greece and Romania are the ones with the most reports about CRKP. Since 2007, KPCs are the dominant carbapenemases in both countries. KPC-2 and NDM-1-producing K. pneumoniae strains have been identified as the most frequent CRKP in Croatia, Bulgaria, Serbia, and Slovenia. OXA-48 enzyme has been identified in most Balkan countries. In addition, since 2018, CRKP sequence type 11 (ST11) seems to have replaced ST258 in Balkan Peninsula, while ST15 continues to thrive throughout the years. Not only efficacy of colistin against CRKP has decreased dramatically during the last ten years but colistin resistance mechanism is based on alterations of chromosomal mgrB gene, rather than the already known mcr genes.Moreover, ceftazidime-avibactam-resistant CRKP were detected mostly in Greece. Emergence of CRKP poses a severe threat to the Balkan countries. Due to the narrow therapeutic window, it is essential to prevent the spread of multiresistant K. pneumoniae strains.

Comprehensive study reveals phenotypic heterogeneity in Klebsiella pneumoniae species complex isolates

Here, we conducted a comprehensive analysis of 356 Klebsiella pneumoniae species complex (KpSC) isolates that were classified as classical (cl), presumptive hypervirulent (p-hv) and hypermucoviscous-like (hmv-like). Overall, K. pneumoniae (82.3%), K. variicola (2.5%) and K. quasipneumoniae (2.5%) were identified. These isolates comprised 321 cl-KpSC, 7 p-hv-KpSC and 18 hmv-like-KpSC. A large proportion of cl-KpSC isolates were extended-spectrum-β-lactamases (ESBLs)-producers (64.4%) and 3.4% of isolates were colistin-resistant carrying carbapenemase and ESBL genes. All p-hv-KpSC showed an antibiotic susceptible phenotype and hmv-like isolates were found to be ESBL-producers (8/18). Assays for capsule production and capsule-dependent virulence phenotypes and whole-genome sequencing (WGS) were performed in a subset of isolates. Capsule amount differed in all p-hv strains and hmv-like produced higher capsule amounts than cl strains; these variations had important implications in phagocytosis and virulence. Murine sepsis model showed that most cl strains were nonlethal and the hmv-like caused 100% mortality with 3 × 108 CFUs. Unexpectedly, 3/7 (42.9%) of p-hv strains required 108 CFUs to cause 100% mortality (atypical hypervirulent), and 4/7 (57.1%) strains were considered truly hypervirulent (hv). Genomic analyses confirmed the diverse population, including isolates belonging to hv clonal groups (CG) CG23, CG86, CG380 and CG25 (this corresponded to the ST3999 a novel hv clone) and MDR clones such as CG258 and CG147 (ST392) among others. We noted that the hmv-like and hv-ST3999 isolates showed a close phylogenetic relationship with cl-MDR K. pneumoniae. The information collected here is important to understand the evolution of clinically important phenotypes such as hypervirulent and ESBL-producing-hypermucoviscous-like amongst the KpSC in Mexican healthcare settings. Likewise, this study shows that mgrB inactivation is the main mechanism of colistin resistance in K. pneumoniae isolates from Mexico.

Molecular epidemiology and mechanisms of carbapenem and colistin resistance in Klebsiella and other Enterobacterales from treated wastewater in Croatia

Among the most problematic bacteria with clinical relevance are the carbapenem-resistant Enterobacterales (CRE), as there are very limited options for their treatment. Treated wastewater can be a route for the release of these bacteria into the environment and the population. The aim of this study was to isolate CRE from treated wastewater from the Zagreb wastewater treatment plant and to determine their phenotypic and genomic characteristics. A total of 200 suspected CRE were isolated, 148 of which were confirmed as Enterobacterales by MALDI-TOF MS. The predominant species was Klebsiella spp. (n = 47), followed by Citrobacter spp. (n = 40) and Enterobacter cloacae complex (cplx.) (n = 35). All 148 isolates were carbapenemase producers with a multidrug-resistant phenotype. Using multi-locus sequence typing and whole-genome sequencing (WGS), 18 different sequence types were identified among these isolates, 14 of which were associated with human-associated clones. The virulence gene analysis of the sequenced Klebsiella isolates (n = 7) revealed their potential pathogenicity. PCR and WGS showed that the most frequent carbapenemase genes in K. pneumoniae were blaOXA-48 and blaNDM-1, which frequently occurred together, while blaKPC-2 together with blaNDM-1 was mainly detected in K. oxytoca, E. cloacae cplx. and Citrobacter spp. Colistin resistance was observed in 40% of Klebsiella and 57% of Enterobacter isolates. Underlying mechanisms identified by WGS include known and potentially novel intrinsic mechanisms (point mutations in the pmrA/B, phoP/Q, mgrB and crrB genes) and acquired mechanisms (mcr-4.3 gene). The mcr-4.3 gene was identified for the first time in K. pneumoniae and is probably located on the conjugative IncHI1B plasmid. In addition, WGS analysis of 13 isolates revealed various virulence genes and resistance genes to other clinically relevant antibiotics as well as different plasmids possibly associated with carbapenemase genes. Our study demonstrates the important role that treated municipal wastewater plays in harboring and spreading enterobacterial pathogens that are resistant to last-resort antibiotics.

Molecular Characteristics of Colistin Resistance in Acinetobacter baumannii and the Activity of Antimicrobial Combination Therapy in a Tertiary Care Medical Center in Lebanon.

(1) Background: Infections with pan-drug-resistant (PDR) bacteria, such as A. baumannii, are becoming increasingly common, especially in healthcare facilities. In this study, we selected 15 colistin-resistant clinical A. baumannii isolates from a hospital in Beirut, Lebanon, to test combination therapies and determine their sequence types (STs) and the mechanism of colistin resistance using whole-genome sequencing (WGS). (2) Methods: Antimicrobial susceptibility testing via broth microdilution against 12 antimicrobials from different classes and growth rate assays were performed. A checkerboard assay was conducted on PDR isolates using six different antimicrobials, each in combination with colistin. Genomic DNA was extracted from all isolates and subjected to WGS. (3) Results: All isolates were resistant to all tested antimicrobials with the one exception that was susceptible to gentamicin. Combining colistin with either meropenem, ceftolozane-tazobactam, or teicoplanin showed synergistic activity. Sequencing data revealed that 67% of the isolates belonged to Pasteur ST2 and 33% to ST187. Furthermore, these isolates harbored a number of resistance genes, including blaOXA-23. Mutations in the pmrC gene were behind colistin resistance. (4) Conclusions: With the rise in antimicrobial resistance and the absence of novel antimicrobial production, alternative treatments must be found. The combination therapy results from this study suggest treatment options for PDR ST2 A. baumannii-infected patients.

The first report of the mobile colistin resistance gene, mcr-10.1, in Kenya and a novel mutation in the phoQ gene (S244T) in a colistin-resistant Enterobacter cloacae clinical isolate.

This study describes the identification of the mcr-10.1 gene in a clinical isolate of an ST1 Enterobacter cloacae isolate cultured in 2015 in Kenya. The isolate was multidrug resistant, phenotypically non-susceptible to various antibiotics, including colistin. Whole genome sequence analyses indicated carriage of chromosomally encoded antimicrobial resistance genes and the colistin-resistant gene mcr-10.1 located on a 72-kb plasmid designated pECC011b with an IncFIA(HI1) replicon directly adjacent to tyrosine recombinase gene, xerC, and downstream of an ISKPn26 insertion sequence. Studies have shown that expression of mcr-10.1 may not be sufficient to confer colistin resistance, but a novel non-synonymous mutation (S244T) was identified in the phoQ gene known to influence colistin resistance within lipid modification pathways, which could have complemented the mcr-10.1 resistance mechanism. In silico analysis of the mutant phoQ protein shows the location of the mutation to be at the Histidine kinases, Adenyl cyclases, Methyl-accepting proteins and Phosphatases (HAMP) region, which plays a crucial role in the protein's activity. This study and our previous report of mcr-8 in Klebsiella pneumoniae indicate the presence of mobile mcr genes in the Enterobacterales order of bacteria in Kenya. The study points to the importance of regulation of colistin in the animal industry and enhancing surveillance in both human and animal health to curb the spread of mcr genes and accurately assess the risks posed by these mobile genetic elements in both sectors.IMPORTANCEThis paper reports the detection of new colistin resistance mechanisms in Kenya in a clinical isolate of Enterobacter cloacae in a patient with a healthcare-associated infection. The plasmid-mediated resistance gene, mcr-10.1, and a novel amino acid mutation S244T in the phoQ gene, located in a region of the protein involved in membrane cationic stability contributing to colistin resistance, were detected. Colistin is a critical last-line drug for multidrug-resistant (MDR) gram-negative human infections and is used for treatment and growth promotion in the animal industry. The emergence of the resistance mechanisms points to the potential overuse of colistin in the animal sector in Kenya, which enhances resistance, threatens the utility of colistin, and limits treatment options for MDR infections. This study highlights the need to enhance surveillance of colistin resistance across sectors and strengthen One Health policies that ensure antimicrobial stewardship and implementation of strategies to mitigate the spread of antibiotic resistance.

Genomic investigation unveils colistin resistance mechanism in carbapenem-resistant Acinetobacter baumannii clinical isolates.

Colistin resistance in Acinetobacter baumannii is mediated by multiple mechanisms. Recently, mutations within pmrABC two-component system and overexpression of eptA gene due to upstream insertion of ISAba1 have been shown to play a major role. Thus, the aim of our study is to characterize colistin resistance mechanisms among the clinical isolates of A. baumannii in India. A total of 207 clinical isolates of A. baumannii collected from 2016 to 2019 were included in this study. Mutations within lipid A biosynthesis and pmrABC genes were characterized by whole-genome shotgun sequencing. Twenty-eight complete genomes were further characterized by hybrid assembly approach to study insertional inactivation of lpx genes and the association of ISAba1-eptA. Several single point mutations (SNPs), like M12I in pmrA, A138T and A444V in pmrB, and E117K in lpxD, were identified. We are the first to report two novel SNPs (T7I and V383I) in the pmrC gene. Among the five colistin-resistant A. baumannii isolates where complete genome was available, the analysis showed that three of the five isolates had ISAba1 insertion upstream of eptA. No mcr genes were identified among the isolates. We mapped the SNPs on the respective protein structures to understand the effect on the protein activity. We found that majority of the SNPs had little effect on the putative protein function; however, some SNPs might destabilize the local structure. Our study highlights the diversity of colistin resistance mechanisms occurring in A. baumannii, and ISAba1-driven eptA overexpression is responsible for colistin resistance among the Indian isolates.IMPORTANCEAcinetobacter baumannii is a Gram-negative, emerging and opportunistic bacterial pathogen that is often associated with a wide range of nosocomial infections. The treatment of these infections is hindered by increase in the occurrence of A. baumannii strains that are resistant to most of the existing antibiotics. The current drug of choice to treat the infection caused by A. baumannii is colistin, but unfortunately, the bacteria started to show resistance to the last-resort antibiotic. The loss of lipopolysaccharides and mutations in lipid A biosynthesis genes are the main reasons for the colistin resistance. The present study characterized 207 A. baumannii clinical isolates and constructed complete genomes of 28 isolates to recognize the mechanisms of colistin resistance. We showed the mutations in the colistin-resistant variants within genes essential for lipid A biosynthesis and that cause these isolates to lose the ability to produce lipopolysaccharides.

Roles of crrAB two-component regulatory system in Klebsiella pneumoniae: growth yield, survival in initial colistin treatment stage, and virulence

Alternation of the colistin resistance-regulating two-component regulatory system (crrAB) is a colistin-resistance mechanism in Klebsiella pneumoniae (K. pneumoniae), but its role in bacteria is not fully understood. Twelve colistin-susceptible K. pneumoniae clinical isolates were included in this study: six crrAB-positive and six crrAB-negative. We deleted the crrAB genes from two crrAB-positive isolates and complemented them. We measured the growth yields by determining growth curves in lysogeny broth and minimal media with or without Fe2+. In vitro selection rates for colistin resistance were determined by exposure to colistin, and survival rates against high concentrations of colistin (20 mg/L) at the early stage of growth (20 min) were investigated. Virulence was determined using a serum bactericidal assay and Galleria mellonella larval infection. The presence of crrAB was not associated with colistin resistance and did not increase the in vitro selection rate of colistin resistance after exposure. The growth yield of crrAB-positive isolates was higher in lysogeny broth media and increased when Fe2+ was added to minimal media. The crrAB-positive isolates showed higher survival rates in the early stages of exposure to high colistin concentrations. Decreased serum resistance was identified in the crrAB-deleted mutants. More G. mellonella larvae survived when infected by crrAB-deleted mutants, and higher survival rates of bacteria were identified within the larvae infected with wild-type than crrAB-deletant isolates. Through rapid response to external signals, crrAB would provide advantages for K. pneumoniae survival by increasing the final growth yield and initial survival against colistin treatment. This may partly contribute to the bacterial virulence.

Disrupting iron homeostasis can potentiate colistin activity and overcome colistin resistance mechanisms in Gram-Negative Bacteria

Acinetobacter baumannii is a Gram-negative priority pathogen that can readily overcome antibiotic treatment through a range of intrinsic and acquired resistance mechanisms. Treatment of carbapenem-resistant A. baumannii largely relies on the use of colistin in cases where other treatment options have been exhausted. However, the emergence of resistance against this last-line drug has significantly increased amongst clinical strains. In this study, we identify the phytochemical kaempferol as a potentiator of colistin activity. When administered singularly, kaempferol has no effect on growth but does impact biofilm formation. Nonetheless, co-administration of kaempferol with sub-inhibitory concentrations of colistin exposes bacteria to a metabolic Achilles heel, whereby kaempferol-induced dysregulation of iron homeostasis leads to bacterial killing. We demonstrate that this effect is due to the disruption of Fenton’s reaction, and therefore to a lethal build-up of toxic reactive oxygen species in the cell. Furthermore, we show that this vulnerability can be exploited to overcome both intrinsic and acquired colistin resistance in clinical strains of A. baumannii and E. coli in vitro and in the Galleria mellonella model of infection. Overall, our findings provide a proof-of-principle demonstration that targeting iron homeostasis is a promising strategy for enhancing the efficacy of colistin and overcoming colistin-resistant infections.

Genomic characterization of colistin-resistant Klebsiella pneumoniae isolated from intensive care unit patients in Egypt

BackgroundEgypt has witnessed elevated incidence rates of multidrug-resistant Klebsiella pneumoniae infections in intensive care units (ICUs). The treatment of these infections is becoming more challenging whilst colistin-carbapenem-resistant K. pneumoniae is upsurging. Due to the insufficiently available data on the genomic features of colistin-resistant K. pneumoniae in Egypt, it was important to fill in the gap and explore the genomic characteristics, as well as the antimicrobial resistance, the virulence determinants, and the molecular mechanisms of colistin resistance in such a lethal pathogen.MethodsSeventeen colistin-resistant clinical K. pneumoniae isolates were collected from ICUs in Alexandria, Egypt in a 6-month period in 2020. Colistin resistance was phenotypically detected by modified rapid polymyxin Nordmann/Poirel and broth microdilution techniques. The isolates susceptibility to 20 antimicrobials was determined using Kirby-Bauer disk diffusion method. Whole genome sequencing and bioinformatic analysis were employed for exploring the virulome, resistome, and the genetic basis of colistin resistance mechanisms.ResultsOut of the tested K. pneumoniae isolates, 82.35% were extensively drug-resistant and 17.65% were multidrug-resistant. Promising susceptibility levels towards tigecycline (88.24%) and doxycycline (52.94%) were detected. Population structure analysis revealed seven sequence types (ST) and K-types: ST383-K30, ST147-K64, ST17-K25, ST111-K63, ST11-K15, ST14-K2, and ST525-K45. Virulome analysis revealed yersiniabactin, aerobactin, and salmochelin siderophore systems in ˃ 50% of the population. Hypervirulence biomarkers, iucA (52.94%) and rmpA/A2 (5.88%) were detected. Extended-spectrum β-lactamase- and carbapenemase-producers accounted for 94.12% of the population, with blaCTX-M-15, blaNDM-5, and blaOXA-48 reaching 64.71%, 82.35%, and 82.35%, respectively. Chromosomal alterations in mgrB (82.35%) were the most prevailing colistin resistance-associated genetic change followed by deleterious mutations in ArnT (23.53%, L54H and G164S), PmrA (11.76%, G53V and D86E), PmrB (11.76%, T89P and T134P), PmrC (11.76%, S257L), PhoQ (5.88%, L322Q and Q435H), and ArnB (5.88%, G47D) along with the acquisition of mcr-1.1 by a single isolate of ST525.ConclusionsIn this study, we present the genotypic colistin resistance mechanisms in K. pneumoniae isolated in Egypt. More effective antibiotic stewardship protocols must be implemented by Egyptian health authorities to restrain this hazard and safeguard the future utility of colistin. This is the first characterization of a complete sequence of mcr-1.1-bearing IncHI2/IncHI2A plasmid recovered from K. pneumoniae clinical isolate belonging to the emerging high-risk clone ST525.

Chromosome-Mediated Colistin Resistance in Clinical Isolates of Klebsiella pneumoniae and Escherichia coli: Mutation Analysis in the Light of Genetic Background.

Colistin resistance mechanisms involving mutations in chromosomal genes associated with LPS modification are not completely understood. Mutations in genes coding for the MgrB regulator frequently account for colistin resistance in Klebsiella pneumoniae, whereas mutations in genes coding for PhoPQ and PmrAB are frequent in E. coli. Our aim was to perform a genetic analysis of chromosomal mutations in colistin-resistant (MIC ≥4 µg/mL) clinical isolates of K. pneumoniae (n = 8) and E. coli (n = 7) of different STs. Isolates were obtained in a 3-year period in a university hospital in Santiago, Chile. Susceptibility to colistin, aminoglycosides, cephalosporins, carbapenems and ciprofloxacin was determined through broth microdilution. Whole genome sequencing was performed for all isolates and chromosomal gene sequences were compared with sequences of colistin-susceptible isolates of the same sequence types. None of the isolates carried mcr genes. Most of the isolates were susceptible to all the antibiotics analyzed. E. coli isolates were ST69, ST127, ST59, ST131 and ST14, and K. pneumoniae isolates were ST454, ST45, ST6293, ST380 and ST25. All the isolates had mutations in chromosomal genes analyzed. K. pneumoniae had mutations mainly in mgrB gene, whereas E. coli had mutations in pmrA, pmrB and pmrE genes. Most of the amino acid changes in LPS-modifying enzymes of colistin-resistant isolates were found in colistin-susceptible isolates of the same and/or different ST. Eleven of them were found only in colistin-resistant isolates. Colistin resistance mechanisms depend on genetic background, and are due to chromosomal mutations, which implies a lower risk of transmission than plasmid-mediated genes. Colistin resistance is not associated with multidrug-resistance, nor to high-risk sequence types.

Within-host acquisition of colistin-resistance of an NDM-producing Klebsiella quasipneumoniae subsp. similipneumoniae strain through the insertion sequence-903B-mediated inactivation of mgrB gene in a lung transplant child in China.

Colistin, as the antibiotic of "last resort" for carbapenem-resistant Klebsiella, develop resistance during administration of this antimicrobial agent. We identified an NDM-1-producing Klebsiella quasipneumonuae subsp. similipneumoniae (KQSS) strain KQ20605 recovered from a child, which developed resistance to colistin (KQ20786) through acquiring an IS903B element between the -27th and -26th bp of mgrB promoter region after 6-day colistin usage. The aim of this study is to explore the source of IS903B in the disruptive mgrB gene and its underlying mechanisms. Antibiotics susceptibility testing was conducted via microbroth dilution method. The in vitro colistin-induced experiment of KQ20605 was performed to mimic the in vivo transition from colistin-sensitive to resistant. Whole-genome sequencing was used to molecular identification of colistin resistance mechanism. The IS903B element integrated into mgrB gene of KQ20786 had a 100% nucleotide identity and coverage match with one IS903B on plasmid IncR, and only 95.1% (1005/1057) identity to those on chromosome. In vitro, upon the pressure of colistin, KQ20605 could also switch its phenotype from colistin-sensitive to resistant with IS elements (e.g., IS903B and IS26) frequently inserted into mgrB gene at "hotspots", with the insertion site of IS903B nearly identical to that of KQ20786. Furthermore, IS26 elements in this isolate were only encoded by plasmids, including IncR and conjugative plasmid IncN harboring bla NDM. Mobilizable IS elements on plasmids tend to be activated and integrated into mgrB gene at "hotspots" in this KQSS, thereby causing the colistin resistance emergence and further dissemination.

Characteristics and genomic epidemiology of colistin-resistant Enterobacterales from farmers, swine, and hospitalized patients in Thailand, 2014–2017

BackgroundColistin is one of the last resort therapeutic options for treating carbapenemase-producing Enterobacterales, which are resistant to a broad range of beta-lactam antibiotics. However, the increased use of colistin in clinical and livestock farming settings in Thailand and China, has led to the inevitable emergence of colistin resistance. To better understand the rise of colistin-resistant strains in each of these settings, we characterized colistin-resistant Enterobacterales isolated from farmers, swine, and hospitalized patients in Thailand.MethodsEnterobacterales were isolated from 149 stool samples or rectal swabs collected from farmers, pigs, and hospitalized patients in Thailand between November 2014–December 2017. Confirmed colistin-resistant isolates were sequenced. Genomic analyses included species identification, multilocus sequence typing, and detection of antimicrobial resistance determinants and plasmids.ResultsThe overall colistin-resistant Enterobacterales colonization rate was 26.2% (n = 39/149). The plasmid-mediated colistin-resistance gene (mcr) was detected in all 25 Escherichia coli isolates and 9 of 14 (64.3%) Klebsiella spp. isolates. Five novel mcr allelic variants were also identified: mcr-2.3, mcr-3.21, mcr-3.22, mcr-3.23, and mcr-3.24, that were only detected in E. coli and Klebsiella spp. isolates from farmed pigs.ConclusionOur data confirmed the presence of colistin-resistance genes in combination with extended spectrum beta-lactamase genes in bacterial isolates from farmers, swine, and patients in Thailand. Differences between the colistin-resistance mechanisms of Escherichia coli and Klebsiella pneumoniae in hospitalized patients were observed, as expected. Additionally, we identified mobile colistin-resistance mcr-1.1 genes from swine and patient isolates belonging to plasmids of the same incompatibility group. This supported the possibility that horizontal transmission of bacterial strains or plasmid-mediated colistin-resistance genes occurs between humans and swine.

The occurrence and molecular detection of mcr-1 and mcr-5 genes in Enterobacteriaceae isolated from poultry and poultry meats in Malaysia.

The advent of antimicrobials-resistant (AMR), including colistin-resistant bacteria, poses a significant challenge to animal and human health, food safety, socio-economic growth, and the global environment. This study aimed to ascertain the colistin resistance prevalence and molecular mechanisms of colistin resistance in Enterobacteriaceae. The colistin resistance was determined using broth microdilution assay, PCR; and Sanger sequencing of mcr genes responsible for colistin resistance in Enterobacteriaceae (n = 627), including Escherichia coli (436), Salmonella spp. (n = 140), and Klebsiella pneumoniae (n = 51), obtained from chicken and chicken meats. Out of 627 Enterobacteriaceae, 8.6% of isolates exhibited colistin resistance phenotypically. Among these colistin resistant isolates, 9.3% (n = 37) were isolated from chicken meat, 7.2% (n = 11) from the cloacal swab of chicken and 7.9% (n = 6) from the litter samples. Overall, 12.96% of colistin-resistant isolates were positive with mcr genes, in which mcr-1 and mcr-5 genes were determined in 11.11% and 1.85% of colistin-resistant isolates, respectively. The E. coli isolates obtained from chicken meats, cloacal swabs and litter samples were found positive for mcr-1, and Salmonella spp. originated from the chicken meat sample was observed with mcr-5, whereas no mcr genes were observed in K. pneumoniae strains isolated from any of the collected samples. The other colistin resistance genes, including mcr-2, mcr-3, mcr-4, mcr-6, mcr-7, mcr-8, mcr-9, and mcr-10 were not detected in the studied samples. The mcr-1 and mcr-5 genes were sequenced and found to be 100% identical to the mcr-1 and mcr-5 gene sequences available in the NCBI database. This is the first report of colistin resistance mcr-5 gene in Malaysia which could portend the emergence of mcr-5 harboring bacterial strains for infection. Further studies are needed to characterize the mr-5 harbouring bacteria for the determination of plasmid associated with mcr-5 gene.

Colistin Resistance in Aquaculture Environment: An Emerging Challenge

AbstractThe emergence of colistin resistance is one of the most serious future issues since it is a last‐resort antibiotic used to treat infections caused by multidrug‐resistant (MDR) gram‐negative bacteria, particularly those resistant to carbapenems. The aquatic environment acts as a reservoir for the emergence and spread of colistin resistant pathogens. The emergence and transmission of a plasmid mediated mcr gene is one of the most important mechanism of colistin resistance in gram negative bacteria. Several mcr variants have been identified in zoonotic pathogens of aquatic origin that may easily transmit this gene to human pathogens. Currently, very few studies have been conducted to test colistin susceptibility in aquaculture associated bacteria. This may be attributed to a limited technical resource for screening colistin‐resistant microorganisms. As a result, to reduce the emergence and spread of colistin resistance in human and animal health, as well as in the environment, the One Health strategy and laboratory methodologies must be expanded.

Insight into Antibiotic Synergy Combinations for Eliminating Colistin Heteroresistant Klebsiella pneumoniae.

Colistin heteroresistance has been identified in several bacterial species, including Escherichia coli and Klebsiella pneumoniae, and may underlie antibiotic therapy failures since it most often goes undetected by conventional antimicrobial susceptibility tests. This study utilizes population analysis profiling (PAP) and time-kill assay for the detection of heteroresistance in K. pneumoniae and for evaluating the association between in vitro regrowth and heteroresistance. The mechanisms of colistin resistance and the ability of combination therapies to suppress resistance selection were also analysed. In total, 3 (18%) of the 16 colistin-susceptible strains (MIC ≤ 2 mg/L) were confirmed to be heteroresistant to colistin by PAP assay. In contrast to the colistin-susceptible control strains, all three heteroresistant strains showed regrowth when exposed to colistin after 24 h following a rapid bactericidal action. Colistin resistance in all the resistant subpopulations was due to the disruption of the mgrB gene by various insertion elements such as ISKpn14 of the IS1 family and IS903B of the IS5 family. Colistin combined with carbapenems (imipenem, meropenem), aminoglycosides (amikacin, gentamicin) or tigecycline was found to elicit in vitro synergistic effects against these colistin heteroresistant strains. Our experimental results showcase the potential of combination therapies for treatment of K. pneumoniae infections associated with colistin heteroresistance.

Heteroresistance to Colistin in Clinical Isolates of Klebsiella pneumoniae Producing OXA-48.

Heteroresistance to colistin can be defined as the presence of resistant subpopulations in an isolate that is susceptible to this antibiotic. Colistin resistance in Gram-negative bacteria is more frequently related to chromosomal mutations and insertions. This work aimed to study heteroresistance in nine clinical isolates of Klebsiella pneumoniae producing OXA-48 and to describe genomic changes in mutants with acquired resistance in vitro. Antimicrobial susceptibility was determined by broth microdilution (BMD) and heteroresistance by population analysis profiling (PAP). The proteins related to colistin resistance were analyzed for the presence of mutations. Additionally, PCR of the mgrB gene was performed to identify the presence of insertions. In the nine parental isolates, the PAP method showed colistin heteroresistance of colonies growing on plates with concentrations of up to 64 mg/L, corresponding to stable mutant subpopulations. The MICs of some mutants from the PAP plate containing 4×MIC of colistin had absolute values of ≤2 mg/L that were higher than the parental MICs and were defined as persistent variants. PCR of the mgrB gene identified an insertion sequence that inactivated the gene in 21 mutants. Other substitutions in the investigated mutants were found in PhoP, PhoQ, PmrB, PmrC, CrrA and CrrB proteins. Colistin heteroresistance in K. pneumoniae isolates was attributed mainly to insertions in the mgrB gene and point mutations in colistin resistance proteins. The results of this study will improve understanding regarding the mechanisms of colistin resistance in mutants of K. pneumoniae producing OXA-48.

Prevalence and molecular characteristics of colistin-resistant isolates among clinically isolated carbapenem-resistant Klebsiella pneumoniae in China

Colistin resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP) poses health challenges. To investigate the prevalence and molecular characteristics of colistin-resistant CRKP, 708 isolates were collected consecutively from 28 tertiary hospitals in China from 2018 to 2019, and 14 colistin-resistant CRKP were identified. Two-component systems (TCSs) related to colistin resistance (PmrA/B, PhoP/Q, and CrrA/B), the negative regulator mgrB gene and mcr genes, were analysed using genomic sequencing. The relative expression of TCSs genes along with their downstream pmrC and pmrK genes was determined using quantitative real-time PCR (qRT‒PCR). A novel point mutation in PhoQ was confirmed by site-directed mutagenesis, and the subsequent transcriptome changes were analysed by RNA sequencing (RNA-Seq). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to detect modifications in lipid A. The results showed that only one isolate carried the mcr-8.1 gene, nine exhibited MgrB inactivation or absence, and three exhibited mutations in PmrB. One novel point mutation, L247P, in PhoQ was found to lead to a 64-fold increase in the minimum inhibitory concentration (MIC) of colistin. qRT‒PCR revealed overexpression of phoP/Q and pmrK in isolates with or without MgrB inactivation, and pmrB mutation resulted in overexpression of pmrA and pmrC. Furthermore, transcriptome analysis revealed that the PhoQ L247P novel point mutation caused upregulated expression of phoP/Q and its downstream operon pmrHFIJKLM. Meanwhile, the pmrA/B regulatory pathway did not evolve colistin resistance. Mass spectrometry analysis showed the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to lipid A in colistin-resistant isolates with absence of MgrB. These findings illustrate that the molecular mechanisms of colistin resistance in CRKP isolates are complex, and that MgrB inactivation or absence is the predominant molecular mechanism. Interventions should be initiated to monitor and control colistin resistance.

Lipid A modification-induced colistin-resistant Klebsiella variicola from healthy adults.

Background. Klebsiella variicola was once recognised as a benign plant-endosymbiont but recent case reports suggest that it is a newly emerging Gram-negative pathogen related to opportunistic infection of multiple sites in humans.Methods. Antimicrobial susceptibility testing was performed using broth microdilution method. To identify colistin resistance mechanisms, phoPQ, pmrAB, and mgrB were sequenced and their mRNA expression was analysed using quantitative real-time PCR. In addition, we tried to detect crrAB and mcr. The lipid A moieties of colistin-susceptible and -resistant isolates were analysed using MALDI-TOF.Results. Among the two K. variicola isolates, one is colistin-resistant, and another is colistin-susceptible. The colistin-resistant K. variicola isolate showed no mutations in phoPQ, pmrAB, and mgrB, and crrAB and mcr were not identified. However, its phoQ and pbgP expression was significantly higher and amino-arabinosylated lipid A with hexa-acylated species in lipopolysaccharide was identified.Conclusions. We found that colistin resistance in K. variicola was mediated by the modification of lipid A. Although the isolate was obtained from faecal samples of healthy adults, colistin-resistant K. variicola challenges public health as an opportunistic pathogen.