Genetic and structural basis of colistin resistance in Klebsiella pneumoniae: Unravelling the molecular mechanisms.

OBJECTIVE: Antimicrobial resistance (AMR), together with multidrug resistance (MDR), mainly among Gram-negative bacteria, has been on the rise. Colistin (polymyxin E) remains one of the primary available last resorts to treat infections caused by MDR bacteria during the rapid emergence of global resistance. As the exact mechanism of bacterial resistance to colistin remains undetermined, this study warranted elucidation of the underlying mechanisms of colistin resistance and heteroresistance among carbapenem-resistant Klebsiella pneumoniae isolates. METHODS: Molecular analysis was carried out on the resistant isolates using a genome-wide characterisation approach, as well as MALDI-TOF mass spectrometry, to identify lipid A. RESULTS: Among the 32 carbapenem-resistant K. pneumoniae isolates, several isolates showed resistance and intermediate resistance to colistin. The seven isolates with intermediate resistance exhibited the "skip-well" phenomenon, attributed to the presence of resistant subpopulations. The three isolates with full resistance to colistin showed ions using MALDI-TOF mass spectrometry at m/z of 1840 and 1824 representing bisphosphorylated and hexa-acylated lipid A, respectively, with or without hydroxylation at position C'-2 of the fatty acyl chain. Studying the genetic environment of mgrB locus revealed the presence of two insertion sequences that disrupted the mgrB locus in the three colistin-resistant isolates: IS1R and IS903B. CONCLUSIONS: Our findings show that colistin resistance/heteroresistance was inducible with mutations in chromosomal regulatory networks controlling the lipid A moiety and insertion sequences disrupting the mgrB gene, leading to elevated minimum inhibitory concentration values and treatment failure. Different treatment strategies should be employed to avoid colistin heteroresistance-linked treatment failures, mainly through combination therapy using colistin with carbapenems, aminoglycosides, or tigecycline.

Investigation of the Rapid Emergence of Colistin Resistance in a Newborn Infected with KPC-2-Producing Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae.

OBJECTIVES: Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKp) poses a significant threat to public health. This study reports an infection related to hv-CRKp in a premature infant and reveals its colistin resistance and evolutionary mechanisms within the host. METHODS: Three KPC-producing CRKp strains were isolated from a patient with sepsis and CRKp osteoarthritis who had been receiving colistin antimicrobial therapy. The minimum inhibitory concentrations (MICs) of ceftazidime, ceftazidime-avibactam (CAZ-AVI), meropenem, imipenem, tigecycline, amikacin, minocycline, sulfamethoxazole/trimethoprim, ciprofloxacin, levofloxacin, aztreonam, cefepime, cefoperazone/sulbactam, piperacillin/tazobactam, and colistin were determined using the microbroth dilution method. The whole-genome sequencing analysis was conducted to determine the sequence types (STs), virulence genes, and antibiotic resistance genes of the three CRKp strains. RESULTS: Whole-genome sequencing revealed that all three CRKp strains belonged to the ST11 clone and carried a plasmid encoding blaKPC-2. The three strains all possessed the iucABCDiutA virulence cluster, peg-344 gene, and rmpA/rmpA2 genes, defining them as hv-CRKp. Further experiments and whole-genome analysis revealed that a strain of K. pneuomniae had developed resistance to colistin. The mechanism found to be responsible for colistin resistance was a deletion mutation of approximately 9000 bp including the mgrB gene. CONCLUSION: This study characterizes colistin resistance of the ST11 clone hv-CRKp during colistin treatment and its rapid evolution within the host.

Global prevalence of mutation in the mgrB gene among clinical isolates of colistin-resistant Klebsiella pneumoniae: a systematic review and meta-analysis.

Background: Colistin is used as a last resort for managing infections caused by multidrug-resistant bacteria. However, the high emergence of colistin-resistant strains has restricted the clinical use of this antibiotic in the clinical setting. In the present study, we evaluated the global prevalence of the mutation in the mgrB gene, one of the most important mechanisms of colistin resistance in Klebsiella pneumoniae. Methods: Several databases, including Scopus, Medline (via PubMed), and Web of Science, were searched (until August 2023) to identify those studies that address the mgrB mutation in clinical isolates of K. pneumoniae. Using Stata software, the pooled prevalence of mgrB mutation and subgroup analyses for the year of publication, country, continent, mgrB mutation types, and detection methods of mgrB mutation were analyzed. Results: Out of the 115 studies included in the analysis, the prevalence of mgrB mutations in colistin-resistant K. pneumoniae isolates was estimated at 65% of isolates, and mgrB variations with insertional inactivation had the highest prevalence among the five investigated mutations with 69%. The year subgroup analysis indicated an increase in mutated mgrB from 46% in 2014 to 61% in 2022. Europe had the highest prevalence of mutated mgrB at 73%, while Africa had the lowest at 54%. Conclusion: Mutations in the mgrB gene are reported as one of the most common mechanisms of colistin resistance in K. pneumoniae, and the results of the present study showed that 65% of the reported colistin-resistant K. pneumoniae had a mutation in this gene.

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

BACKGROUND: The 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. RESULTS: In 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. CONCLUSION: While 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.

Assessment of Colistin Heteroresistance among Multidrug-Resistant Klebsiella pneumoniae Isolated from Intensive Care Patients in Europe.

Heteroresistance (HR) to colistin is especially concerning in settings where multi-drug-resistant (MDR) K. pneumoniae are prevalent and empiric use of colistin might lead to treatment failures. This study aimed to assess the frequency of occurrence of colistin HR (CHR) among (MDR) K. pneumoniae (n = 676) isolated from patients hospitalized in 13 intensive care units (ICUs) in six European countries in a clinical trial assessing the impact of decolonization strategies. All isolates were whole-genome-sequenced and studied for in vitro colistin susceptibility. The majority were colistin-susceptible (CS) (n = 597, MIC ≤ 2 µg/mL), and 79 were fully colistin-resistant (CR) (MIC > 2 µg/mL). A total of 288 CS isolates were randomly selected for population analysis profiling (PAP) to assess CHR prevalence. CHR was detected in 108/288 CS K. pneumoniae. No significant association was found between the occurrence of CHR and country, MIC-value, K-antigen type, and O-antigen type. Overall, 92% (617/671) of the K. pneumoniae were MDR with high prevalence among CS (91%, 539/592) and CR (98.7%, 78/79) isolates. In contrast, the proportion of carbapenemase-producing K. pneumoniae (CP-Kpn) was higher among CR (72.2%, 57/79) than CS isolates (29.3%, 174/594). The proportions of MDR and CP-Kpn were similar among CHR (MDR: 85%, 91/107; CP-Kpn: 29.9%, 32/107) and selected CS isolates (MDR: 84.7%, 244/288; CP-Kpn: 28.1%, 80/285). WGS analysis of PAP isolates showed diverse insertion elements in mgrB or even among technical replicates underscoring the stochasticity of the CHR phenotype. CHR isolates showed high sequence type (ST) diversity (Simpson's diversity index, SDI: 0.97, in 52 of the 85 STs tested). CR (SDI: 0.85) isolates were highly associated with specific STs (ST101, ST147, ST258/ST512, p ≤ 0.003). The widespread nature of CHR among MDR K. pneumoniae in our study urge the development of rapid HR detection methods to inform on the need for combination regimens.

An Intra-Hospital Spread of Colistin-Resistant K. pneumoniae Isolates-Epidemiological, Clinical, and Genetic Analysis.

Background and Objective: Klebsiella pneumoniae appears to be a significant problem due to its ability to accumulate antibiotic-resistance genes. After 2013, alarming colistin resistance rates among carbapenem-resistant K. pneumoniae have been reported in the Balkans. The study aims to perform an epidemiological, clinical, and genetic analysis of a local outbreak of COLr CR-Kp. Material and Methods: All carbapenem-resistant and colistin-resistant K. pneumoniae isolates observed among patients in the ICU unit of Military Medical Academy, Sofia, from 1 January to 31 October 2023, were included. The results were analyzed according to the EUCAST criteria. All isolates were screened for blaVIM, blaIMP, blaKPC, blaNDM, and blaOXA-48. Genetic similarity was determined using the Dice coefficient as a similarity measure and the unweighted pair group method with arithmetic mean (UPGMA). mgrB genes and plasmid-mediated colistin resistance determinants (mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5) were investigated. Results: There was a total of 379 multidrug-resistant K. pneumoniae isolates, 88% of which were carbapenem-resistant. Of these, there were nine (2.7%) colistin-resistant isolates in six patients. A time and space cluster for five patients was found. Epidemiology typing showed that two isolates belonged to clone A (pts. 1, 5) and the rest to clone B (pts. 2-4) with 69% similarity. Clone A isolates were coproducers of blaNDM-like and blaOXA-48-like and had mgrB-mediated colistin resistance (40%). Clone B isolates had only blaOXA-48-like and intact mgrB genes. All isolates were negative for mcr-1, -2, -3, -4, and -5 genes. Conclusions: The study describes a within-hospital spread of two clones of COLr CR-Kp with a 60% mortality rate. Clone A isolates were coproducers of NDM-like and OXA-48-like enzymes and had mgrB-mediated colistin resistance. Clone B isolates had only OXA-48-like enzymes and intact mgrB genes. No plasmid-mediated resistance was found. The extremely high mortality rate and limited treatment options warrant strict measures to prevent outbreaks.

Heteroresistance to colistin in wild-type Klebsiella pneumoniae isolates from clinical origin.

IMPORTANCE: Colistin is one of the last remaining therapeutic options for dealing with Enterobacteriaceae. Unfortunately, heteroresistance to colistin is also rapidly increasing. We described the prevalence of colistin heteroresistance in a variety of wild-type strains of Klebsiella pneumoniae and the evolution of these strains with colistin heteroresistance to a resistant phenotype after colistin exposure and withdrawal. Resistant mutants were characterized at the molecular level, and numerous mutations in genes related to lipopolysaccharide formation were observed. In colistin-treated patients, the evolution of K. pneumoniae heteroresistance to resistance phenotype could lead to higher rates of therapeutic failure.

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

Purpose: 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. Methods: 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. Results: 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. Conclusion: 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.

Role of the ISKpn element in mediating mgrB gene mutations in ST11 hypervirulent colistin-resistant Klebsiella pneumoniae.

Background: Colistin has emerged as a last-resort therapeutic against antibiotic-resistant bacterial infections, particularly those attributed to carbapenem-resistant Enterobacteriaceae (CRE) like CRKP. Yet, alarmingly, approximately 45% of multidrug-resistant Klebsiella pneumoniae strains now manifest resistance to colistin. Through our study, we discerned that the synergy between carbapenemase and IS elements amplifies resistance in Klebsiella pneumoniae, thereby narrowing the existing therapeutic avenues. This underscores the instrumental role of IS elements in enhancing colistin resistance through mgrB disruption. Methods: From 2021 to 2023, 127 colistin-resistant Klebsiella pneumoniae isolates underwent meticulous examination. We embarked on an exhaustive genetic probe, targeting genes associated with both plasmid-mediated mobile resistance-encompassing blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48-like, and mcr-1 to mcr-8-and chromosome-mediated resistance systems, including PhoP/Q, PmrA/B, and mgrB. PCR amplification revealed the presence of virulence-associated genes from the pLVPK plasmid, such as rmpA, rmpA2, iucA, iroB, and peg344. mgrB sequencing was delegated to Sangon Biotech, Shanghai, and the sequences procured were validated using BLAST. Our search for IS elements was navigated through the IS finder portal. Phenotypically, we harnessed broth microdilution (BMD) to ascertain the MICs of colistin. To sketch the clonal lineage of mgrB-mutated CoR-Kp isolates, sophisticated methodologies like MLST and PFGE were deployed. S1-PFGE unraveled the intrinsic plasmids in these isolates. Our battery of virulence assessment techniques ranged from the string test and capsular serotyping to the serum killing assay and the Galleria mellonella larval infection model. Results: Among the 127 analyzed isolates, 20 showed an enlarged mgrB PCR amplicon compared to wild-type strains. These emerged over a three-year period: three in 2021, thirteen in 2022, and four in 2023. Antimicrobial susceptibility tests revealed that these isolates consistently resisted several drugs, notably TCC, TZP, CAZ, and COL. Additionally, 85% resisted both DOX and TOB. The MICs for colistin across these strains ranged between 16 to 64 mg/L, with a median of 40 mg/L. From a genetic perspective, MLST unanimously categorized these mgrB-mutated CoR-hvKp isolates as ST11. PFGE further delineated them into six distinct clusters, with clusters A and D being predominant. This distribution suggests potential horizontal and clonal genetic transmission. Intriguingly, every mgrB-mutated CoR-hvKP isolate possessed at least two virulence genes akin to the pLVPK-like virulence plasmid, with iroB and rmpA2 standing out. Their virulence was empirically validated both in vitro and in vivo. A pivotal discovery was the identification of three distinct insertion sequence (IS) elements within or near the mgrB gene. These were:ISKpn26 in eleven isolates, mainly in cluster A, with various insertion sites including +74, +125, and an upstream -35.ISKpn14 in four isolates with insertions at +93, -35, and two upstream at -60.IS903B present in five isolates, marking positions like +74, +125, +116, and -35 in the promoter region. These diverse insertions, spanning six unique locations in or near the mgrB gene, underscore its remarkable adaptability. Conclusion: Our exploration spotlights the ISKpn element's paramount role in fostering mgrB gene mutations in ST11 hypervirulent colistin-resistant Klebsiella pneumoniae. Employing MLST and PFGE, we unearthed two primary genetic conduits: clonal and horizontal. A striking observation was the ubiquitous presence of the KPC carbapenemase gene in all the evaluated ST11 hypervirulent colistin-resistant Klebsiella pneumoniae strains, with a majority also harboring the NDM gene. The myriad mgrB gene insertion locales accentuate its flexibility and the overarching influence of IS elements, notably the pervasive IS5-like variants ISKpn26 and IS903B. Our revelations illuminate the escalating role of IS elements in antibiotic resistance within ST11 hypervirulent colistin-resistant Klebsiella pneumoniae, advocating for innovative interventions to counteract these burgeoning resistance paradigms given their profound ramifications for prevailing treatment modalities.

Polymyxin resistance caused by large-scale genomic inversion due to IS26 intramolecular translocation in Klebsiella pneumoniae.

Polymyxin B and polymyxin E (colistin) are presently considered the last line of defense against human infections caused by multidrug-resistant Gram-negative organisms such as carbapenemase-producer Enterobacterales, Acinetobacter baumannii, and Klebsiella pneumoniae. Yet resistance to this last-line drugs is a major public health threat and is rapidly increasing. Polymyxin S2 (S2) is a polymyxin B analogue previously synthesized in our institute with obviously high antibacterial activity and lower toxicity than polymyxin B and colistin. To predict the possible resistant mechanism of S2 for wide clinical application, we experimentally induced bacterial resistant mutants and studied the preliminary resistance mechanisms. Mut-S, a resistant mutant of K. pneumoniae ATCC BAA-2146 (Kpn2146) induced by S2, was analyzed by whole genome sequencing, transcriptomics, mass spectrometry and complementation experiment. Surprisingly, large-scale genomic inversion (LSGI) of approximately 1.1 Mbp in the chromosome caused by IS26 mediated intramolecular transposition was found in Mut-S, which led to mgrB truncation, lipid A modification and hence S2 resistance. The resistance can be complemented by plasmid carrying intact mgrB. The same mechanism was also found in polymyxin B and colistin induced drug-resistant mutants of Kpn2146 (Mut-B and Mut-E, respectively). This is the first report of polymyxin resistance caused by IS26 intramolecular transposition mediated mgrB truncation in chromosome in K. pneumoniae. The findings broaden our scope of knowledge for polymyxin resistance and enriched our understanding of how bacteria can manage to survive in the presence of antibiotics.

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.

Background: 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. Objectives: The aim of this study is to explore the source of IS903B in the disruptive mgrB gene and its underlying mechanisms. Materials and methods: 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. Results: 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. Conclusion: 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.

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

BACKGROUND: Egypt 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. METHODS: Seventeen 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. RESULTS: Out 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 ß-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. CONCLUSIONS: In 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.

Molecular characterization of colistin resistance genes in a high-risk ST101/KPC-2 clone of Klebsiella pneumoniae in a University Hospital of Split, Croatia / Caracterización molecular de genes de resistencia a la colistina en un clon ST101/KPC-2 de alto riesgo de Klebsiella pneumoniae en un Hospital Universitario de Split, Croacia

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has become a major concern worldwide due to multidrug resistance and the ability to spread locally and globally. Infections caused by KPC-KP are great challenge in the healthcare systems because these are associated with longer hospitalization and high mortality. The emergence of colistin resistance has significantly reduced already limited treatment options. This study describes the molecular background of colistin-resistant KPC-KP isolates in the largest hospital in southern Croatia. Thirty-four non-duplicate colistin-resistant KPC-KP isolates were collected during routine work from April 2019 to January 2020 and from February to May 2021. Antimicrobial susceptibility was determined using disk diffusion, broth microdilution, and the gradient strip method. Carbapenemase was detected with an immunochromatographic test. Identification of blaKPC and mcr genes or mutations in pmrA, pmrB, mgrB, phoP, and phoQ genes were performed by polymerase chain reaction (PCR) and positive products were sequenced. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. All isolates were multidrug-resistant, with colistin minimum inhibitory concentrations (MICs) from 4 to >16 mg/L, and all harbored blaKPC-2 and had a single point mutation in the mgrB gene resulting in a premature stop codon, with the exception of one isolate with four point mutations corresponding to stop codons. All isolates were negative for mcr genes. PFGE analysis identified a single genetic cluster, and MLST revealed that all isolates belonged to sequence type 101 (ST101). These results show emergence of the high-risk ST101/KPC-2 clone of K. pneumoniae in Croatia as well as appearance of colistin resistance due to mutations in the mgrB gene. Molecular analysis of epidemiology and possible resistance mechanisms are important to develop further strategies to combat such threats.(AU)

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.

Genetic Diversity of Polymyxin-Resistance Mechanisms in Clinical Isolates of Carbapenem-Resistant Klebsiella pneumoniae: a Multicenter Study in China.

Polymyxin has been the last resort to treat multidrug-resistant Klebsiella pneumonia. However, recent studies have revealed that polymyxin-resistant carbapenem-resistant Klebsiella pneumonia (PR-CRKP) emerged due to the mutations in chromosomal genes or the plasmid-harboring mcr gene, leading to lipopolysaccharide modification or efflux of polymyxin through pumps. Further surveillance was required. In the present study we collected PR-CRKP strains from 8 hospitals in 6 provinces/cities across China to identify the carbapenemase and polymyxin resistance genes and epidemiological features by whole-genome sequencing (WGS). The broth microdilution method (BMD) was performed to determine the MIC of polymyxin. Of 662 nonduplicate CRKP strains, 15.26% (101/662) were defined as PR-CRKP; 10 (9.90%) were confirmed as Klebsiella quasipneumoniae by WGS. The strains were further classified into 21 individual sequence types (STs) by using multilocus sequence typing (MLST), with ST11 being prevalent (68/101, 67.33%). Five carbapenemase types were identified among 92 CR-PRKP, blaKPC-2 (66.67%), blaNDM-1 (16.83%), blaNDM-5 (0.99%), blaIMP-4 (4.95%), and blaIMP-38 (0.99%). Notably, 2 PR-CRKP strains harbored both blaKPC-2 and blaNDM-1. The inactivation of mgrB, associated significantly with high-level polymyxin resistance, was mainly caused by the insertion sequence (IS) insertion (62.96%, 17/27). Furthermore, acrR was inserted coincidently by ISkpn26 (67/101, 66.33%). The deletion or splicing mutations of crrCAB were significantly associated with ST11 and KL47 (capsule locus types), and diverse mutations of the ramR gene were identified. Only one strain carried the mcr gene. In summary, the high IS-inserted mgrB inactivation, the close relationship between ST11 and the deletion or splicing mutations of the crrCAB, and the specific features of PR-K. quasipneumoniae constituted notable features of our PR-CRKP strains in China. IMPORTANCE Polymyxin-resistant CRKP is a serious public health threat whose resistance mechanisms should be under continuous surveillance. Here, we collected 662 nonduplicate CRKP strains across China to identify the carbapenemase and polymyxin resistance genes and epidemiological features. Polymyxin resistance mechanism in 101 PR-CRKP strains in China were also investigated, 9.8% of which (10/101) were K. quasipneumoniae, as determined via WGS, and inactivation of mgrB remained the most crucial polymyxin resistance mechanism, significantly related to high-level resistance. Deletion or splicing mutations of crrCAB were significantly associated with ST11 and KL47. Diverse mutations of the ramR gene were identified. The plasmid complementation experiment and mRNA expression analysis further confirmed that the mgrB promoter and ramR played a critical role in polymyxin resistance. This multicenter study contributed to the understanding of antibiotic resistance forms in China.

Within-host resistance evolution of a fatal ST11 hypervirulent carbapenem-resistant Klebsiella pneumoniae.

OBJECTIVES: Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKp) has become a great threat to public health. This study reported an hv-CRKp-associated fatal infection and revealed its mechanisms of antimicrobial resistance and within-host evolution. METHODS: A carbapenem-susceptible K. pneumoniae (CSKp) and 11 KPC-producing CRKp strains were isolated from a lung transplant recipient receiving continual antimicrobial therapy for 1.5 years. Pulsed-field gel electrophoresis (PFGE) separated two clusters between CSKp and CRKp. RESULTS: Further whole genome sequencing analysis found that all 11 CRKp were ST11-KL64 clones, while the CSKp was ST412-KL57. Among these 11 CRKp strains, three and one were resistant to colistin and ceftazidime/avibactam (CAZ/AVI), respectively. Three different mechanisms were found to be responsible for the colistin resistance, including the insertions of two different IS (ISKpn74 and IS903B) into the same position of mgrB and one related to the efflux pump system. CAZ/AVI resistance was associated with blaKPC-2 mutation, and it was also found that increasing blaKPC-2 expression increased the MICs of CAZ/AVI, but not at the resistance level. All these 12 strains had iucABCDiutA virulence cluster and rmpA/rmpA2 genes, with higher siderophore production than a reference classic K. pneumoniae (cKp), which were thought to be hypervirulent K. pneumoniae (hvKp). However, only the CSKp showed higher mucoviscosity according to the mucoviscosity assay. Genomic analysis showed that the rmpA variation (interrupted by ISKpn26) existed in all CRKp strains except the CSKp strain, demonstrating that hypermucoviscous phenotype assays could not accurately identify hvKp. CONCLUSION: This study depicted a rapid and diverse within-host evolution of resistance in hv-CRKp of ST11-KL64 clone.

Genomic characterization of colistin resistance in Klebsiella spp. under the pressure of colistin.

Introduction. Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a serious threat to global public health. Colistin is regarded as the last-resort antibiotic for CRKP infections, but colistin resistance among CRKP is increasingly being reported, making clinical treatment for CRKP infections more difficult.Hypothesis/Gap Statement. The molecular mechanisms of colistin resistance in Klebsiella spp. under the pressure of colistin have not been fully investigated.Aim. We aimed to investigate the phenotypic and genetic variation in two colistin-susceptible Klebsiella spp. strains under selective pressure of colistin.Methodology. One hundred microlitres of overnight cultures of the CRKP clinical strain CRKP12-130 and of ATCC 700603 was spread on five Mueller-Hinton Agar (MHA) plates with colistin concentrations of 2, 4, 8, 16 and 32 µg ml-1, and growth of colonies was observed for five consecutive days. Colonies collected from plates were passaged daily for 10 days on MHA plates without colistin and susceptibility testing of colistin was performed by broth microdilution. Thirty-four colistin-resistant strains randomly selected were submitted to whole genome sequencing (WGS). Transcriptional levels of genes involved in colistin resistance (mgrB, phoP, phoQ, pmrA, pmrB, pmrD, pmrE and pmrK) were measured by quantitative real-time PCR.Results. A total of 114 and 119 colistin-resistant colonies were obtained from CRKP12-130 and ATCC 700603 in this study, among which 16 and 18 colonies were submitted to WGS, respectively. Among these 34 sequenced isolates, mutation in phoQ (13/16, 81.25 %) was the main genetic factor mediating colistin resistance in strains from CRKP12-130, while for strains from ATCC 700603, mutation associated with mgrB (8/18, 44.44 %) was found to be the commonest. Mutation of mgrB led to a significant increase in the MIC for colistin (from 64 to >128 µg ml-1), and a novel mutation C28R in mgrB was first reported in this study.Conclusion. Colistin-resistant Klebsiella spp. could be easily selected under pressure of different concentrations of colistin. Mutations of mgrB, phoP, phoQ and pmrB genes were the main mechanisms leading to chromosomally mediated colistin resistance in Klebsiella spp.

Molecular characterization of colistin resistance genes in a high-risk ST101/KPC-2 clone of Klebsiella pneumoniae in a University Hospital of Split, Croatia.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has become a major concern worldwide due to multidrug resistance and the ability to spread locally and globally. Infections caused by KPC-KP are great challenge in the healthcare systems because these are associated with longer hospitalization and high mortality. The emergence of colistin resistance has significantly reduced already limited treatment options. This study describes the molecular background of colistin-resistant KPC-KP isolates in the largest hospital in southern Croatia. Thirty-four non-duplicate colistin-resistant KPC-KP isolates were collected during routine work from April 2019 to January 2020 and from February to May 2021. Antimicrobial susceptibility was determined using disk diffusion, broth microdilution, and the gradient strip method. Carbapenemase was detected with an immunochromatographic test. Identification of blaKPC and mcr genes or mutations in pmrA, pmrB, mgrB, phoP, and phoQ genes were performed by polymerase chain reaction (PCR) and positive products were sequenced. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. All isolates were multidrug-resistant, with colistin minimum inhibitory concentrations (MICs) from 4 to >16 mg/L, and all harbored blaKPC-2 and had a single point mutation in the mgrB gene resulting in a premature stop codon, with the exception of one isolate with four point mutations corresponding to stop codons. All isolates were negative for mcr genes. PFGE analysis identified a single genetic cluster, and MLST revealed that all isolates belonged to sequence type 101 (ST101). These results show emergence of the high-risk ST101/KPC-2 clone of K. pneumoniae in Croatia as well as appearance of colistin resistance due to mutations in the mgrB gene. Molecular analysis of epidemiology and possible resistance mechanisms are important to develop further strategies to combat such threats.

Polymyxin resistance caused by large-scale genomic inversion due to IS26 intramolecular translocation in Klebsiella pneumoniae

Polymyxin B and polymyxin E (colistin) are presently considered the last line of defense against human infections caused by multidrug-resistant Gram-negative organisms such as carbapenemase-producer Enterobacterales, Acinetobacter baumannii, and Klebsiella pneumoniae. Yet resistance to this last-line drugs is a major public health threat and is rapidly increasing. Polymyxin S2 (S2) is a polymyxin B analogue previously synthesized in our institute with obviously high antibacterial activity and lower toxicity than polymyxin B and colistin. To predict the possible resistant mechanism of S2 for wide clinical application, we experimentally induced bacterial resistant mutants and studied the preliminary resistance mechanisms. Mut-S, a resistant mutant of K. pneumoniae ATCC BAA-2146 (Kpn2146) induced by S2, was analyzed by whole genome sequencing, transcriptomics, mass spectrometry and complementation experiment. Surprisingly, large-scale genomic inversion (LSGI) of approximately 1.1 Mbp in the chromosome caused by IS26 mediated intramolecular transposition was found in Mut-S, which led to mgrB truncation, lipid A modification and hence S2 resistance. The resistance can be complemented by plasmid carrying intact mgrB. The same mechanism was also found in polymyxin B and colistin induced drug-resistant mutants of Kpn2146 (Mut-B and Mut-E, respectively). This is the first report of polymyxin resistance caused by IS26 intramolecular transposition mediated mgrB truncation in chromosome in K. pneumoniae. The findings broaden our scope of knowledge for polymyxin resistance and enriched our understanding of how bacteria can manage to survive in the presence of antibiotics.

Colistin Resistance Mechanisms in Human and Veterinary Klebsiella pneumoniae Isolates.

Colistin (polymyxin E) is increasingly used as a last-resort antibiotic for the treatment of severe infections with multidrug-resistant Gram-negative bacteria. In contrast to human medicine, colistin is also used in veterinary medicine for metaphylaxis. Our objective was to decipher common colistin resistance mechanisms in Klebsiella pneumoniae isolates from animals. In total, 276 veterinary K. pneumoniae isolates, derived from companion animals or livestock, and 12 isolates from human patients were included for comparison. Six out of 276 veterinary isolates were colistin resistant (2.2%). Human isolates belonging to high-risk clonal lineages (e.g., ST15, ST101, ST258), displayed multidrug-resistant phenotypes and harboured many resistance genes compared to the veterinary isolates. However, the common colistin resistance mechanism in both human and animal K. pneumoniae isolates were diverse alterations of MgrB, a critical regulator of lipid A modification. Additionally, deleterious variations of lipopolysaccharide (LPS)-associated proteins (e.g., PmrB P95L, PmrE P89L, LpxB A152T) were identified. Phylogenetic analysis and mutation patterns in genes encoding LPS-associated proteins indicated that colistin resistance mechanisms developed independently in human and animal isolates. Since only very few antibiotics remain to treat infections with MDR bacteria, it is important to further analyse resistance mechanisms and the dissemination within different isolates and sources.