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Multidrug-resistant Klebsiella pneumoniae: genetic diversity, mechanisms of resistance to polymyxins and clinical outcomes in a tertiary teaching hospital in Brazil.

ABSTRACT: Increased resistance to polymyxin in Klebsiella pneumoniae (ColRKP) has been observed. Molecular epidemiology, as well as the clinical impact of these difficult to treat pathogens need to be better characterized. We present the clinical outcomes of 28 patients infected by ColRKP in a tertiary hospital. Isolates with MIC >2 by Vitek 2 were confirmed by the microdilution broth test. Polymerase chain reaction (PCR) was performed for blaKPC, blaNDM, blaOXA-48 and blamcr-1 genes in the isolates, and Whole Genome Sequencing (WGS) was performed in six isolates. Seventeen (61%) patients were female and the mean age was 50 years old. In-hospital and 30-day mortality were 64% (18/28) and 53% (15/28), respectively. Central line-associated bloodstream infection in addition to bacteremia episodes due to other sources were the most frequent (61%). Mean APACHE and Charlson comorbidity index were 16 and 5, respectively. Twenty patients (71%) received at least one active drug and ten (35%) received two drugs: tigecycline 46% (13/28); amikacin 21% (6/28) and fosfomycin 3% (1 case). Twenty-six out of 28 tested cases were positive for blaKPC. Eight different clusters were identified. Four STs were detected (ST11, ST23, ST340, and ST437). Mutations on pmrA, arnB, udg, and yciM genes were present in all six isolates submitted to WGS; lpxMand mgrB mutations were also detected in all but one isolate. In conclusion, we observed resistance to polymyxin in severely ill patients mostly from intensive care units and/or immunosuppressed patients with high mortality rates in whom a diversity of ColRKP clusters was identified and might indicate selective pressure.

SUBMITTER: Boszczowski I

PROVIDER: S-EPMC6592011 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Multidrug-resistant Klebsiella pneumoniae: genetic diversity, mechanisms of resistance to polymyxins and clinical outcomes in a tertiary teaching hospital in Brazil.

Boszczowski Icaro I Salomão Matias Chiarastelli MC Moura Maria Luísa ML Freire Maristela Pinheiro MP Guimarães Thais T Cury Ana Paula AP Rossi Flávia F Rizek Camila Fonseca CF Martins Roberta Cristina Ruedas RCR Costa Silvia Figueiredo SF

Revista do Instituto de Medicina Tropical de Sao Paulo 20190619

Increased resistance to polymyxin in Klebsiella pneumoniae (ColRKP) has been observed. Molecular epidemiology, as well as the clinical impact of these difficult to treat pathogens need to be better characterized. We present the clinical outcomes of 28 patients infected by ColRKP in a tertiary hospital. Isolates with MIC >2 by Vitek 2 were confirmed by the microdilution broth test. Polymerase chain reaction (PCR) was performed for blaKPC, blaNDM, blaOXA-48 and blamcr-1 genes in the isolates, and ...[more]

PMID: 31241658

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Project description:Multidrug-resistant (MDR) Klebsiella pneumoniae (Kp) is a major bacterial pathogen responsible for hospital outbreaks worldwide, mainly via the spread of high-risk clones and epidemic resistance plasmids. In this study, we evaluated the molecular epidemiology and β-lactam resistance mechanisms of MDR-Kp strains isolated in a Brazilian academic care hospital. We used whole-genome sequencing to study drug resistance mechanisms and their relationships with a K. pneumoniae carbapenemase-producing (KPC) Kp outbreak. Forty-three Kp strains were collected between 2003 and 2012. Antimicrobial susceptibility testing was performed for 15 antimicrobial agents, and polymerase chain reaction (PCR) was used to detect 32 resistance genes. Mutations in ompk35, ompk36, and ompk37 were evaluated by PCR and DNA sequencing. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were carried out to differentiate the strains. Based on distinct epidemiological periods, six Kp strains were subjected to whole-genome sequencing. β-lactamase coding genes were widely distributed among isolates. Almost all isolates had mutations in porin genes, particularly ompk35. The presence of bla KPC promoted a very high increase in carbapenem minimum inhibitory concentration only when ompk35 and ompk36 were interrupted by insertion sequences. A major cluster was identified by PFGE analysis and all isolates from this cluster belonged to clonal group (CG) 258. We have also identified a large repertoire of resistance genes in the sequenced isolates. A bla KPC-2-bearing plasmid (pUFPRA2) was also identified, which was very similar to a plasmid previously described in the first Brazilian KPC-Kp (2005). We found high-risk clones (CG258) and an epidemic resistance plasmid throughout the duration of the study (2003 to 2012), emphasizing a persistent presence of MDR-Kp strains in the hospital setting. Finally, we found that horizontal transfer of resistance genes between clones may have played a key role in the evolution of the outbreak.

Project description:BackgroundOver the last two decades, the prevalence of colistin resistance among the members of Enterobacteriaceae has been increasing, particularly among Klebsiella pneumoniae isolates; this limits the potential use of colistin and leads to worsened clinical outcomes.MethodsWe investigated the prevalence and genetic characteristics of colistin-resistant K. pneumoniae (COLR-KP) in clinical isolates using genomic sequencing.ResultsIn total, 53 K. pneumoniae isolates (4.5%, 53/1,171) were confirmed as COLR-KP, of which eight isolates carried mobile colistin-resistant (mcr) gene. Although the overall prevalence rate (0.7%, 8/1,171) of mcr-like genes in clinical K. pneumoniae remained relatively low, the presence of mcr (15.1%, 8/53) among the COLR-KP isolates indicated that the mobile resistance gene was already widespread among K. pneumoniae isolates in hospital setting. We randomly selected 13 COLR-KP isolates (four mcr-bearing and nine non-mcr-bearing isolates) for whole-genome sequencing, including two pandrug-resistant and four sequence type 11 (ST11) isolates. Phylogenetic analysis revealed that all COLR-KP isolates were genetically diverse. Among the four mcr-bearing isolates, three (KP4, KP18, and KP30) were positive for mcr-1 and one (KP23) for mcr-8; none of the other mcr genes were detected. The mcr-1 in the KP4 and KP30 isolates were located in an IncX4 plasmid (approximately 33 kb) and could be successfully transferred to Escherichia coli J53AZR. In contrast, for the mcr-8-bearing plasmid in KP23 (IncFII), colistin resistance could not be transferred by conjugation. The mcr-1-producing isolate KP18 coexists a novel plasmid-carried tigecycline resistance gene tmexCD1-toprJ1. The most common chromosomal mutation associated with colistin resistance was a T246A amino acid substitution in PmrB, which was identified in most COLR-KP isolates (11/13, 84.6%). All ST11 isolates additionally had an R256G amino acid substitution. Critical virulence factors associated with hypervirulent K. pneumoniae were detected in four COLR-KP isolates; these virulence factors included aerobactin, salmochelin, and yersiniabactin.ConclusionWe found that mcr-bearing COLR-KP emerged in our hospital and was growing at an increasing rate. Simultaneous emergence of hypervirulence and colistin-tigecycline-carbapenem resistance in the epidemic clone ST11 K. pneumoniae was also observed; this highlights the significance of active and continuous surveillance.

Project description:Multidrug-resistant gram-negative bacteria, such as carbapenem and colistin-resistant Klebsiella pneumoniae (ColR-CRKP), represent a major problem for health systems worldwide and have high lethality. This study investigated the genetic relationship, antimicrobial susceptibility profile, and resistance mechanisms to ColR-CRKP isolates from patients infected/colonized in a tertiary hospital in Salvador, Bahia/Brazil. From September 2016 to January 2018, 46 patients (56 ColR-CRKP positive cultures) were enrolled in the investigation but clinical and demographic data were obtained from 31 patients. Most of them were men (67.7%) and elderly (median age of 62 years old), and the median Charlson score was 3. The main comorbidities were systemic arterial hypertension (38.7%), diabetes (32.2%), and cerebrovascular disease (25.8%). The average hospitalization stay until ColR-CRKP identification in days were 35.12. A total of 90.6% used mechanical ventilation and 93.7% used a central venous catheter. Of the 31 patients who had the data evaluated, 12 had ColR-CRKP infection, and seven died (58.4%). Previous use of polymyxins was identified in 32.2% of the cases, and carbapenems were identified in 70.9%. The minimum inhibitory concentration (MIC) for colistin was > 16 μg/mL, with more than half of the isolates (55%) having a MIC of 256 μg/mL. The bla KPC gene was detected in 94.7% of the isolates, bla NDM in 16.0%, and bla GES in 1.7%. The bla OXA-48, bla VIM, and bla IMP genes were not detected. The mcr-1 test was negative in all 56 isolates. Alteration of the mgrB gene was detected in 87.5% (n = 49/56) of the isolates, and of these, 49.0% (24/49) had alteration in size probably due to IS903B, 22.4% (11/49) did not have the mgrB gene detected, 20.4% (10/49) presented the IS903B, 6.1% (3/49) had a premature stop codon (Q30*), and 2.1% (1/49) presented a thymine deletion at position 104 - 104delT (F35fs). The PFGE profile showed a monoclonal profile in 84.7% of the isolates in different hospital sectors, with ST11 (CC-258) being the most frequent sequence type. This study presents a prolonged outbreak of ColR-CRKP in which 83.9% of the isolates belonged to the same cluster, and 67.6% of the patients evaluated had not used polymyxin, suggesting the possibility of cross-transmission of ColR-CRKP isolates.

Project description:BackgroundAntibiotic-resistant Klebsiella pneumoniae has emerged as a critical public health threat worldwide. Understanding the antimicrobial resistance mechanisms of multidrug-resistant K. pneumoniae (MDR-Kp) and its prevalence in time and space would provide clinical significance for managing pathogen infection.MethodsEighteen clinical MDR-Kp strains were analyzed by whole genome sequencing (WGS), and the antimicrobial resistance genes and associated resistance mechanisms were compared with results obtained from the conventional microbiological test (CMT). The sequence homology across strains in our study and those previously collected over time from a wide geographical region was assessed by phylogenetic analysis.ResultsMDR-Kp strains were collected from eighteen patients who had received empirical treatment before strain collection, with sputum (83.3%, 15/18) being the primary source of clinical samples. The commonly received treatments include β-lactamase inhibitors (55.6%, 10/18) and carbapenems (50%, 9/18). Using CMT, we found that all 18 strains were resistant to aztreonam and ciprofloxacin, while 14 (77.8%) showed resistance to carbapenem. Polymyxin B and tigecycline were the only antibiotics to which MDR-Kp strains were sensitive. A total of 42 antimicrobial resistance mechanisms were identified by WGS, surpassing the 40 detected by the conventional method, with 25 mechanisms shared between the two techniques. Despite a 100% accuracy rate of WGS in detecting penicillin-resistant strains, the accuracy in detecting cephalosporin-resistant strains was only at 60%. Among all resistance genes identified by WGS, Klebsiella pneumoniae carbapenemase-2 (KPC-2) was present in all 14 carbapenem-resistant strains. Phenotypic analysis indicated that sequence type (ST) 11 isolates were the primary cause of these MDR-Kp infections. Additionally, phylogenic clustering analysis, encompassing both the clinical and MDR-Kp strains previously reported in China, revealed four distinct subgroups. No significant difference was observed in the sequence homology between K. pneumoniae strains in our study and those previously collected in East China over time.ConclusionThe application of WGS in identifying potential antimicrobial-resistant genes of MDR-Kp has demonstrated promising clinical significance. Comprehensive genomic information revealed by WGS holds the promise of guiding treatment decisions, enabling surveillance, and serving as a crucial asset in understanding antibiotic resistance.

Dataset Information

Multidrug-resistant Klebsiella pneumoniae: genetic diversity, mechanisms of resistance to polymyxins and clinical outcomes in a tertiary teaching hospital in Brazil.

Publications

Multidrug-resistant Klebsiella pneumoniae: genetic diversity, mechanisms of resistance to polymyxins and clinical outcomes in a tertiary teaching hospital in Brazil.

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