Novel diagnostic methods for rapid characterization of extended-spectrum beta-lactamase- and carbapenemase-producing Enterobacteriaceae

Klebsiella pneumoniae and Escherichia coli are two of the most clinically important pathogens of the family Enterobacteriaceae. Both can cause severe infections including urinary tract infections (UTI), bloodstream infections (BSI) and pneumonia. Both are also involved in dissemination of antibiotic resistance. The prevalence of extended-spectrum β-lactamase(ESBL) producing Enterobacteriaceae (EPE) and carbapenemase-producing Enterobacteriaceae (CPE) are increasing worldwide. This is a severe threat to public health, as antibiotic treatment becomes limited. The general aim of the thesis was to investigate the clonal diversity and prevalence of ESBL in invasive isolates of Klebsiella pneumoniae from the Stockholm area, the acquisition of EPE/CPE in healthy Swedish tourists travelling to regions with high prevalence of EPE/CPE, and finally to evaluate rapid detection and characterization methods for EPE and CPE. In Paper I, we studied the clonal structure of K. pneumoniae from BSI patients in a well-defined geographical area, as well as the prevalence of ESBL in these strains. We also characterized the clinical isolates to investigate the association between phylogroups, bacterial virulence factors, 30-day mortality and comorbidity. A total of 139 samples were collected from the Department of clinical microbiology at Karolinska University Hospital, Stockholm, Sweden. Only five out of 139 isolates were detected as multidrug-resistant (MDR) and all five isolates were EPE. Only one isolate was detected as a CPE. Further, all isolates were tested for mucoid pheno types, serotypes (K1, K2, K5, K20, K54 and K57) and screened for virulence genes using real-time PCR. To identify risk factors, we retrieved data from medical records including age, sex, time to adequate treatment, hospital acquired infections, comorbidity and mortality. The 30-day mortality was primary end-point. All isolates were further subjected to multilocus sequence typing (MLST) for phylogenetic analysis. We found that all isolates were divided into three distinct phylogroups; KpI (consisting of 96 isolates of K. pneumoniae) followed by phylogroup KpIII (consisting of 34 isolates of K. variicola) and phylogroup KpII (consisting of 9 isolates of K. quasipneumoniae). We observed 24/139 (17.3%) overall 30-day mortality. We also observed that K. variicola KpIII (29.4%) were highly associated with 30-day mortality compared to K. pneumoniae KpI isolates (13.5%). We also observed that several comorbidities; malignancy, diabetes mellitus, cirrhosis, biliary tract disorders and alcoholism among patients may influence the mortality. In Paper II, we evaluated a commercial version of RAPIDEC CARBA NP for rapid detection of carbapenemases against 276 Gram-negative bacilli. According to the manufacturer’s protocol RAPIDEC CARBA NP assay was performed on 138 carbapenemase-producers and 138 non-carbapenemase-producers. Carbapenemase detection was also performed by using conventional and real-time PCR. A total of 135 out of 138 carbapenemase-producers were detected successfully by the RAPIDEC CARBA NP assay. The RAPIDEC CARBA NP assay was unable to detect one OXA-48-producing K. pneumoniae and two Acinetobacter baumannii producing OXA-23 and OXA-24 carbapenemases. The RAPIDEC CARBA NP assay successfully detected 135 non-carbapenemase producers. A total of two false positives; one Pseudomonas aeruginosa with OprD loss and efflux, one Enterobacter cloacae with impermeability were detected by the RAPIDEC CARBA NP assay. The overall sensitivity and specificity of the RAPIDEC CARBA NP assay was 97.8% and 98.5%, respectively. In Paper III, we investigated the acquisition of EPE and CPE among healthy Swedish travellers visiting regions with high prevalence of EPE and CPE; Southeast Asia, Indian subcontinent, North Africa and the Middle East. We studied 188 healthy adult travellers and excluded 13 pre-travel colonized tourists from the analysis. Molecular characterization was performed using real-time PCR and survey data were collected for analysis. We detected a total of 67/175 tourist were colonized by EPE strains and most dominant species was E. coli (n=65), followed by K. pneumoniae (n=1) and Citrobacter freundii (n=1). CTX-M type (CTX-M-1 n=49, CTX-M-9 n=12) was the most prevalent EPE type detected in 61 isolates but carbapenemases were absent. Only six travellers had strains with the virulence factor pyelonephritis-associated pili-encoding genes (pap) genes, and all of those had travelled to India. One traveller to Thailand was colonized with colistin resistance gene mcr-1. The highest colonization rate was detected among the travellers to the Indian subcontinent (49%) and northern Africa (44%) whereas lower rates were detected in travellers to Southeast Asia (19%) and Turkey (10%). EPE colonization was associated with diarrhea and antibiotic treatment during the trip but was not associated with sex, age, duration of trip, intake of proton-pump inhibitors, or use of oral cholera vaccine. In paper IV, we assessed a CRISPR/Cas assisted optical DNA mapping method for rapid identification and characterization of plasmids during an EPE outbreak. We studied a total of 17 neonates initially colonized with ESBL-producing K. pneumoniae (ESBL-KP). In follow-up samples we observed that some of them were colonized with ESBL-producing E. coli. This method successfully detected two plasmids of different sizes; small size (80 kbp) and larger plasmid (162-222 kbp) in all ESBL-KP isolates. Using this method, we also observed that the blaCTX-M-15 gene was located on the smaller size plasmid. In follow-up samples we also observed that the ESBL-KP clone was present and blaCTX-M-15 was stable up to two years. This method demonstrated that plasmids can change over time: three deletions were observed in three plasmids of three isolates with different sizes (~ 5 kbp), (~ 55 kbp) and (~ 31 kbp) and an inversion of 31 kbp in another isolate. This method also confirmed that resistance plasmids were not transferred from one patient to another. In conclusion, this thesis provides new knowledge on the prevalence of EPE and CPE among BSI patients and healthy Swedish tourists, along with describing the molecular features of ESBLand carbapenemase-producing Enterobacteriaceae. This thesis also demonstrates the performance of a rapid phenotypic carbapenemase detection assay, as well as a novel CRISPR/Cas-assisted optical DNA mapping method for rapid plasmid characterization. LIST OF SCIENTIFIC PAPERS I. Makaoui Matallah, Malin Vading, Muhammad Humaun Kabir, Amina Bakhrouf, Mats Kalin, Pontus Naucler, Sylvain Brisse, Christian G Giske. Klebsiella variicola is a frequent cause of bloodstream infection in the Stockholm area, and associated with higher mortality compared to K. pneumoniae. PLoS One. 2014;9(11):e113539 II. Muhammad Humaun Kabir, Daniele Meubier, Katie L. Hopkins, Christian G. Giske and Neil Woodford. A two-centre evaluation of RAPIDEC CARBA NP for carbapenemase detection in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp. J Antimicrob Chemother. 2015; 71(5):1213-6. III. Malin Vading, Muhammad Humaun Kabir, Mats Kalin, Aina Iversen, Susanne Wiklund, Pontus Naucler, Christian G. Giske. Frequent acquisition of low-virulent strains of ESBL-producing Escherichia coli in travellers. J Antimicrob Chemother. 2016;71(12):3548-3555. IV. Santosh K. Bikkarolla, Viveka Nordberg, Fredrika Rajer, Vilhelm Müller, Muhammad Humaun Kabir, Sriram Kesarimangalam, Albertas Dvirnas, Tobias Ambjörnsson, Christian G. Giske, Lars Navér, Linus Sandegren, Fredrik Westerlund. Optical DNA mapping combined with Cas9-targeted resistance gene identification for rapid tracking of resistance plasmids in a neonatal intensive care unit outbreak. MBio 2019;10(4). pii: e00347-19.