Antibiotic resistance in Gram-negative bacilli : with focus on cephalosporin resistance mechanisms in Escherichia coli and Klebsiella pneumoniae

Objectives: The objectives of this thesis were to study mechanisms conferring cefuroxime resistance in clinical non-ESBL (extended-spectrum beta-lactamase) producing isolates of Escherichia coli and Klebsiella pneumoniae, as well as the presence of co-resistance to other antibiotics in the K. pneumoniae isolates. Further, the objectives were to study findings of and resistance patterns of Gram-negative bacilli isolated from patients seeking medical advice in Stockholm after the tsunami catastrophe.

Materials and methods: In paper I 30 clinical non-ESBL blood culture isolates of E. coli with various degrees of cefuroxime susceptibility were examined. Cefuroxime susceptibility was determined in the presence of the efflux pump inhibitor phenylalanine arginine beta-naphthylamide (PAbetaN). Organic solvent tolerance (OST) was determined, as a phenotypic method for detecting efflux. In paper II 14 clinical non-ESBL isolates of E. coli with various degrees of cefuroxime susceptibility were examined. Transcription levels of acrA (encoding AcrA, part of the AcrAB-TolC efflux pump) and ompF (encoding the porin OmpF) were determined with quantitative RTPCR. AmpC activity was determined with spectrophotometry. In paper III an epidemiological survey was made of findings of and resistance patterns of Gramnegative bacilli from patients seeking medical advice in Stockholm after the tsunami catastrophe. In paper IV ten blood culture isolates of K. pneumoniae, all cefuroximeresistant, but cefotaxime susceptible and a multiply antibiotic resistant (MAR) laboratory strain (selected by chloramphenicol) were examined. Transcription levels of acrA, ompK35 (the homologue of ompF), and of the regulatory genes ramA, marA and soxS were determined with quantitative RT-PCR. Antibiotic susceptibility was also determined in the presence of PAbetaN.

Results: In paper I was shown that the organic solvent tolerant isolates had significantly decreased susceptibility to cefuroxime compared to the non-tolerant isolates. Further, the susceptibility to cefuroxime was increased by PAbetaN for thecyclohexane tolerant isolates. In paper II, increased acrA transcription was seen in seven of the eleven cefuroxime resistant isolates. Very low ompF transcription levels were seen in three and increased AmpC activity in two of the cefuroxime resistant isolates. Paper III showed that findings of various Gram-negative bacilli were common in secretion cultures from the tsunami victims and that resistant isolates of E. coli, K. pneumoniae, Proteus mirabilis and Acinetobacter spp. were more common in cultures from tsunami victims compared to the Swedish reference material, as was not the case for Pseudomonas aeruginosa. In paper IV all examined isolates and the laboratory strain showed similar antibiograms with decreased susceptibility to cefuroxime, chloramphenicol, nalidixic acid and tigecycline. All strains also had increased acrA transcription and decreased ompK35 transcription. The laboratory strain and all the clinical isolates except one displayed increased ramA transcription. PAbetaN increased susceptibility to chloramphenicol, nalidixic acid and tigecycline, but not to cefuroxime.

Conclusions: In clinical non-ESBL cefuroxime resistant isolates of E. coli several resistance mechanisms (efflux of the AcrAB-TolC complex, lack of OmpF and increased AmpC activity) seem to contribute to cefuroxime resistance. In K. pneumoniae a multidrug resistant (MDR) phenotype was observed in all the examined isolates, with decreased susceptibility to cefuroxime, chloramphenicol, nalidixic acid and tigecycline. Further, the same resistance mechanisms were detected in almost all clinical isolates. The findings of various Gram-negative bacteria from tsunami victims affect which empirical antibiotic treatment should be given when these kinds of traumatic infections are treated.

List of scientific papers

I. Källman O, Fendukly F, Karlsson I, Kronvall G (2003). Contribution of efflux to cefuroxime resistance in clinical isolates of Escherichia coli. Scand J Infect Dis. 35(8): 464-70.
https://pubmed.ncbi.nlm.nih.gov/14514145

II. Källman O, Giske CG, Samuelsen Ø, Wretlind B, Kalin M and Olsson-Liljequist B (2008). Interplay of efflux, impermeability and AmpC activity contributes to cefuroxime resistance in clinical, non-ESBL isolates of Escherichia coli. [Submitted]

III. Källman O, Lundberg C, Wretlind B, Ortqvist A (2006). Gram-negative bacteria from patients seeking medical advice in Stockholm after the tsunami catastrophe. Scand J Infect Dis. 38(6-7): 448-50.
https://pubmed.ncbi.nlm.nih.gov/16798691

IV. Källman O, Motakefi A, Wretlind B, Kalin M, Olsson-Liljequist B, Giske CG (2008). Cefuroxime non-susceptibility in multidrug-resistant Klebsiella pneumoniae overexpressing ramA and acrA and expressing ompK35 at reduced levels. J Antimicrob Chemother. Jul 21: Epub ahead of print
https://pubmed.ncbi.nlm.nih.gov/18647746