Susceptibility and bactericidal activity of five biocides on Klebsiella pneumoniae and its association with efflux pump genes and antibiotic resistance

Abstract

Klebsiella pneumoniae is one of the top eight pathogens in hospitals, causing around 10% of hospital-acquired infections (nosocomial infections). It often produces extended-spectrum β-lactamase enzymes (ESBLs). This has led to numerous outbreaks, especially in intensive care, neonatal and surgical wards, associated with increases in morbidity and mortality. In order to reduce the number of infections caused by multi-resistant K. pneumoniae and improve standards of infection control within hospitals, there is extensive use of biocides as disinfectants and antiseptics. However this raises concerns that intensive exposure of hospital pathogens to biocides may result in the emergence of resistance not just to themselves but also to antibiotics. The reduced susceptibility to biocides and their relationship with resistance to antibiotics was assessed in this thesis. The susceptibility of 64 isolates of K. pneumoniae to five biocides preparations, Chlorhexidine (CHX), Benzalkonium chloride (BZK), Trigene, MediHex-4 (MH-4), Mediscrub (MS) and 17 antibiotics, were tested. The isolates of K. pneumoniae were collected from Royal Infirmary Hospital in Edinburgh (RIE) between 2006 and 2008 from different sites of infection. Antimicrobial susceptibility was tested by the agar double dilution method (DDM) and disc diffusion methods following the British Society for Antimicrobial Chemotherapy (BSAC) guidelines. A few isolates of K. pneumoniae showed insusceptibility to cephalosporins, colistin, rifampicin, trimethoprim and penicillin but not to carbapenems. Biocide susceptibility testing showed that 57, 55 and 61 strains had reduced susceptibility to Chlorhexidine, Trigene and Benzalkonium chloride, respectively, but not to MediHex-4 and Mediscrub. The effect of efflux pumps were determined by carbonyl cyanide m-chlorophenylhydrazone (CCCP) (10mg/L), which decreased the MICs of Chlorhexidine and Medihex-4 by 2 – 128 fold but had no impact on the MICs of Benzalkonium chloride, Trigene and Mediscrub. Six isolates of K. pneumoniae were chosen for their varying sensitivity to Chlorhexidine (CHX), and were tested for their minimum bactericidal concentration (MBC) to biocides. The high MBCs of Mediscrub and Trigene, over 500-fold greater than the minimum inhibitory concentration (MICs), indicates that these compounds are mainly bacteriostatic. Conversely, the MBCs of Chlorhexidine and MediHex-4, which contains chlorhexidine, were less than 10-times the MIC value indicating they are effective in killing the organism. However, this thesis showed how the killing capability of Chlorhexidine was hindered by the presence of organic matter, which compromised its effect. The relationship between reduced susceptibility to biocides and the carriage of antiseptic resistance genes, cepA, qacΔE1 and qacE was determined by polymerase chain reaction. The antiseptic resistance genes cepA, qacΔE1 and qacE were found in 56, 34 and 1 isolates respectively, and the levels of gene expression were detected by the reverse transcription polymerase chain reaction (RT-PCR). These results have shown that there was a close link between carriage of efflux pump genes, cepA, qacΔE1 and qacE genes and reduced susceptibility to biocides. Most strains showed decreased susceptibility to Chlorhexidine, Trigene and Benzalkonium chloride and this correlated with the carriage of the cepA, qacΔE1 and qacE genes encoding efflux. There was no correlation between the reduced susceptibility to biocides and antibiotic resistance in clinical isolates of K. pneumoniae.