Klebsiella pneumoniae is a one of the most important hospital-acquired pathogens causing a wide range of infections such as pneumonia, urinary tract infections and septicaemia (De Oliveira et al., 2008). The overuse of expanded-spectrum cephalosporins for the treatment of these organisms has led to the emergence of ESBLs production in K. pneumoniae strains reported worldwide (Ali et al., 2018; Keynan & Rubinstein, 2007; Ben Hamouda et al., 2003; Romero et al., 2007). In Malaysia, Palasubramaniam et al. (2005) reported the first nosocomial outbreak of ESBL- producing K. pneumoniae associated with SHV-5 ESBL enzyme. Other ESBL types have also been reported between 2009–2019 (CTX-M, SHV, TEM) (Lim et al., 2009; Al Marzooq et al., 2015; Low et al., 2017, Mobasseri et al., 2020).
The cell envelope of Gram-negative bacterium consists of three layers: the outer membrane, the peptidoglycan cell wall, and the inner membrane. The outer membrane porin (Omp) is a trimeric membrane protein that functions as water-filled protein channels for transportation of small hydrophilic molecules such as iron, nutrients, and antibiotics (including β-lactams) across the outer membrane. Porins also function as the receptors for phages, bacteriocins and in conjunction with peptidoglycan and lipopolysaccharide (LPS) in maintaining the structure the cells (Tsai et al., 2011).
The role of porins in Gram-negative bacteria has been studied intensively. Two major porins, OmpC and OmpF which are found in E. coli and Salmonella serovars, have been known as homologue to OmpK35 and OmpK36 in K. pneumoniae, (Doménech-Sánchez et al., 2009; Tsai et al., 2011). In general, OmpF has slightly larger pore than OmpC allowing more molecules to pass through (Jiang et al., 2009; Tsai et al., 2011). In ESBL-producing K. pneumoniae, the expression of OmpK36 was significantly higher than OmpK35. In some strains, the OmpK35 porin has not been expressed at all. The low or non-expression of ompK35 has increased the antibiotic resistance of ESBL-producing K. pneumoniae (Palasubramaniam et al., 2009). In addition, the role of OmpK36 in carbapenem-resistance has been elucidated in reports worldwide (Uz-Zaman et al., 2014; Malek et al., 2019; Wise et al., 2018).
Energy-dependent efflux is also a contributing factor to antibiotic resistance in K. pneumoniae (Padilla et al., 2010). One of the efflux systems involved in this resistance phenotype is the AcrAB multidrug efflux system that encoded by the AcrRAB operon. In AcrRAB operon, AcrAB repressor is encoded by acrR, while acrA and acrB encode a periplasmic lipoprotein, AcrB connects with TolC, an outer membrane protein which belongs to a family of envelope proteins and are found in all Gram-negative bacteria (Padilla et al., 2010). The increasing AcrAB efflux pump expression in MDR-resistant K. pneumoniae strains was reported to be caused by mutation in AcrR, AcrAB repressor, or overexpression of RamA (transcriptional regulator) (Schneiders et al., 2003). Correlation between reduced susceptibility to antibiotics and AcrA overexpression have been reported in several studies (Mazzariol et al., 2002; Schneiders et al., 2003; Hasdemir et al., 2004; Padilla et al., 2010).
To better manage the increase of the antimicrobial resistance in worldwide, there is a need to investigate other mechanisms that might contribute to resistance. Therefore, the objectives of this study were to determine the correlation between reduced susceptibility and overexpression of efflux genes among 12 ESBL-producing K. pneumoniae isolated from a hospital in Johor Bahru, Malaysia. In addition, the effects of the presence and expressions of OMPs on the resistance levels of the strains were also investigated.