Association of Eux Pump and OMPs with Antibiotic Susceptibility Among ESBL-Producing Klebsiella Pneumoniae Clinical Strains in Malaysia

Multidrug-resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) poses a serious public health threat. K. pneumoniae strains that produce extended-spectrum beta-lactamases (ESBL) are becoming increasingly reported in nosocomial and community-acquired infections. Besides resistance genes, integrons, and plasmids, altered membrane permeability caused by porin loss and energy-dependent eux have also contributed to antibiotic resistance in K. pneumoniae. The objective of this study was to determine the correlation between the reduction of antibiotic susceptibility and overexpression of eux pump as well as the lack of outer membrane proteins (OMPs) among clinical ESBLs resistant K. pneumoniae. The expression levels of ramA, acrA, ompK35 and ompK36 in 12 MDR K. pneumoniae strains with varying MICs levels were analyzed using quantitative real time-Polymerase Chain Reaction (qRT-PCR). The role of eux pump on antibiotic resistance was also studied by using minimum inhibitory concentration (MICs) method with//without eux pump inhibitor. The result indicated that the strains with highest resistance to cefotaxime showed the lowest level of ompK35 and ompK36 genes expression while the strains with lowest MIC level of resistance to cefotaxime showed the highest level of expression of acrA and ramA. Our nding also revealed the effect of eux pump inhibitor phenyl-arginine-b-naphthylamide (PAβN) on the MIC levels of ceftazidime, amoxicillin-clavulanate and cefotaxime which were signicantly reduced around 1–7 folds MIC levels. These results suggest that Eux pump system and deciently of OMPs contributing role in antibiotic susceptibility which should be taken seriously to prevent the treatment failure due to antimicrobial resistance.


Introduction
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  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-lled 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 signi cantly 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 Energy-dependent e ux is also a contributing factor to antibiotic resistance in K. pneumoniae (Padilla et al., 2010). One of the e ux systems involved in this resistance phenotype is the AcrAB multidrug e ux 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., 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 e ux 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.

Materials And Methods
Bacterial strains and PCR detection of OmpK35, OmpK36, acrA and ramA genes Twelve Malaysian K. pneumoniae strains were previously isolated and con rmed as ESBL-producer phenotypically and genotypically (Mobasseri et al., 2020). Genomic DNAs of these 12 K. pneumoniae strains was extracted by using genomic DNA extraction kit (YEASTERN Biotech Co., Ltd.). PCR detection for ompK35, ompK36, acrA and ramA genes was performed using established primers (Ruzin et al., 2008; Landman et al., 2009). All PCR ampli ed products were puri ed and sent for DNA sequencing to con rm their identity. Total RNA for 12 selected ESBL-producing strains were obtained from late-exponential-phase cultures using RNeasy kit (Qiagen, Germany), the extracted RNA was quanti ed using NanoDrop (IMPLEN, Germany) and adjusted to 25 ng. The RNA was then subjected to transcription using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Foster City, California, USA) and gene expression for ompK35, ompK36, ramA and acrA as previously described by Ruzin et al. In this study, we showed correlation between overexpression of e ux pump and lacking of OMPs with ESBLs resistance in twelve ESBL-producing K. pneumoniae strains.
The minimal inhibitory concentrations (MIC) for ceftazidime, cefotaxime and amoxicillin-clavulanate for the 12 ESBL-producing strains with and without PAβN (e ux pumps inhibitor) are summarized in Table 1. MIC results for ESBL-producing strains showed the range of resistance to cefotaxime (MIC from > 256 µg/mL to 16 µg/mL), cefotaxime + PAβN (MIC from > 128 µg/mL to 1 µg/mL), ceftazidime (MIC from > 256 µg/mL to > 8 µg/mL), ceftazidime + PAβN (MIC from > 96 µg/mL to > 0.75 µg/mL), amoxicillinclavulanate (MIC from > 256 µg/mL to > 4 µg/mL), and amoxicillin-clavulanate + PAβN (MIC from > 32 µg/mL to > 2 µg/mL). Overall, the MIC levels in these ESBL-producing strains were reduced 2-5 folds for cefotaxime and ceftazidime and 1-7 folds for amoxicillin-clavulanate (Table 1)  All targeted e ux pump and porin-associated genes were observed in the 12 ESBL-producing strains used in the determination of gene expression levels in this study. The results of the qRT-PCR analysis for acrA and ramA gene expression levels clearly indicated the correlation between reduced susceptibility to cefotaxime and AcrA, ramA gene overexpression. Strain KP2014C19 (cefotaxime MIC ranged 16 (µg/mL)) showed lowest level of gene expression for acrA and ramA (7.51 ΔCT, 6.07 ΔCT, respectively), while in KP2014C68 (cefotaxime MIC ranged > 256 (µg/mL), gene expression levels of acrA and ramA dramatically increased to highest level of gene expression (38.24 ΔCT, 23.7 ΔCT, respectively) (Table 2) (Fig. 2). These data concurred with studies by Mazzariol  In this study, the OmpK35 and OmpK36 gene expression pro les showed that the strain KP2014C37 (cefotaxime MIC ranged 16 (µg/mL)) showed highest level of gene expression for OmpK35 and OmpK36 (1.02 ΔCT, 1.31 ΔCT, respectively), while gene expression levels of OmpK35 and OmpK36 have dramatically decreased to lowest level of gene expression (0.29 ΔCT, 0.35 ΔCT, respectively) in KP2014C13 (cefotaxime MIC ranged > 256 (µg/mL)) (Table 2) (Fig. 2). The expression of ompK35, ompK36 genes is a major factor in conferring resistance against ESBLs in K. pneumoniae (Uz-Zaman et al., 2014). The lack of OmpK35 has been reported as the major porin through which ceftazidime penetrates the Gram-negative outer membrane in many ESBL-producing K. pneumoniae strains while the absence or reduced expression of the two major porins (OmpK35 and OmpK36) in K. pneumoniae in combination with various β-lactamases has been implicated in carbapenem resistance by previous studies ( In conclusion, this study showed the correlation between the reduction of antibiotic susceptibility and expression of e ux pump and OMPs as well as role of e ux pump inhibitor among ESBL-producing K. pneumoniae. The presence of e ux pump inhibitor PAβN, has direct association with MIC levels of CAZ, CTX and AMC which is more signi cant in amoxicillin-clavulanate MIC levels (reduced 1-7 folds) followed by 2-5 folds reduction in ceftazidime, these results suggested that the major ESBL-resistance mechanism found in these strains is a PAβN-sensitive mechanism, namely, a drug e ux mechanism (Hasdemir et al., 2004). Studies on mechanisms and structure-function association of bacterial OMPs and e ux systems as well as the interactions between the pumps and other resistance mechanisms are needed to monitor the usage of antibiotics in hospital settings to control and prevent antimicrobial resistance issue.