Molecular Docking of Multidrug Resistant Klebsiella pneumoniae from River Water of Klang Valley, Malaysia

Malaysia Klang River covering states of Selangor and Kuala Lumpur, has been severely polluted from urbanization, industrialization and increased hospitals. By serving as reservoir for bacterial gene transformation, Multidrug Resistant (MDR) strains especially Extended Spectrum Beta Lactamase (ESBL) resistant strains of Klebsiella pneumoniae (K. pneumoniae) have increased. To identify MDR K.pneumoniae; phenotypically determine ESBL resistant traits and molecular characterize these strains, 50 water samples were collected along Klang River and their physicochemical properties were determined. Gram-negative K. pneumoniae isolates were tested for multidrug and ESBL resistance using Kirby-Bauer method. DNA of ESBL resistant K. pneumoniae were extracted and amplified using Polymerase Chain Reaction (PCR); and conferred using blaTEM and blaCTX-M genes. Through β-lactamase gene sequencing and docking studies, the effectiveness of drugs against ESBL resistant K.pneumoniae were determined. A total of 31 (62.0%) K.pneumoniae were isolated with 12 (38.7%) positive MDR strains and 5 (41.7%) ESBL resistant strains. Only 1 (20%) blaTEM gene and 4 (80%) blaCTX-M genes were detected with Ceftriaxone (CRO) as the most effective drug showing highest binding energy (-9.2Kcal/mol) against ESBL resistant K. pneumoniae. With high prevalence of CTX-M-type ESBL resistant K.pneumoniae in Klang River, effective environmental and antibiotic controls should be adopted.


INTRODUCTION
Klebsiella pneumoniae (K.pneumoniae) from Enterobacteriaceae family is a gram negative bacteria residing in about 40% of human and animals' intestinal tract. It is a type of normal flora localised in mouth, skin and intestine but also an opportunistic human pathogen responsible for 10% of nosocomial infections and 30-50% of exacerbations in hospitalized patients with immunosuppression. This is because K.pneumoniae not only able to colonize human skin as normal flora, it also has a persistent long survival rate in hospitals and environmental surface. Thus, transmission is relatively easy among patients in hospitals through hand contact with hospital care personnel [1][2][3] . To control the wide transmission rate and easy outbreak of K.pneumoniae, many antibiotics have been used as treatment. However, uncontrolled use of antibiotics lead to occurrence of bacterial selection pressure and caused a rise in Multidrug Resistant (MDR) strains that resist to more than one type of antibiotics [4][5] . MDR K.pneumoniae generally resistant to the Extended Spectrum Beta Lactamase (ESBL) antibiotics followed by Carbapenems, Aminoglycosides and Fluoroquinolones 6 . ESBL are plasmid mediated enzymes encoded by genes which are located mainly in the mobile genetic elements of K.pneumoniae 7 . According to Diagbouga and Ozturk, ESBL resistant K.pneumoniae are found to be commonly against ‫-ג‬lactam antibiotics such as Penicillins, Monobactams (Aztreonam); and oxyimino Cephalosporins (Cefotaxime, Ceftazidime, Ceftriaxone, Cefuroxime, Cefepime) in which Cephalosporins are responsible for most of the ESBL antibiotic resistance in K.pneumoniae [8][9] . The common genes that responsible for this ESBL resistance in K. pneumoniae are CTX-M, TEM and SHV families with CTX-M β-lactamases as the most predominant and heterogeneous type 5,10 . The major mechanism involved in transmitting these genes is horizontal gene transfer where ESBL resistant clones are spread by sharing of plasmids across bacteria 11,5,2 .
The rise in resistance towards different types of antibiotics in MDR strains has become a critical issue when the resistance genes are spread into environmental bacteria especially those in highly polluted aquatic ecosystem which serve as reservoirs. Bacteria can evolves and transmits resistance among each other when in contact with human microbiota 12,5 . For example, resistant genes are spread into environment bacteria through discharges especially hospital waste water which contains 8 times more antibiotic resistant bacteria than domestic discharges 13 . The antibiotics discharged exert a selection favouring resistant bacteria by killing or inhibiting growth of susceptible bacteria. Resistant bacteria then adapt to environmental conditions and become vectors for transmission of antibiotic resistance 12 . Also, construction or industries effluents into aquatic ecosystem can affect the physical, chemical and biological properties of water bodies resulting in poor water quality that facilitates the evolution and transmission of resistance to environmental bacteria 14 . These pollutions are especially common in Klang River which covers the most densely populated areas in Malaysia including many hospitals, household areas, industries and constructions sites. The environmental degradation has gradually reduced the water quality in Klang River and the water acts as reservoir for bacterial gene transformation where environmental bacteria inter-communicates with bacteria of human or animals by horizontal gene transfer resulting in the rise of MDR strains 15 . Although there are many studies on the high occurrence of MDR strains in river water samples, studies on physicochemical and bacteriological status of the river are limited [16][17] . Thus, MDR pattern particularly ESBL resistance of K.pneumoniae in water bodies along with the physicochemical properties of Klang River are focused with TEM and CTX-M genes to be identified. Also, the most effective drug against mutated Klebsiella pneumoniae are determined.

Sample site description and collection
Malaysia Klang River covered a total of 11 major tributaries: rivers of Gombak, Batu, Kerayong, Damansara, Keruh, Kuyoh, Penchala and Kerayong, Ampang, Gisir and Kemusing with a length of 120km and drained a basin of approximately 1,288km 2 . 18 Klang River with geographical location of 3° 13' 01.22", 101° 40' 54.92" was sourced from Klang Dam Gates located in Gombak District and ended at Port Klang, the principal port in Selangor. The upstream of Klang River in Gombak District was covered with mountains and tropical forest. The unpolluted water then flowed into the middle stream of Klang River covering the highly populated urban areas within Kuala Lumpur then towards downstream areas including the Selangor Districts of Petaling and Klang that saturated with industries and residences. From the Federal Territory of Kuala Lumpur to part of the state of Selangor in Malaysia, the river water will eventually flowed into the Straits of Malacca. The average annual temperature of Klang River was ranged between 29°C and 32°C and highest temperature was observed in March to June [19][20] . 50 water samples were collected from upstream (unpolluted) to downstream (extremely polluted) of Klang River (Fig.1). Each water samples was collected along Klang River approximately 2km away on weekly basis and only when there was no rain to avoid any miscellaneous results. All samples were collected into 100ml sterile containers and 300ml dark amber bottles respectively for the estimation of Dissolved Oxygen (DO) and Biological Oxygen Demand (BOD). The bottles were tightly capped to avoid entry of air bubbles.

Physicochemical analysis
Physicochemical parameters of water samples including physical properties, pH, temperature ( 0 C), DO (mg/L) and BOD (mg/L) were determined on site 14 .

Bacterial isolation and characterization
Water samples were serially diluted by 10-folds and spread plated on MacConkey (MAC) agar. Pure isolates resembled K.pneumoniae were obtained by further streaking on Eosin Methylene Blue (EMB) agar. After incubation of plates at 37°C for 24 hours, picked colonies were transferred to Nutrient agar slants and stored in 50% glycerol solution at -80°C 17,21-22 .

Bacterial identification
Pure colonies resembled K.pneumoniae were inoculated in saline water and adjusted to a standard of 0.

Phenotypic tests
MDR isolates equated to McFarland standard 0.5 were used to make a bacterial lawn across MHA and E.coli ATCC 25922 was used as negative control. ESBL production of K.pneumoniae was screened by Double Disc Synergy Test (DDST) using discs of AMC (20/10µg), CRO (30µg), CTX (30µg), CAZ (30µg) and CPD (30µg); and confirmed by Combination Disc Test with discs of CAZ (30µg) and Ceftazidime/Clavulanic Acid, CAZ/CA (30/10µg) according to CLSI guidelines 26 .

DNA extraction, PCR amplification and genotype detection
Genomic DNA from ESBL resistant K.pneumoniae were extracted using innuPREP Bacterial DNA Kit (Biometra, Germany) following manufacturer's instructions. Extracted DNA were then amplified in a Thermal Cycler (Eppendorf, Germany) using Polymerase Chain Reaction (PCR) kit-MyTaq Mix (Bioline, United Kingdom). PCR amplification was performed using forward and reverse primers to detect TEM and CTX-M genes under specific conditions 27 (Table 1). PCR products were detected using 1% gel electrophoresis and positive strains containing TEM or CTX-M gene were directed to b-lactam gene sequencing 28 .

Molecular docking
The forward and reverse genes' sequence obtained were assembled into single gene sequence using BioEdit Tool then transcripted to mRNA sequence using Sequence Manipulating Suite and visualized in a 3-D structure with  SWISSMODEL Server 29 . After determining the potential binding sites via CASTp Server and obtaining the 3-D structure of ligand (antibiotics) through PubChem database, virtual screening was performed using AutoDock-Vina to study the protein-ligand interactions with eight b-lactam antibiotics: AMP, CXM, CTX, CAZ, CRO, CPD, FEP, and ATM. A binding score (Kcal/mol) was obtained to determine drug effectiveness. The higher the binding score, the more effective was the drug 30 .

RESULTS AND DISCUSSION Physicochemical analysis
Of all 50 water samples, 31 (62.0%) water samples that were yellowish to brown, with foul smell, contained few or no debris; have Journal of Pure and Applied Microbiology a temperature of 28-32°C; and pH of 6.8-7.6 ( Table  2) showed presence of K.pneumoniae. Most of the yellowish or brown, foully water samples were collected from middle and end of Klang River saturated with hospitals, housing areas, industries and animal farms. This showed that the river water was gradually contaminated from clear, odourless to yellowish or brown with fouled smell. These water samples showed a higher mean BOD (1.50±1.31mg/L; 2.54±1.94mg/L; 5.30±4.35mg/L) compared to upstream water samples that were clear or slightly clear with mean BOD (0.80±0.21mg/L; 2.95±2.04mg/L; 0.89±0.88mg/L). The highest BOD (8.85mg/L) was observed in water sample collected from end of Klang River (Port Klang) and lowest BOD (0.00mg/L) was observed in clear or slightly clear water samples collected from upstream of Klang River. When the BOD is higher compared to lower limit (2.4mg/L), the DO also showed significantly lower. The higher BOD and lower DO observed indicated the midstream, particularly end of Klang River were more polluted and have poorer water quality due to increase in total nutrients in water from discharges that encouraged growth of bacteria. Rise in amount of bacteria depleted the oxygen dissolved in water for breakdown of organic matter thus lowering DO.
These results also proved the close relationship of BOD and DO, where a higher BOD will have a lower DO and vice versa. The pH of overall water samples collected (6.8-7.6) was within range of 6.5-9.0 as suggested by Gandaseca 31 , still feasible for aquatic life. The temperature of water samples (28-32°C) was also within the range of average annual temperature of Klang River (28-30°C) and highest temperature (32°C) observed was common as the time of sample collection was between March-June.

MDR and ESBL resistant determinants
Among 31 K.pneumoniae isolated, 12 (38.7%) were MDR strains and most of the isolates were resistant to AMP showing a high prevalence of MDR K.pneumoniae (21,27.6%) towards ‫-ג‬lactam antibiotics (Fig.2 & Fig.3). Through phenotypic DDST and CDT, 5 ESBL strains (41.7%) were isolated. The ESBL production was identified from presence of 'key-hole' inhibition towards AMC in DDST; or a ≥5mm increase in inhibition zone of CAZ/CA compared to CAZ in CDT with reference to negative control strain E.coli ATCC 25922 (Fig.4a-d). These results confirmed K.pneumoniae was one of the most common MDR and ESBL producing bacteria to be isolated from river water samples as correlated with similar  (Fig.5a-d). Higher frequency of recovering CTX-M gene in this research showed that CTX-M was a predominant ESBL gene compared to other common ESBL genes such as TEM and SHV families .

Molecular docking
From the molecular docking study of CTX-M gene in K35, SER73, ASN107, TYR108, SER133, ASN135, PRO170, SER240, ASP242, SER275 and ARG277 (Table 3) showed to be the amino acids that were more involved in binding to eight common β-lactam antibiotics including most of the 3rd and 4th generation Cephalosporins which were the main cause of ESBL resistance in K.pneumoniae. These findings were essential particularly in designing new drugs based on existing antibiotics where functional groups in the antibiotics that allowed binding of respective amino acids can be removed, prohibiting proteinligand interactions that encouraged enzyme hydrolysis resulting in ESBL resistance. This method is more recommended and convenient instead of changing the amino acids in mutated structure by point mutations to prohibit protein-ligand binding interactions. This is because point mutations are more challenging, time consuming and not cost effective.
Effectiveness of drug were also determined by comparing the energy released during proteinligand interactions (binding energy). CRO with the highest binding energy (-9.2Kcal/mol) followed by FEP (-8.7Kcal/mol), CXM (-8.4Kcal/mol), CPD and CAZ (-7.5Kcal/mol), CTX (-7.4Kcal/mol), ATM (-7.0Kcal/mol) and AMP (-6.5Kcal/mol) suggested more energy was released and weakest activity of b-lactamase with CRO ( Fig.6a and Fig.6b). Thus, CRO was most unlikely to be hydrolyzed by the β-lactamase, making the drug effective.  In conclusion, Klang River with major pollutions has harbored high prevalence of MDR resistant (38.7%), especially ESBL resistant K.pneumoniae (41.7%) with CTX-M gene as the predominant type (80%) due to uncontrolled use of antibiotics and discharges that increase bacterial resistance by selection pressure. In order to overcome the rise of MDR strains particularly  ESBL resistance in bacteria, effective waste treatment management and antibiotic control should be adapted. For example, reduce the use of AMP with replaced with CRO which is a more effective drug. From overall findings of research, the objectives were achieved and hypothesis accepted.
In future studies, modified DDST instead of general DDST method should be used to reduce risk of false negative results from interference of AmpC. In addition, more ESBL genes types should be identified to determine its prevalence rate. Besides, more molecular techniques such as Real Time PCR (qPCR) was suggested to further confirmed presence of ESBL resistance genes by identifying the exact size of DNA fragments. Location of resistance genes (plasmids, transposons or integrons) can also be determined.
A wider range of antibiotics should also be used in docking studies to confirm the most effective drug.