Bioprospecting Studies of Actinomycete Bioactive Compound on Bla CTX-M -Extended -Spectrum Beta- Lactamase (ESBL) Producing Escherichia Coli and Klebsiella Species

Forty one Extended Spectrum Beta Lactamase (ESBL) producing E. coli and Klebsiella species were analyzed for the presence of bla CTX-M genes by Uniplex Polymerase Chain Reaction (PCR) in which 15 isolates harbored bla CTX-M genes. Transconjugation studies were carried out to demonstrate the transfer of antibiotic resistance using E. coli K12J62-2 (F – rif r lac - ) strain among which UTEC31 and UTK2 strains showed successful transconjugation. Sequence analysis revealed the presence of bla CTX-M-15 genes. Antibacterial activity of Streptomyces sp PUT23 was evaluated against CTX-M ESBL producing E. coli and Klebsiella sp. was determined. The ethyl acetate extract showed inhibitory activity and Reversed phase thin layer chromatography (RP-TLC) analysis revealed two fractions, EFI and EF2. Bioautogram of EF2 fraction demonstrated inhibitory activity with a minimum inhibitory concentration (MIC) value 156 μg/ml. The EF2 fraction was identied as a avonoid upon chemical characterization. Sequencing of 16S rRNA gene and phylogenetic analysis of Streptomyces sp. strain PUT23 revealed its close relatedness to S. griseus. This study revealed the rapid spread and dominance of CTX-M producing E. coli and Klebsiella sp. in the suburban community of South Chennai, Tamil Nadu which has to be considered as a serious public health issue. There is an urgent need for newer and effective antimicrobials and bioactive compounds derived from Streptomyces species proved to be a promising and ecient resource. Further characterization and purication of EF2 fraction are needed and could be used in the formulation of new therapeutics against ESBL mediated infections.


Introduction
Infections caused by multidrug resistant bacterial pathogens are considered to be the important cause of morbidity and mortality [1]. Though antibacterial agents have been a major factor in treating bacterial infections, the phenomenon of microbial drug resistance has rendered most of the antibiotics useless, especially multidrug resistance among gram negative bacteria represents a unique and immediate threat [2].
The production of extended spectrum beta lactamases (ESBLs) is one of the vitriolic mechanisms of antimicrobial resistance (AMR). Reports on ESBL producing uropathogenic bacteria as a leading cause of morbidity and mortality are of major concern. Moreover, their ability to disseminate into the community is an emerging public health problem globally [3].
Broad spectrum antibiotics are frequently given to treat UTIs in place of a narrow spectrum antibiotic which may be adequate. Recent reports suggest that the global antibiotic consumption from the year 2000 to 2015 had increased by 65% while a 39% increase was reported on the rate of antibiotic consumption for de ned daily doses (DDDs) per 1000 people [4].
The reasons for global antibiotic resistance are complex and multifactorial. They may be attributed to the indiscriminate and irresponsible use of antibiotics, mutations in bacteria, acquisition of resistance genes, overuse and misuse of antibiotics, selection pressure sustaining resistant bacteria and competition among physicians prescribing antibiotics unnecessarily to retain patients, lack of awareness among people all contribute to this global problem [5]. Metagenomic analysis has revealed the widespread prevalence of genes encoding antibiotic resistance in the environment [6].
The AWaRe classi cation of antibiotics [7] was introduced by the WHO as a tool for antibiotic stewardship with the aim of reducing AMR and ESBL producing pathogens are included in the Priority 1 -Critical group list [8] for which the WHO cautions appropriate use of existing antibiotics and also emphasizes the need of new antimicrobials.
Actinobacteria are widely exploited for their bioactive compounds and are excellent resources for the control of ESBL producing pathogens [9]. These groups of lamentous gram positive bacteria are recognized for their pivotal role and genetic capability in the production of useful secondary metabolites like antibiotics, antitumors etc. [10].
Among many actinobacterial genera, Streptomyces species are renowned and eulogized for their production of diverse array of antibiotics. These accounts for over two thirds of the clinically used natural antibiotics produced by Streptomyces alone and about 75% of the marketable and therapeutically useful antibiotics are derived from this genus [11]. The ability of the bioactive compounds of actinomycetes in controlling multi drug resistant pathogens is well known [12]. The problem of antimicrobial resistance (AMR) is increasing while the production of newer antibiotics is slow [13]. Hence newer potential antibiotics are needed, to curb the problem of multidrug resistance, especially from natural habitats rather than their synthetic counterparts.
This study was instigated to determine the prevalence of bla CTX−M ESBL producing E. coli and Klebsiella species in the southern semi urban regions of Chennai. This study also reported the antibacterial properties of Streptomyces sp. against the ESBL producing organisms.

Methods
Identi cation of bla CTX-M mediated ESBL production in E. coli and Klebsiella species by uniplex polymerase chain reaction (PCR) In this study, 41 ESBL producing bacterial cultures [14] including 27 E. coli and 14 Klebsiella species were used for the determination of bla CTX-M genes. Plasmid DNA was isolated from the strains following the alkaline lysis method [15]. PCR ampli cation of bla CTX-M alleles was carried out for all ESBL positive bacterial cultures using the set of universal primers CTX-M-F-5'-ATG TGC AGY ACC AGT AAR GT -3' and CTX-M-R-5'-TGG GTR AAR TAR GTS ACC AGA -3' [16]. The strain E. coli MTCC 443 was used as negative control. The Y, R, S represent the standard nucleotide combinations where Y is C or T, R is A or G and S is C or G. This primer combination is designed to accommodate sequence variation in CTX-M type enzymes.
Demonstration of horizontal transfer of antibiotic resistant genes by transconjugation The isolates of E. coli and Klebsiella species that harbored bla CTX-M genes were analyzed for capability of gene transfer in the recipient strain E. coli K12 J62-2 (Frif r lac -). Mating experiment was carried out where the isolates and the recipient were grown in Brain Heart Infusion broth for overnight and were mixed in the ratio of 1:10 to match McFarland turbidity 0.5 [17]. They were then incubated at 37°C overnight and selected on MacConkey agar supplemented with rifampicin (2.5 mg/ml) and cefotaxime (2 μg/ml). The transconjugants were subjected to antibiotic sensitivity testing against ceftazidime disc and plasmid pro le analysis.
Sequencing of bla CTX-M plasmid DNA The ESBL positive E. coli and Klebsiella species that demonstrated transferrable resistance were subjected to DNA sequence analysis (Beckman Coulter CEQ 8000 auto analyzer). Molecular characterization of the ESBL coding gene bla CTXM-15 using the same set of primers mentioned above was analyzed from the selected strains UTEC31 and UTK2 [18].
The sequenced nucleotide was subjected to NCBI BLAST (Basic Local Alignment Search Tool) analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The nucleotide BLAST was run using megablast (high similar sequence) mode with maximum target of 50 for con rmation of the sequence. After con rming the sequence, the sequences were submitted to GenBank/NCBI to obtain the accession number through "Sequin" submission tool. Further, the Open Reading Frame (ORF) of the sequences was predicted. The ORF and the initiation codon was predicted using tool, ORF nder related strains was carried out to predict the evolutionary relationship with similar sequences. Dendrogram was constructed with MEGA 4.0 using neighborhood joining method from 500 resamplings [19].
In vitro testing of Streptomyces extract against ESBL pathogens

Description of actinobacterial strain
The actinobacterial strain PUT23, previously isolated from agricultural soil sample was obtained from the Actinobacterial Research Laboratory, Department of Microbiology, Periyar University, Salem, Tamil Nadu.
Viability of the strain PUT23 was maintained on yeast extract malt extract (YEME) agar slants at 4 o C as well as in 20% glycerol broth at -80 o C.
The thermal cycling conditions included an initial denaturation of the target DNA at 94°C for 4 minutes followed by 30 cycles at 94°C for 1 minute, primer annealing at 52°C for one minute, primer extension at 72°C for one minute. The reaction mixture was then held at 72°C for 10 minutes and cooled to 4°C. The ampli ed product was detected by 1% agarose gel electrophoresis and was visualized by Gel Documentation System.
Sanger's Dideoxy Chain Terminator Sequencing was performed using DTCS Quick Start Dye Terminator Kit (Beckman Coulter) and Beckman Coulter CEQ 8000 auto analyzer. The cycle sequencing was carried out using the same primers used in PCR ampli cation. Sequence alignment was done using Clustal-X2 Version 2.1 and phylogenetic tree was constructed using MEGA Version 5.0 [19] and BLAST analysis of the strain was carried out to determine the evolutionary relationship of the strain with closely related strains derived from the NCBI database. The deduced 16S rRNA sequence of the strain PUT23 was submitted to GenBank/NCBI to get the accession number.

Production and extraction of the bioactive compounds
Extraction of the bioactive compound from the PUT23 strain was done through fermentation following the method from a previous study [22] with modi cations. Brie y, pure cultures of actively growing Streptomyces sp. was inoculated into 100 ml of YEME broth and incubated at 30°C for 3 days. About 2% of the YMD broth culture was again inoculated in about 1 L of International Streptomyces Project 4 (ISP4) broth and incubated at 30°C for 5 days. After fermentation at 30°C for 5 days, the cell free supernatant was collected by ltration. Extraction of the crude compound from the culture ltrate was carried out by liquid-liquid extraction method with equal volumes of ethyl acetate (1:1 v/v) by vigorous shaking for 1 hour and concentrated by evaporation. The crude extract was then tested for anti-ESBL activity.

Determination of antibacterial activity
The antibacterial activity of the ethyl acetate extract against the ESBL producing E. coli and Klebsiella species was determined by agar well diffusion method [23] with ethyl acetate (50 mg/ml) and cipro oxacin (5 μg/ml) as controls. The plates were examined for the zones of inhibition and results were compared and graded by measuring the zone diameter. E.coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were used as controls. Antimicrobial e cacy was graded based on the zone diameter as high activity (≥15 mm), moderate activity (10-14 mm), trace activity (5-9 mm) and no activity (≤ 4 mm) [24].
Puri cation of the bioactive compound by reversed phase thin layer chromatography (RP-TLC) Partial puri cation and characterization of the ethyl acetate extract of Streptomyces PUT23 was attempted by RP-TLC also known as Double Ascending TLC [25] using readymade silica gel coated TLC sheets (7.5 x 2.5 cm) (Silica Gel 60 F 254nm) using the mobile phase n-hexane: ethyl acetate: acetic acid in the ratio 7:2.5:0.5. The solvent ascent was xed to 5.8 cm for calculating R f values. The chamber saturation time for the mobile phase was 15 min at room temperature (22 ± 1°C). Double ascending TLC was performed and the plates were dried at room temperature. The spots were detected in the presence of iodine vapor. The Retardation Factor (R f ) value was calculated with values of the R l (R f of the Leading Front) and R t (R f of the Trailing Front) using the relation R f = 0.5 (R l + R t ).

Bioautography of puri ed ethyl acetate extract
The anti-ESBL activity of the separated ethyl acetate fractions namely EF1 and EF2 in RP-TLC method was analyzed by contact or direct bioautography [26] by placing the chromatogram face down onto Mueller-Hinton agar seeded with bla CTX-M producing E. coli and Klebsiella species and incubated at 37°C for 18-24 hours. The zones of inhibition on the agar surface, corresponding to the spots in chromatographic plates were indicative of the bioactive fraction.
The active fraction which showed inhibitory activity in the bioautography was further puri ed by preparative TLC by scrapping the fraction with silica gel in analytical TLC and repeated extraction with methanol. The puri ed bioactive compound present in the methanol portion was collected by centrifugation at 10,000 rpm for 10 minutes and ltration. The obtained ltrate was concentrated at 45°C using eppendorf concentrator.
Determination of MIC of the bioactive fraction against bla CTX-M producing ESBL E. coli and Klebsiella species The minimum inhibitory concentration (MIC) was determined for the bioactive compound using the dilutions at 5, 2.5, 1.25, 0.625, 0.312 and 0.156 mg/ml prepared in DMSO [24]. About 50 μl of the bioactive compound from each dilution with cipro oxacin (8 µg/ml) as control were loaded in wells of Mueller-Hinton agar plates seeded with a lawn culture of bla CTX-M positive bacteria. Solvent controls of 1% DMSO and 1% ethyl acetate were also included to exclude the possibility of their antagonistic activity. MIC was determined after incubating the plates at 37°C for 18-24 hours by observing for the zones of inhibition.

Preliminary characterization of active compound
Appearance and solubility analysis Colour and consistency of the bioactive fraction was determined by visual inspection. Solubility of the compound was tested by dissolving 1 mg of puri ed compound in the solution containing water, 2.5 M Sodium hydroxide (NaOH), 0.6 M Sodium bicarbonate (NaHCO 3 ), 1.5 M Hydrochloric acid (HCl) and concentrated sulphuric acid (H 2 SO 4 ) to resolve the functional groups present in the partially puri ed bioactive compound [27].
Solubility of the same was determined by dissolving 1 mg of puri ed compound in 10 ml of solvents such as water, methanol, acetone, chloroform, dichloromethane, diethyl ether, ethyl acetate, n-hexane, dimethyl sulfoxide.

Chemical screening of the bioactive fraction
To determine the chemical group to which the puri ed pigment belongs, the following qualitative biochemical reactions were performed [28]. About 10 mg/ml stock solution of the bioactive fraction was prepared using methanol for the analysis. Tests were performed to detect the presence of terpenoids, diterpenes, alkaloids, carotenoids, avonoids, antroquinones and glycosides.

Results And Discussion
Isolation of plasmid DNA from ESBL positive E. coli and Klebsiella species Plasmid pro le analysis of ESBL positive isolates revealed the presence of plasmids in all 27 E. coli and 14 Klebsiella sp. and few isolates harbored multiple plasmids ranging from 2 to 5 numbers. This nding is in full accordance with earlier studies reporting multiple plasmids among ESBL producing bacteria. [29], [30]. Among E. coli isolates, strains UTEC22, -24, -45, -48 harbored 5 plasmids ranging between 6000 bp and >1 kb and UTEC4, -5 ,-29, -31, -40, -53, -57 demonstrated 4 plasmids from 3000 bp to >1 kb. Eight isolates showed three plasmids and three isolates harbored two plasmids while the remaining isolates showed single plasmid.
Identi cation of bla CTX-M mediated ESBL production in E. coli and Klebsiella species by uniplex polymerase chain reaction (PCR) The CTX-M genotypes have gained clinical and epidemiological importance in the context of their ability to disseminate and causing outbreaks [31]. In India, the bla CTX-M-15 genotype is reported to be found exclusively [32]. Hence the bla CTX-M gene mediated ESBL production among the isolates was determined by using uniplex PCR. The overall prevalence of bla CTX-M genes in the isolates was 36.58% where 9 out of 27 E. coli (33.33%) and 6 out of 14 Klebsiella sp. (42.85%) carried the bla CTX-M genes. The size of the amplicons ranged between 500-600 bp (Fig. 2).
The present study had identi ed 42.85% prevalence of CTX-M ESBLs and is in full accordance with previous report [33]. An earlier Chennai-based study [34] revealed CTX-M type ESBLs to be prevalent in 75% of the isolates followed by TEM enzymes (73%) and SHV ESBLs (42%), while another recent study reported 88% prevalence rate in Chennai [35].
This variation could be related to the shortening of the boundaries between hospitals and the community, accelerated transfer of the antibiotic resistant genes by e cient mobile elements, clonal expansion of the isolates and selective advantage for survival of the resistant isolates ideal for dissemination [32], [36].

Demonstration of horizontal transfer of antibiotic resistant genes by transconjugation
In the transconjugation analysis, 2 out of 15 donor strains (13.33%), namely, UTEC31 and UTK2 showed successful transconjugation in the recipient strain of E. coli K12J62-2 (Frif r lac -). The recipient demonstrated acquired antibiotic resistance against ceftazidime and the acquisition of plasmids (Fig. 3). The rate of transferability was lower in this study when compared to other reports [37], [38].
It is well known that conjugative plasmids are major tools for intra-and inter-species and also intergeneric gene transfers. ESBLs encoded on plasmids isolated from clinically resistant uropathogenic bacteria are constitutively expressed. This is an important observation because plasmid mediated ESBLs possibly inhabit environments in which antibiotics are found in high concentrations [39]. It was proposed that only compatible plasmids could be rescued in transconjugation [40]. However, bla CTX-M genes are reported to be linked to plasmids of incompatibility groups which include IncF, IncN, IncN2, IncI1, IncHI2, IncL/M, IncA/C, IncK, IncX4, IncU and RCR [32].
Determination of transferable resistance in this study emulates the dissemination of resistant genes in the environment and will be of clinical and epidemiological signi cance for two reasons. Firstly, the isolates belonged to community settings and hence possess the vitriolic potential to disseminate within community. Secondly, they are problematic because of conferring resistance to various antibiotics providing a survival advantage under pressure of exposure to various antibiotics. This makes the treatment options not only limited but also di cult. The ORF nder online tool predicted different open reading frames but the vast size reading frame was considered as beta-lactamase ORF. The ORF consisted of 543 base pairs corresponding to 179 amino acids in E. coli and 568 base pairs in size coding for 156 amino acids in Klebsiella sp. In both the cases AUG was the initiation codon for methionine. Since there was no termination codon in this frame for both sequences, the sequence might be a partial sequence of bla CTX-M-15 gene. In order to nd out the amino acid sequences, the ORF sequences were subjected to amino acid translation through ExPAsy online tool.
The number of amino acid sequences consisted with the results of ORF nding results.
CTX-M-15 is included under the CTX-M-3 cluster based on its amino acid identity and phylogeny. It differs from other members of CTX-M-3 groups by a single amino acid substitution, D240G, where aspartic acid is substituted with glycine at position 240 [41]. This mutation in the gene is responsible for its increased hydrolytic activity against antibiotics such as ceftazidime by the isolates [42]. The amplicon size of the 16S rRNA gene of Streptomyces sp. strain PUT23 was around 1000 base pairs and BLAST analysis showed 100% identity to 16S rRNA sequences of Streptomyces griseus strains con rming the strain PUT23 to belong to the genus Streptomyces. The sequence was submitted to GenBank with accession number KU356755. Based on the 16S rRNA sequence, the BLAST analysis further revealed the phylogenetic relatedness of the test strain PUT23 to its closely related strains of Streptomyces species obtained from the NCBI database (Fig. 6).
Determination of antibacterial activity of the bioactive compound by agar well diffusion method Approximately, 1.94 g of crude extract was obtained in solvent extraction with ethyl acetate. Testing for anti-ESBL activity of the extract showed an inhibition zone between 13 -17 mm (Table 2) and the result is consistent with other studies [11], [21].
Puri cation of the bioactive compound by reversed phase thin layer chromatography (RP-TLC) The RP-TLC analysis of the ethyl acetate extract resulted in the separation of 2 fractions, namely EF1 and EF2 (R f values -0.58 and 0.73, respectively). Double ascending TLC or Reversed Phase TLC offers added advantage in organizing hydrocarbon ligands on the silica surfaces, interaction of solvent into the bonded layers and aid in separation of pharmaceuticals like surface active pharmaceutical molecules [25].

Bioautography of puri ed ethyl acetate extract
In bioautography, the second fraction, EF2, (R f value -0.73) showed activity against the bla CTX-M positive uropathogens tested (Fig. 7). Fraction EF1 did not show any activity against the test organisms. This technique is simple, easy to perform, inexpensive and is a useful means for the screening of compounds from natural source using developed TLC. Prediction of results is made easy indicated by the development of zones of inhibition on the agar surface near the spots in the chromatogram relative to the bioactive fractions [43]. In the preparative TLC, 1 gm of ethyl acetate extract yielded 62 mg of fraction EF2.
Determination of MIC of the bioactive fraction against bla CTX-M ESBL uropathogens.
The MIC of the ethyl acetate extract of bioactive component was found to be 156 µg/ml against bla CTX-M-15 producing E. coli and Klebsiella sp. which was found to increase with increasing concentrations (Fig.  8). Thus a concentration <156 μg/ml may also be inhibitory to ESBL producing pathogens. This is higher than the ndings of a previous study reporting the MIC to be as low as 25 μg/ml against K. pneumoniae [44].
Determination of the functional group of bioactive fraction by chemical screening The bioactive compound EF2 was brown powdery and was insoluble in water, 2.5 M NaOH and 0.6 M NaHCO 3 but soluble in 1.5 M HCl and concentrated sulphuric acid. The original brown colour of the compound changed to red colour upon the addition of 1.5 M HCl and concentrated sulphuric acid. This indicated that the compound EF2 might contain amine, ester, alkene and/or alcohol as functional groups. It was soluble in methanol, DMSO, less soluble in n-hexane and insoluble in water.

Chemical screening of bioactive fraction
In the various screening tests performed for the detection of chemical group, compound EF2 was found to belong to avonoid class ( Table 3). Some of the classes of avonoids that possess antimicrobial activity include Flavones, Iso avones, Flavonols and their glycosides, Flavan-3-ols, Flavanones, Flavans etc., [45]. The anti-ESBL activity of the ethyl acetate extract is ascribed to the presence of avonoids as evident in the bioautography and this nding is supported by other reports [46], [47].
The inhibitory activity of the extract could be attributed to the presence of avonoid group which has ability to complex with bacterial cell wall, extracellular and soluble proteins and probably causing membrane disruption [48]. Flavonoids conjugated with nanoparticles have been proved to be a promising alternative for antibiotics [49].
Reports on ESBL prevalence from southern suburban regions of Chennai are scarce. This study revealed the rapid emergence and dominance of CTX-M producing E. coli and Klebsiella sp. in the suburban community of South Chennai, Tamil Nadu which has to be considered as a serious public health issue.
Hence the ndings in this work are important due to the fact that more residential area and industrial setups are being laid in the region. The study had also highlighted the inhibitory activity of the avonoid produced by Streptomyces PUT23 strain against ESBL pathogens and its potential to be developed into antimicrobial agent, especially in the biocontrol of multidrug resistant bacteria. To put in the words of Baltz, (2008) [50], we are in the early stages of a renaissance in the antibiotic discovery from actinomycetes. Funding: Not applicable. There was no funding received for this work.     The amplicon size of the 16S rRNA gene of Streptomyces sp. strain PUT23 was around 1000 base pairs and BLAST analysis showed 100% identity to 16S rRNA sequences of Streptomyces griseus strains con rming the strain PUT23 to belong to the genus Streptomyces. The sequence was submitted to GenBank with accession number KU356755. Based on the 16S rRNA sequence, the BLAST analysis further revealed the phylogenetic relatedness of the test strain PUT23 to its closely related strains of Streptomyces species obtained from the NCBI database The MIC of the ethyl acetate extract of bioactive component was found to be 156 µg/ml against bla CTX-M-15 producing E. coli and Klebsiella sp. which was found to increase with increasing concentrations