Isolation of plasmid DNA from ESBL positive E. coli and Klebsiella species
Plasmid profile 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 finding is in full accordance with earlier studies reporting multiple plasmids among ESBL producing bacteria. , . 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.
Plasmid profile of Klebsiella species demonstrated the presence of 4 plasmids among five isolates namely UTK12, -21, -22, -36, -38 and three plasmids among the isolates UTK1, -2, -15, -20, and -30. Three isolates namely UTK3, -16, -29 harbored two plasmids while UTK33 showed a single plasmid. The plasmids were in the range between 4000 bp and >1 kb (Table 1 and Fig. 1).
Identification 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 . In India, the bla CTX-M-15 genotype is reported to be found exclusively . 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 identified 42.85% prevalence of CTX-M ESBLs and is in full accordance with previous report . An earlier Chennai-based study  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 .
This variation could be related to the shortening of the boundaries between hospitals and the community, accelerated transfer of the antibiotic resistant genes by efficient mobile elements, clonal expansion of the isolates and selective advantage for survival of the resistant isolates ideal for dissemination , .
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 (F - rif 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 , .
It is well known that conjugative plasmids are major tools for intra- and inter- species and also inter-generic 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 . It was proposed that only compatible plasmids could be rescued in transconjugation . 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 .
Determination of transferable resistance in this study emulates the dissemination of resistant genes in the environment and will be of clinical and epidemiological significance 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 difficult.
Sequencing of bla CTX-M plasmid DNA of ESBL positive E. coli and Klebsiella species Molecular characterization of ESBL coding gene
The two isolates UTEC31 and UTK2 which showed successful transconjugation were subjected to sequencing of bla CTX-M gene. Strains UTEC31 and UTK2 showed the amplified DNA product of bla CTX-M-15 genes of 500 and 600 bp.
Sequencing of plasmid DNA revealed the nucleotide results averaging 543 nucleotides for E. coli UTEC31 (GenBank Accession No. KJ746672). For Klebsiella sp. UTK2, it was about 568 nucleotides (GenBank Accession No. KJ746673). The BLAST results revealed that the bla CTX-M-15 sequence obtained from E. coli demonstrated 100% identity with bla CTX-M-15 partial sequence of E. coli (Genbank accession KF723004, KF723003 etc.) and the bla CTX-M-15 sequence obtained from Klebsiella sp. paired 100% with bla CTX-M-15 partial sequence of Klebsiella species (Genbank accession JQ686199, JQ686198 etc.) under 100% query coverage.
The ORF finder 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 find 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 finding 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 . This mutation in the gene is responsible for its increased hydrolytic activity against antibiotics such as ceftazidime by the isolates .
Phylogenetic analysis of bla CTX-M-15 gene of E. coli strain UTEC31 using neighborhood joining method with a boot strap value of 500 showed similarity with bla CTX-M-15 genes of Escherichia coli ESBL370B15, Escherichia coli ESBL490B15, Escherichia coli ESBL360 strains. Additionally, this gene also showed similarity with bla CTX-M-15 genes of Escherichia coli strain C21, E. coli E5, E. coli E12, E. coli E11 strains. Similarly, phylogenetic analysis of bla CTX-M-15 of Klebsiella sp. UTK2 based on the neighborhood joining method with a boot strap value of 500 revealed that the sequence showed similarity with bla CTX-M-15 gene of K. pneumoniae KP34C. Additionally this gene of Klebsiella sp. UTK2 also showed similarity with bla CTX-M-15 genes of K. neumoniae ST15, K. pneumoniae KP9, K. pneumoniae L4, K. pneumoniae 639 and K. pneumoniae 51 (Fig. 4 and Fig. 5).
Molecular analysis and taxonomy of the potential Streptomyces sp. PUT23 strain
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 confirming 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 , .
Purification 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 (Rf 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 .
Bioautography of purified ethyl acetate extract
In bioautography, the second fraction, EF2, (Rf 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 . 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 findings of a previous study reporting the MIC to be as low as 25 μg/ml against K. pneumoniae .
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 NaHCO3 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 flavonoid class (Table 3). Some of the classes of flavonoids that possess antimicrobial activity include Flavones, Isoflavones, Flavonols and their glycosides, Flavan-3-ols, Flavanones, Flavans etc., . The anti-ESBL activity of the ethyl acetate extract is ascribed to the presence of flavonoids as evident in the bioautography and this finding is supported by other reports , .
The inhibitory activity of the extract could be attributed to the presence of flavonoid group which has ability to complex with bacterial cell wall, extracellular and soluble proteins and probably causing membrane disruption . Flavonoids conjugated with nanoparticles have been proved to be a promising alternative for antibiotics .
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 findings in this work are important due to the fact that more residential area and industrial set-ups are being laid in the region. The study had also highlighted the inhibitory activity of the flavonoid 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) , we are in the early stages of a renaissance in the antibiotic discovery from actinomycetes.