ABSTRACT
Streptococcus pyogenes causes a variety of diseases ranging from mild diseases to severe invasive infections which result in significant morbidity and mortality. This study focuses on the antibiotic resistance of S. pyogenes and their interaction with cysteine protease. Around 36 beta-hemolytic isolates were collected from the clinical lab, of which seven isolates (19.4%) were identified as Streptococcus pyogenes. One of the seven isolates was collected from a urinary tract infection, which was identified by antibody agglutination and MALTI-TOF-MS, and it is designated as S. pyogenes NBMKU12. Around 8.3 to 66.6 % of the isolates were found to be resistant to one or more antimicrobial agents, especially, penicillin-G resistance was exhibited by 29.1% of the isolates. In the NBMKU12 isolate, the beta lactem (TEM) gene was detected among the 13 antibiotic genes for which it was tested. Furthermore, when analysis for presence of 13 virulence genes were carried out in NBMKU12 isolate, only speJ and speB were detected. The speB (streptococcal pyrogenic exotoxin B) encoding cysteine protease gene was cloned. This was followed by performing DNA sequencing to understand the putative cysteine protease interaction with antibiotics, inhibitors, and substrate. The speB gene consists of 1197 nucleotides and encodes a protein with multiple domains, including a signal peptide (aa 1-22), an inhibitor region (aa 27-156), and a catalytic cysteine domain (aa 160-367). The signal peptide cleavage site is predicted between Ala22 and Asn23. The putative 398 amino acid residues were found to have a theoretical pI of 8.76 and a molecular mass of 43,204.36 Da. The tested culture supernatants of NBMKU12 isolate exhibited the proteolytic activity against casein, papaya and pineapple used as substrates. The proteolytic activity suggests the expression of speB gene. Molecular docking analysis of cysteine protease showed that erythromycin (bond length 2.41 Å), followed by chloramphenicol (2.51 Å), exhibited a strong interaction; while penicillin-G (3.24 Å) exhibited a weak interaction, and this factor could be considered as a cause for penicillin-G resistance. The present study contributes to a better understanding of speB gene encoding cysteine protease, antibiotic resistance, and their interaction in the isolate, S. pyogenes NBMKU12. The antibiotics and cysteine protease interaction study confirms the resistance or sensitivity of S. pyogenes. Hence, it could be hypothesized that the isolate NBMKU12 is resistant to most of the tested antibiotics, and this resistance might be a cause for mutation.
ABSTRACT
BACKGROUND: Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-beta-lactamase 1 (NDM-1) are potentially a major global health problem. We investigated the prevalence of NDM-1, in multidrug-resistant Enterobacteriaceae in India, Pakistan, and the UK. METHODS: Enterobacteriaceae isolates were studied from two major centres in India--Chennai (south India), Haryana (north India)--and those referred to the UK's national reference laboratory. Antibiotic susceptibilities were assessed, and the presence of the carbapenem resistance gene bla(NDM-1) was established by PCR. Isolates were typed by pulsed-field gel electrophoresis of XbaI-restricted genomic DNA. Plasmids were analysed by S1 nuclease digestion and PCR typing. Case data for UK patients were reviewed for evidence of travel and recent admission to hospitals in India or Pakistan. FINDINGS: We identified 44 isolates with NDM-1 in Chennai, 26 in Haryana, 37 in the UK, and 73 in other sites in India and Pakistan. NDM-1 was mostly found among Escherichia coli (36) and Klebsiella pneumoniae (111), which were highly resistant to all antibiotics except to tigecycline and colistin. K pneumoniae isolates from Haryana were clonal but NDM-1 producers from the UK and Chennai were clonally diverse. Most isolates carried the NDM-1 gene on plasmids: those from UK and Chennai were readily transferable whereas those from Haryana were not conjugative. Many of the UK NDM-1 positive patients had travelled to India or Pakistan within the past year, or had links with these countries. INTERPRETATION: The potential of NDM-1 to be a worldwide public health problem is great, and co-ordinated international surveillance is needed.
