Cloning, expression, homology modelling and molecular dynamics simulation of four domain-containing α-amylase from Streptomyces griseus.

In the present study, α-amylase from Streptomyces griseus TBG19NRA1 was amplified, cloned and successfully expressed in E. coli BL21/DE3. Sequence analysis of S. griseus α-amylase (SGAmy) revealed the presence of four domains (A, B, C and E). Alpha-amylases with E domain (also known as carbohydrate binding module 20 (CBM20)) are capable of degrading raw starch and this property holds great potential for application in starch processing industries. Though α-amylase is a well-studied and characterized enzyme, there is no experimental structure available for this four domain-containing α-amylases. To gain more insight about SGAmy structure and function, homology modelling was performed using a multi-template method. The template α-amylase from Pseudoalteromonas haloplanktis (PDB ID 1AQH) and E domain of Cyclodextrin glucanotransferase from Bacillus circulans (PDB ID 1CGY) was found to have significant similarity with the complete target sequence of SGAmy. Therefore, homology model for SGAmy was generated from the crystal structure of 1AQH and 1CGY and the resulting structure was subjected to 10 ns molecular dynamics (MD) simulation. Remarkably, CBM20 domain of SGAmy showed greater flexibility in MD simulation than other three domains. This observation is highly rational as this part of SGAmy is strongly implicated in substrate (raw starch) binding. Thus, conformational plasticity at CBM20 is functionally beneficial.Communicated by Ramaswamy H. Sarma.

Comparative analysis of emm types, superantigen gene profiles and antibiotic resistance genes among Streptococcus pyogenes isolates from ocular infections, pharyngitis and asymptomatic children in south India.

Group A Streptococcus (Streptococcus pyogenes) is responsible for a wide array of infections and incidence is high in developing countries like India. Although distribution of emm types of S. pyogenes in India has been described, its association with the virulence genes and ocular isolates is less concentrated. In the present study emm type surveillance as well as its association with toxin gene profile was analyzed. Ocular infected cases such as lacrimal abscess, corneal ulcers, mucocoele showed the presence of 20 S. pyogenes isolates. For noninvasive isolates, we screened 370 pharyngitis cases and 400 asymptomatic school children and recovered 33 pharyngitis and 14 carrier isolates respectively. 14 Emm type distributions were observed in ocular isolates, 11 emm types each in pharyngitis and asymptomatic carrier isolates. The two dominant emm types, emm49 and emm63 were accounted for 33% of the total S. pyogenes isolates. Among ocular isolates, slo, smeZ, speB and speG were found in >50% of isolates, in pharyngitis smeZ (48%), speB (45%) and speG (42%) genes were found to be prevalent. Alarmingly, carrier isolates showed more prevalence to virulence genes than the ocular and pharyngitis isolates with speF (79%), speB, speG (64%), slo and sil (64%). Among the three groups, pharyngitis isolates harbored more prtF1 (33%) and prtF2 (94%) than the asymptomatic carriers (28% and 71%) and the ocular isolates (45% and 40%). 450bp Size band in prtF1 and 350bp size band in prtF2 showed dominance. Among the three groups tested, the distribution of ermB and mefA was high in pharyngitis isolates (30%) where 10 isolates showed the presence of both genes. None of the isolates showed the presence of ermA and tetO genes. Dendrogram generated based on the virulence and antibiotic resistance gene profiles revealed that except one cluster, all other clusters showed some correlation with ocular, pharyngitis and asymptomatic carrier isolates, irrespective of their emm types.

Effect of subinhibitory concentrations of fluoroquinolones on biofilm production by clinical isolates of Streptococcus pyogenes.

BACKGROUND & OBJECTIVES: Subinhibitory concentrations (sub-MICs) of antibiotics, although not able to kill bacteria, but influence bacterial virulence significantly. Fluoroquinolones (FQs) which are used against other bacterial pathogens creates resistance in non-targeted Streptococcus pyogenes. This study was undertaken to characterize the effect of sub-MICs of FQs on S. pyogenes biofilm formation. METHODS: Biofilm forming six M serotypes M56, st38, M89, M65, M100 and M74 of S. pyogenes clinical isolates were challenged against four FQs namely, ciprofloxacin, ofloxacin, levofloxacin and norfloxacin. The antibiofilm potential of these FQs was analysed at their subinhibitory concentrations (1/2 to 1/64 MIC) using biofilm assay, XTT reduction assay, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). RESULTS: Among the four FQs tested, ofloxacin and levofloxacin at 1/2 MIC showed the maximum inhibition (92%) of biofilm formation against M56 and M74 serotypes. FQs effectively interfered in the microcolony formation of S. pyogenes isolates at 1/2 to 1/8 sub-MICs. Inhibition of biofilm formation was greatly reduced beyond 1/16 MICs and allowed biofilm formation. XTT reduction assay revealed the increase in metabolic activity of S. pyogenes biofilm against the decrease in FQs concentration. SEM and CLSM validated the potential of sub-MICs of FQs against the six S. pyogenes. INTERPRETATION & CONCLUSIONS: Our results showed that the inhibitory effect all four FQs on S. pyogenes biofilm formation was concentration dependent. FQs at proper dosage can be effective against S. pyogenes and lower concentrations may allow the bacteria to form barriers against the antibiotic in the form of biofilm.

Virulence and multidrug resistance patterns of Vibrio cholerae O1 isolates from diarrheal outbreaks of South India during 2006-2009.

A total of 31 Vibrio cholerae O1 (4- Inaba and 27- Ogawa serotype) isolates collected during a three-year period (2006-2009) from acute diarrheal cases in Tamil Nadu, India were analyzed for antibiotic resistance profiling, virulence-associated factors, genetic profiling by enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC PCR), and biofilm-forming ability. Antibiotic resistance profile revealed that most of the strains have become multidrug-resistant strains. All the isolates are resistant to ampicillin and polymyxin B, 97% of the isolates are resistant to nalidixic acid, 90% to co-trimoxazole, 32.3% to norfloxacin and ciprofloxacin, 29% to doxycycline, 10% to gentamicin, whereas only 3% to chloramphenicol. Molecular characterization of virulence-associated genes by multiplex PCR revealed the presence of ace, ctxA, tcpA, toxR, and ompU as 93.5%, followed by ompW with 33.3%. The presence of zot was restricted to only one isolate and hlyA was not encountered in any of the strains. ERIC PCR produced more than 10 bands for each isolate and the dendrogram generated based on the cluster analysis showed the presence of 29 electrophoretic types among the 31 isolates. Isolates from different area or year of isolation are intermingled in all the clusters. With respect to biofilm formation, 24 isolates were found to be biofilm formers and eight of them produced strong biofilm. This study demonstrates the presence of critical virulence factors and antibiotic resistance in the diarrhea isolates, which signifies the importance of routine monitoring and proper treatment to prevent cholera outbreaks.

Emergence of methicillin-resistant, vancomycin-intermediate Staphylococcus aureus among patients associated with group A Streptococcal pharyngitis infection in southern India.

Beyond Staphylococcus aureus being an etiological agent for several serious clinical complications, the foot prints of S. aureus in pharyngitis infection has also been recently recognized. With due response to the fact, a prospective study was conducted between 2009 and 2010 to describe the molecular epidemiology of S. aureus in throat swabs of pharyngitis patients. A total of 63 methicillin-resistant S. aureus (MRSA) and 102 methicillin-susceptible S. aureus (MSSA) isolates were recovered from 265 throat swabs, representing a community-acquired outpatient population from Tamil Nadu, India. Molecular characterization of MRSA was done by two conventional multiplex PCR assays including Panton-Valentine leukocidin (PVL), mecA and nuc genes, and staphylococcal cassette chromosome mec (SCCmec) typing. Among 165 S. aureus isolates, methicillin resistance was observed in 38.2% (n=63), in which 69.8% (n=44/63) of the MRSA along with 55.9% (n=57/102) of MSSA harbored PVL toxin genes. SCCmec typing showed 50.8% of isolates as SCCmec V (n=32), 44.4% as SCCmec III (n=28), and 1.6% as SCCmec types I, II and IVa (n=1). Multilocus sequence typing performed for 26 selected MRSA isolates resulted in 12 different sequence types (ST), including a novel ST2129/SCCmec III, PVL-positive. Ten MRSA isolates were categorized as ST772 (38.5%)/SCCmec V, PVL-positive, and three isolates as ST368 (11.5%)/SCCmec III, PVL-negative. Though the prominent clones of ST772/SCCmec V were multidrug-susceptible worldwide, they were highly multidrug-resistant in the current study, including four clones intermediate to vancomycin. Totally, 10 (15.9%) out of 63 MRSA isolates were documented as vancomycin-intermediate S. aureus (VISA). Collectively, the present study for the first time portrayed the high prevalence of active MRSA pharyngitis infection and also emphasizes an alarming need for discrimination of pharyngeal-asymptomatic carriers of S. aureus from those with an active S. aureus pharyngitis infection.

Comparison of bacterial communities in the throat swabs from healthy subjects and pharyngitis patients by terminal restriction fragment length polymorphism.

Terminal restriction fragment length polymorphism (T-RFLP) analysis was applied to characterize bacterial flora present in the throats of healthy subjects and pharyngitis patients. The 16S rRNA genes of bacteria present in throat metagenome were amplified by PCR with 6-carboxy-fluorescein (6-FAM)-labeled universal forward primer (27 F) and a universal reverse primer (1513R). The 16S rDNAs were digested with restriction enzymes with 4-bp recognition sites (MspI or RsaI) and analyzed by using an automated DNA sequencer. T-RFLP patterns were numerically analyzed using computer programs. From analysis of the throat bacterial community, patterns derived from MspI and RsaI digested samples of healthy subjects and pharyngitis patients were grouped into different clusters, though RsaI digested samples showed some uncertainty. Pharyngitis throats generated an average species richness of 9 [±2.1 (SD)] and 10 (±2.9) for MspI and RsaI digests, respectively, whereas healthy throats generated 6.3 (±1.2) and 6.1 (±1.5) in MspI and RsaI digests, respectively. These results suggest that samples from pharyngitis patients contain an unexpected diversity of causative bacteria. The pharyngitis throats were colonized with a rich diversity of bacterial species than that of healthy throats. Using T-RFLP, we are able to detect a model bacterium, Streptococcus pyogenes SF370, and T-RF patterns were consistent with the Streptococcal T-RFLP patterns. Our study indicates that T-RFLP analysis is useful for the assessment of diversity of throat bacterial flora and rapid comparison of the community structure between subjects with and without pharyngitis.

Characterization of biofilms in different clinical M serotypes of Streptococcus pyogenes.

Streptococcus pyogenes is a notorious human pathogen responsible for a wide array of infections. The ability of S. pyogenes to form biofilms is an innate property during the pathogenesis of invasive infections. From the eleven M serotypes tested: M56, M74, M100, M65, M89 and st38 formed dense biofilms in 48 h. The present study is the first of its kind to report about the biofilm formation in the serotypes M56, M65 M74 M100 and st38. XTT reduction assay of the biofilms showed decreased metabolic activity with increase in incubation time. The surface architecture of the biofilms when observed by scanning electron microscopy (SEM) revealed the microcolony formation. Confocal laser scanning microscopy (CLSM) was used to compare the surface topography and thickness of biofilms between the biofilm formers with and without the addition of glucose. Interestingly a non-biofilm former (st2147) was induced to form biofilms with the addition of glucose. On correlating the drug (erythromycin) resistance of the various M serotypes with their biofilm forming ability we noticed that erythromycin sensitive strains were found to be good biofilm formers. We also noticed that biofilm formation in S. pyogenes is independent of sil gene.

Development of species-specific primers for detection of Streptococcus pyogenes from throat swabs.

A species-specific molecular marker has been developed to detect the human pathogen Streptococcus pyogenes from throat swabs. Streptococcus pyogenes is an important pathogen among Gram-positive organisms. A rapid and simple diagnostic tool is of utmost importance for the identification of this pathogen. The random amplified polymorphic DNA (RAPD) technique was used to differentiate the S. pyogenes strains. A differentially amplified fragment obtained from RAPD profiles was sequenced and characterized, which was developed into a sequence characterized amplified region (SCAR) marker to evaluate the specificity of S. pyogenes from other species of Streptococcus. The sensitivity of the SCAR primers was studied by qualitative PCR and the detection limit was found to be 10(-1) ng of genomic DNA or one to two cells of S. pyogenes. The specificity of the primers was assayed in 270 clinical throat swabs wherein 23 samples turned to be positive, which was highly significant over culture-based methods. This species-specific primer enables accurate detection of S. pyogenes from clinical samples and will be a useful tool in epidemiological studies.

The correction of involutional entropion of eyelid by lateral strip procedure.

AIM: To determine cosmetic and functional outcome following lateral strip procedure (LSP) for involutional entropion. MATERIALS AND METHODS: This study was a prospective analysis of 15 patients (20 eyelids) of involutional entropion, who needed surgical repair. After thorough evaluation, the surgical treatment (LSP) was done in all 15 patients. RESULTS: Cosmetic and functional outcome was excellent in all cases following LSP. No complications and recurrence were encountered in any case. CONCLUSIONS: LSP is simple, physiologic, easy and quick to perform as OPD procedure for involutional entropion under local anesthesia without hospitalization by a general ophthalmologist.