Virulence gene profile and antimicrobial resistance of non-typeable Streptococcus suis isolated from clinically healthy and diseased pigs from North East India.

The present study was conducted to investigate the virulence gene profile and antimicrobial resistance of non-typeable Streptococcus suis isolates circulating in pigs of North East India. Fifty-two non-typeable S. suis isolates from clinically healthy and diseased pigs were screened by using PCR for the presence of the muramidase-released protein (mrp), extracellular factor (epf), hemolysin suilysin (sly), arginine deiminase (arcA), and glutamate dehydrogenase (gdh) genes. Five different virulence gene profiles were observed and the most predominant virulence gene profile found in healthy pigs was mrp- + sly- + arcA- + gdh + + epf- whereas the most predominant virulence gene profile recorded in diseased pigs was mrp+ + sly- + arcA+ + gdh+ + epf-. Significantly lower carrier rate of mrp+ + sly- + arcA+ + gdh+ + epf- virulence gene profile was observed among the isolates from healthy pigs compared to those from diseased pigs (P < 0.05). Antimicrobial resistance patterns of the S. suis isolates revealed fourteen resistance groups (R1 to R14) where 88.46% isolates showed multi-drug resistance. The most predominant resistance pattern observed was CD-COT-E-TE. This is perhaps the first study reporting virulence gene profile and antimicrobial resistance of non-typeable S. suis isolates from pigs in North East India. The occurrence of relatively high levels of resistance of S. suis to some antimicrobials (e.g. macrolides, tetracyclines, and sulphonamides) as observed in the present study may represent a human health concern.

Densitometric analysis of rep-PCR data: Insight into genetic variability and transmission of Clostridium perfringens typed with an improved multiplex PCR.

An epidemiological study was conducted in North-East India (part of Indo-Burma biodiversity hotspot) to better understand the distribution, diversity, and transmission of Clostridium perfringens among livestock, pets, wild animals (captive), and humans. A total of 160 C. perfringens isolates were recovered from 642 diarrhoeic faecal samples with an isolation rate of 24.92%. Isolation rate was the highest among captive wild animals (37.5%) followed by dog (34.6%), human (33.8%), pig (32.7%), cattle (20.8%), goat (18.3%) and poultry (9.3%). Isolates were toxin typed using a seven gene multiplex PCR designed for simultaneous detection of cpa, cpb, cpb2, etx, iap, cpe and netB. The majority of isolates, 128 (80%) were of type A, followed by 17 (10.62%), 5 (3.12%), 4 (2.5%), 3 (1.87%), 2 (1.25%) and 1 (0.63%) isolates of type C, D, E, G, F and B, respectively. Beta 2 toxin gene was present in 65 (50%) of type A isolates, followed by 7 (41.2%), 4 (80%), 1(25%), and 1 (100%) of type C, D, G and B isolates, respectively. Beta 2 toxin has a high prevalence among dogs (28.6%), cattle (27.3%), and pig (20.8%) compared to humans, goat, wild animals, and poultry (1.2-14.3%). The prevalence of CPE and NetB toxin-positive strains was low, with only 3 (1.8%) and 5 (3.1%) isolates, respectively. Association of C. perfringens with diarrhoea in Civet Cat, Golden Langur, and Gray Langur has been reported for the first time. The genetic diversity and transmission of isolates were investigated using automated rep-PCR (Diversilab®, bioMérieux) using two densitometry-based matrices: modified Kullback-Leibler (KL) and Pearson's correlation (PC). The PC and modified KL matrices formed three distinct clusters with 59% and 27.2% similarity, respectively. C. perfringens diversity and transmission were best studied using modified KL matrix that placed more emphasis on the presence of bands rather than intensity. However, the PC method was found to be more suitable for differentiating strains within a toxin type, with slightly higher D-values.

Molecular characteristics of Clostridium difficile isolates from human and animals in the North Eastern region of India.

A total of 1034 samples were collected from different sources and C. difficile was isolated from 18 (9.04%) of 199 human, 9 (4.89%) of 184 cattle, 29 (12.44%) of 233 pig, and from 23 (13.94%) of 165 poultry samples. Variations were observed on the rate of isolation according to age and clinical conditions (diarrhoea). None of the samples from cow, sheep, goat, local chicken, and wild animals yielded any C. difficile. Out of those isolates, 8, 2, 19 and 6 isolates from human, cattle, pig and poultry, respectively were toxigenic. The toxigenic isolates carried both tcdA, and tcdB (A+B+) and most of the human and the pig isolates were also positive for binary toxin genes (cdtA and cdtB). The A+B+ isolates belonged to three different toxinotypes (0, VI and XXXIII). Human and pig A+B+ isolates belonged to three (045, 126 and ACD 019) and four (046, 087, 126 and ACD 011) different ribotypes, respectively and the ribotypes of two cattle isolates were 014 and ACD 010. Six A+B+ avian isolates belonged to six different ribotypes (014, 087, SLO 134, SLO 160, ACD 012, ACD 014). The non-toxigenic isolates from human, cattle, pig and poultry were grouped into 7, 4, 4 and 7 different ribotypes, respectively. PFGE analysis could not differentiate similar ribotypes/toxinotypes of toxigenic isolates. All the toxigenic isolates showed cytopathic effect on Vero and Hela cell monolayers at 1:100 dilutions of cell-free culture supernatants within 18-20 h of inoculation.

Isolation and characterization of Clostridium difficile from pet dogs in Assam, India.

One hundred and seventeen faecal samples from pet dogs (pup = 21 and adult = 96) brought for treatment to a veterinary clinic were examined for Clostridium difficile. A total of 16 (13.67%) samples were positive. Nine (56.25%) isolates were obtained from 17 adult dogs undergoing antibiotic treatment and this was significantly higher (p < 0.01) as compared to isolates from dogs without antibiotic treatment. Ten isolates (62.5%) were toxigenic (all toxinotype 0) and six were non-toxigenic. None of the isolates were positive for binary toxin genes. PCR ribotyping revealed three different ribotypes (012, 014 and 046) among A(+)B(+) isolates and five different ribotypes (010, SLO 131, and ACD 001 to ACD 003) among A(-)B(-) isolates. The PFGE analysis of toxigenic isolates revealed three different pulsotypes corresponding to the PCR ribotypes.