Indranil Samanta

Potential antibacterial activity of berberine against multi drug resistant enterovirulent Escherichia coli isolated from yaks (Poephagus grunniens) with haemorrhagic diarrhoea

OBJECTIVE To evaluate the antimicrobial efficacy of berberine, a plant alkaloid. METHODS Five multi-drug resistant (MDR) STEC/EPEC and five MDR ETEC isolates from yaks with haemorrhagic diarrhoea were selected for the study. Antibacterial activity of berberine was evaluated by broth dilution and disc diffusion methods. The binding kinetics of berberine to DNA and protein was also enumerated. RESULTS For both categories of enterovirulent Escherichia coli (E. coli) isolates, berberine displayed the antibacterial effect in a dose dependent manner. The MIC(50) of berberine chloride for STEC/EPEC isolates varied from 2.07 μM to 3.6 μM with a mean of (2.95 ± 0.33) μM where as for ETEC strains it varied from 1.75 to 1.96 μM with a mean of (1.87 ± 0.03) μM. Berberine bind more tightly with double helix DNA with Bmax and Kd of (24.68±2.62) and (357.8±57.8), respectively. Berberine reacted with protein in comparatively loose manner with Bmax and Kd of (18.9±3.83) and (286.2±113.6), respectively. CONCLUSIONS The results indicate clearly that berberine may serve as a good antibacterial against multi drug resistant E. coli.

Understanding osteomyelitis and its treatment through local drug delivery system

Chronic osteomyelitis is a major challenge in bone surgery. Conventional use of antibiotics is not an effective way to control the malaise due to so many reasons. Determination of optimal treatment strategy becomes difficult for the orthopaedic surgeons and as a consequence, the patients suffer not only from therapeutic failure but also due to adverse side effects of antibiotics and financial loss due to additional stay at hospitals. A wide application of carrier systems, as a medium for local delivery of antibiotics, is being used experimentally and clinically for the treatment of osteomyelitis. This kind of delivery system provides sustained higher concentration of antibiotics at the infection site with reduced possibility of toxicity. This review highlight etiology and pathophysiology of osteomyelitis, current therapeutic options with their limitations, and potentiality of biomaterial based carrier materials impregnated with antibiotics as local delivery approach.

Virulence Repertoire, Characterization, and Antibiotic Resistance Pattern Analysis of Escherichia coli Isolated from Backyard Layers and Their Environment in India

SUMMARY This study was undertaken to observe the prevalence, serogroup, avian pathogenic Escherichia coli (APEC)-associated virulence gene, randomly amplified polymorphic DNA (RAPD) pattern, and antibiotic resistance genes of E. coli in backyard layers and their environment in India. From the 360 samples of healthy layers and their environment, 272 (75.5%) E. coli were isolated. The majority (28.67%) of them were untypeable. Among the studied virulence genes (papC, tsh, iucC, astA), 52 (14.32%) isolates were found to possess astA, including the isolates from the drinking water of the birds (4/272, 1.47%). These strains belonged to 18 different serogroups. Most of the isolates were typeable by RAPD and they produced different patterns. Phenotypic resistance of the isolates was most frequently observed to erythromycin (95.83%), chloramphenicol (87.52%), and cotrimoxazole (78.26%). None of the isolates was found to possess extended-spectrum beta-lactamases (blaTEM, blaSHV, blaCTX-M) or quinolone resistance (qnrA) genes by PCR. The present study was the first attempt in India to assess APEC distribution in backyard poultry production. RESUMEN Repertorio de virulencia, caracterización y análisis de los patrones de resistencia a los antibióticos de Escherichia coli aisladas de gallinas de postura de traspatio y de su medio ambiente en la India. Este estudio se realizó para determinar la prevalencia, el serogrupo, los genes asociados a virulencia de Escherichia coli patógena para las aves (APEC), los patrones de ADN polimórfico amplificado aleatoriamente (RAPD), y los genes de resistencia a los antibióticos de E. coli en gallinas de traspatio y su ambiente en la India. De las 360 muestras de gallinas sanas y de su ambiente, se aislaron 272 cepas de E. coli (75.5%). La mayoría (28.67%) de ellas no fueron tipificables. Entre los genes de virulencia estudiados (papC, tsh, iucC, astA), se encontró que 52 aislamientos (14.32%) poseían el gene astA, incluyendo los aislamientos de agua de bebida de las aves (4/272, 1.47%). Estas cepas pertenecían a 18 serogrupos diferentes. La mayoría de los aislamientos fueron tipificables mediante RAPD y produjeron diferentes patrones. La resistencia fenotípica de los aislamientos se observó con mayor frecuencia contra eritromicina (95.83%), cloranfenicol (87.52%), y contra cotrimoxazol (78.26%). Se encontró mediante PCR que ninguno de los aislamientos poseía genes de un espectro extendido de beta-lactamasas (blaTEM, blaSHV, blaCTX-M), o resistencia contra quinolonas (qnrA). El presente estudio es el primer intento en la India para evaluar la distribución de E. coli patógena para la producción de aves de traspatio.

Isolation, molecular characterization and antibiotic resistance of Shiga Toxin–Producing Escherichia coli (STEC) from buffalo in India

In total, 363 Escherichia coli were isolated from 165 faecal samples of healthy buffaloes in West Bengal, India. Twenty‐four of these isolates (6·61%) were found to carry at least one gene characteristic for Shiga toxin–producing Escherichia coli (STEC). These STEC strains belonged to 13 different O‐serogroups. The stx1 gene was present in 23 (95·8%) of total STEC isolates, whereas 20 (83·3%) STEC isolates carried the gene stx2. Twelve strains of E. coli (50% of total STEC isolates) possessed enterohaemolysin (ehxA) gene in combination with others. Fourteen (58·33%) isolates found to possess saa gene. However, no E. coli was detected harbouring gene for intimin protein (eaeA). Of 23 stx1‐positive isolates, seven (30·43%) were positive for genes of the stx1C subtype. Of the 20 isolates with the stx2 gene, 25% (5/20) possessed stx2C and 10% (2/20) possessed stx2d gene. The phylogenetic analysis after RAPD of STEC strains revealed six major clusters. The isolated STEC strains were resistant most frequently to erythromycin (95·83%), cephalothin (62·5%), amikacin (54·17%), kanamycin (45·83%) and gentamicin (41·67%) group of antibiotics. No ESBL‐producing (blaCTXM, blaTEM, blaSHV) or quinolone resistance gene (qnrA) was detected in the STEC isolates.

Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolate Possessing the Shiga Toxin Gene (stx1) Belonging to the O64 Serogroup Associated with Human Disease in India

Shiga toxin-producing Escherichia coli (STEC) causes a spectrum of human sufferings, like bloody diarrhea and even life-threatening conditions, such as hemolytic uremic syndrome (HUS) ([1][1]). In addition to the serotype O157:H7, several serogroups of STEC have been isolated from severe outbreaks

Co-infection of methicillin-resistant Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus and extended spectrum β-lactamase producing Escherichia coli in bovine mastitis – three cases reported from India

Emergence of antimicrobial resistance among bovine mastitis pathogens is the major cause of frequent therapeutic failure and a cause of concern for veterinary practitioners. This study describes intra-mammary infection of methicillin-resistant Staphylococcus epidermidis (MRSE), methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum β-lactamase (ESBL) producing Escherichia coli in two Holstein Friesian crossbred cows with subclinical mastitis and one non-descript cow with clinical mastitis in two different districts of West Bengal, India. In total, three MRSE, one MRSA and three ESBL producing E. coli were isolated from these cases. Both the crossbreds were detected with MRSE (HFSE1 and HFSE2) and ESBL producing E. coli (HFEC1 and HFEC2), whereas, simultaneous infection of three pathogens viz. MRSA (NDSA1), MRSE (NDSE1) and ESBL producing E. coli (NDEC1) was found in the non-descript cow. The methicillin-resistant isolates possessed mecA gene and exhibited resistance to various antibiotics such as amikacin, tetracycline and glycopeptides. The ESBL producers were positive for blaCTX-M and blaTEM genes; in addition, HFEC1 and HFEC2 were positive for blaSHV and possessed the genes for class I integron (int1), sulphonamide resistance (sul1), quinolone resistance (qnrS) and other virulence factors (papC, iucD and ESTA1). All the ESBL producers exhibited resistance to a variety of antibiotics tested including third- and fourth-generation cephalosporins and were also intermediately resistant to carbapenems. This is the first ever report on simultaneous occurrence of MRSE, MRSA and ESBL producing E. coli in bovine mastitis indicating a major concern for dairy industry and public health as well.

Approaches to characterize extended spectrum beta-lactamase/beta-lactamase producing Escherichia coli in healthy organized vis-a-vis backyard farmed pigs in India

The study was undertaken to investigate the occurrence and to characterize the ESBL/beta-lactamase producing-Escherichia coli in healthy pigs of organized and backyard farms in West Bengal, India. Total 200 rectal swabs were collected randomly from healthy pigs maintained in four organized farms and 10 backyard farms (n=100 each) and 76 isolates were identified as E. coli from organized (48/100, 48%) and backyard pigs (28/100, 28%). Twelve E. coli isolates (6%) in the present study were detected to possess any of the ESBL/beta-lactamase genes studied. ESBL/beta-lactamase producers were isolated with significantly more frequency from backyard pigs than the organized farm pigs (p=0.026). Six of ESBL/beta-lactamase producing isolates were phenotypically confirmed as CTX-M producers and ten of them were confirmed as TEM/SHV producers. PCR and sequencing of the amplified product from representative isolates revealed the presence of blaCTX-M-9, blaSHV-12 and blaTEM-1. No unique combination of the studied beta lactamase genes for organized and backyard farm pig isolates was noted. The ESBL isolates belonged to O13, O55, O133, O153, O157, O158, O166, rough and OUT serogroups. The association of heat labile toxin (elt) (p<0.0005) with organized farm isolates and heat stable toxin (estA) (p=0.0143) with backyard piggery sector was significantly higher. The ESBL/beta-lactamase producers from organized farm (Ak/Ex) and indigenous pigs (Ak/Ex/Te; Ak/CoT/G) showed a characteristic phenotypical antibiotic resistance pattern. Two pairs of isolates from organized and backyard farm pigs showed clonal relationship indicating a possible transmission between the farms which were situated adjacently. Thus the present study revealed backyard farm pigs as major source of ESBL/beta-lactamase producing-E. coli associated with STa and characteristic antibiotic resistance pattern in India.

Effect of black cumin seeds on growth performance, nutrient utilization, immunity, gut health and nitrogen excretion in broiler chickens

BACKGROUND Use of antibiotic growth promoters (AB) as feed additives in broiler chickens poses risks due to cross-resistance amongst pathogens and residues in tissues. The aim of this study was to evaluate the effect of dietary supplementation of black cumin seeds (BCS) as a natural growth promoter in chickens on nutrient utilization, intestinal microbiota and morphology, immunity, antioxidant status, protein deposition in muscles and nitrogen excretion. RESULTS Broiler chickens were fed BCS at 0, 5, 10 and 20 g kg-1 diet. Body weight gain tended to increase (P = 0.10) and daily feed intake increased quadratically with increasing concentrations of BCS in the diets. Supplementation of BCS resulted in a tendency to decrease feed conversion efficiency on days 28-42. Metabolizability of nutrients increased linearly with increasing doses of BCS. Protein deposition in thigh and breast muscles was increased and nitrogen excretion was reduced by BCS and AB compared with the control (CON). Intestinal morphology in the duodenum, jejunum and ileum was not generally affected by BCS. Counts of total bacteria, Escherichia coli, Lactobacillus spp. and Clostridium spp. were not affected by BCS and AB compared with CON, but Salmonella spp. decreased linearly (P = 0.05) with increasing doses of BCS. Antibody titers against Newcastle disease virus on day 35 increased quadratically (P < 0.001) with increasing doses of BCS. Concentrations of glucose and triglyceride in blood were not affected by BCS. Concentrations of cholesterol decreased linearly while the concentration of total protein increased linearly with increasing doses of BCS. CONCLUSION The use of dietary BCS may improve growth performance, immunity and nutrient utilization in broiler chickens.

Effects of dietary supplementation of cinnamaldehyde and formic acid on growth performance, intestinal microbiota and immune response in broiler chickens

An experiment was conducted to investigate the effects of replacing antibiotic growth promoter (AGP) with a combination of essential oil and organic acids (EO + OA) on growth performance, gut microbiota and immune response in broiler chickens. In Experiment 1, 320 day-old broiler chicks were randomly distributed to 32 pens with 10 birds in each pen and the pens were equally allotted to four treatment groups. In Experiment 2, 120 day-old chicks were divided into the same four treatment groups, each group containing three replicated pens with 10 birds in each pen. The groups were (1) Negative Control (NC) without AGP or other growth-promoting feed additives; (2) AGP (NC + enramycin; 125 mg/kg feed; (3) OA (NC + OA; 500 mg/kg feed); and (4) EO + OA (NC + a combination of cinnamaldehyde and calcium formate; 500 mg/kg feed). Experiment 1 lasted for 40 days, whereas Experiment 2 continued for 28 days. In Experiment 2, all birds were orally challenged with Escherichia coli (108 bacteria/bird) on Day 14. Overall intake, growth and feed conversion ratio (FCR) on Day 40 had no difference (P > 0.05) among the groups in Experiment 1. In Experiment 2, growth, feed intake and FCR were not affected by any dietary treatments until Day 14, but after being challenged with E. coli, bodyweight gain and FCR improved (P 0.05) the counts of total bacteria, E. coli and Lactobacillus in the contents of ileum and caecum. However, Salmonella counts in the ileal and caecal contents decreased (P < 0.001) for AGP, OA and EO + OA compared with NC group. Clostridium counts were lower for EO + OA group than for NC and AGP groups in the ileum, and for AGP, OA and EO + OA groups than for NC in the caeca (P < 0.05). Antibody titer on Day 35 against Newcastle disease vaccine was higher in EO + OA group than in NC, AGP and OA groups (P < 0.001). In conclusion, EO + OA did not affect growth and FCR in broilers. However, AGP and EO + AO improved growth performance and FCR after being challenged with E. coli. Moreover, EO + OA was effective in reducing the Clostridium count in the small intestine and caecum and increasing the villus height and antibody titer against Newcastle disease vaccine.

Seroepidemiology of bluetongue in South Bengal

Aim: With the aim of revealing the epidemiological intricacies of bluetongue (BT) in the southern part of West Bengal state, the present study was undertaken to assess seroprevalence of BT along with identification of the vector of the disease, i.e., Culicoides midges available in the region in their breeding season with conducive environmental factors, if any. Materials and Methods: A total of 1509 (sheep-504, goat-1005) samples were collected from three different agroclimatic zones of South Bengal viz. new alluvial, red laterite and coastal saline. To detect anti-BT antibodies in the collected serum samples, indirect-enzyme-linked immunosorbent assay (i-ELISA) was performed. Culicoides midges were collected from those agro-climatic zones of South Bengal for species identification. The meteorological parameters, viz. temperature (maximum and minimum), rainfall and relative humidity of three agro-climatic zones of South Bengal were analyzed for the months of July to December during 2010-2013. Results: The overall seropositivity was 33.13% and 30.24% in sheep and goat, respectively as assessed by i-ELISA. In South Bengal, the predominant species of Culicoides found were Culicoides schultzei, Culicoides palpifer and Culicoides definitus. Conclusion: Since virus transmitting species of Culicoides midges could be detected in South Bengal, besides high seropositivity in ruminants, the possibility of circulating BT virus in South Bengal is quite imminent.