Devi Prasad Isore

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.

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.

Seroprevalence of bluetongue in ruminants of Jharkhand

Aim: This study was carried out to assess the presence of anti-bluetongue (BT) antibodies in sheep, goat and cattle of different agro-climatic zones of Jharkhand. Materials and Methods: Serum samples were collected from apparently healthy as well as suspected sheep, goat and cattle from different districts of Jharkhand covering different agro-climatic zones. Serum samples were screened by indirect enzyme linked immunosorbent assay (iELISA) for detecting anti-BT antibodies. Results: Out of a total of 480 animal serum samples (sheep-190, goats-210 and cattle-80) screened, 83 (43.68%) of sheep, 91 (43.33%) of goat and 46 (57.50%) of cattle sera were found positive. The % positivity ranged between 41% and 51% in different agro-climatic zones. The results showed slight higher seroprevalence, although not significantly, in cattle than sheep and goats in different agro-climatic zones of Jharkhand. Conclusions: The above data indicate widespread prevalence of BT virus antibodies in studied areas. The incidence of BT is not detected officially, so far. The present seroprevalence status of BT in Jharkhand indicates presence of BT infection in the state for the first time.

Detection, characterization, and antibiogram of extended-spectrum beta-lactamase Escherichia coli isolated from bovine milk samples in West Bengal, India

Background: Milk is considered as complete food and an important part of human diet throughout the world including India. Bacterial contamination of milk such as Escherichia coli due to unhygienic condition and poor udder health can cause infections, especially in infants and elders or in immunocompromised persons. Possession of antimicrobial resistance genes by commensal bacteria present in milk makes the issue more serious. Aim: The study was aimed to isolate and characterize extended-spectrum beta-lactamase (ESBL)-producing E. coli from milk samples collected from different parts of West Bengal, India, to assess the potential risk associated with the food. Materials and Methods: Around 182 milk samples were collected from apparently healthy cows reared by organized dairy farms in West Bengal. E. coli was isolated from collected samples as per standard methods followed by serotyping. The detection of ESBL-producing E. coli was done both phenotypically and genotypically by detecting the presence of blaCTX-M gene. Antibiogram of the ESBL-positive isolates was done using common 12 antibiotics by disc diffusion method. Results: A total of 22 (12.1%) samples were found to be positive for E. coli in this study. Different serotypes such as O11, O20, O22, O34, O35, O128, O149, and UT were isolated from the collected samples. 12 (54.5%) E. coli strains showed the capability of producing ESBL, both phenotypically and genotypically with the presence of blaCTX-M gene. Antibiogram of these ESBL-positive isolates revealed the drugs such as colistin (100%), levofloxacin (83.33%), and imipenem (66.67%) to be highly sensitive against this pathogen but drugs such as cefotaxime (100%), ceftazidime (91.67%), amoxicillin/clavulanic acid (83.33%), tetracycline (75.00%), and gentamicin (58.33%) to be very much resistant. Conclusion: More than 50% of the E. coli strains prevalent in the bovine milk samples were positive for ESBL production and are resistant to most of the common antimicrobials which may be alarming for human health.

Characterization of Salmonella Gallinarum isolates from backyard poultry by polymerase chain reaction detection of invasion (invA) and Salmonella plasmid virulence (spvC) genes

Aim: The aim was to characterize Salmonella enterica serovar Gallinarum isolated from backyard poultry by polymerase chain reaction (PCR) detection of virulence genes invasion (invA) and Salmonella plasmid virulence C (spvC). Materials and Methods: Two strains of Salmonella serovar Gallinarum isolates used in this study were obtained from an outbreak of fowl typhoid in backyard Vanaraja fowl. PCR technique was used for detection of invA and spvC genes using standard methodology. The invA PCR product from one representative isolate was sequenced and compared with other related Salmonella serovars in GenBank data. Results: Salmonella Gallinarum produced expected amplicons of invA and spvC gene products. Nucleotide sequence of 285 bp invA gene was deposited in GenBank with accession no. KX788214. Sequence analysis of invA gene was found conserved in Salmonella serovars and demonstrated 100% homology with closely related serovars of Salmonella. Conclusion: Invasion gene (invA) was found to be highly conserved in Salmonella Gallinarum and highly similar with closely related serovars. The isolates also contained plasmid-mediated spvC gene indicating possession of virulence plasmid.