Pankaj Dhaka

Antimicrobial effects of Lactobacillus plantarum and Lactobacillus acidophilus against multidrug-resistant enteroaggregative Escherichia coli

The in vitro and in vivo antimicrobial effects of Lactobacillus plantarum and Lactobacillus acidophilus were evaluated individually and synergistically against multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC). In vitro evaluation of each probiotic strain when co-cultured with MDR-EAEC isolates revealed a reduction in MDR-EAEC counts (eosin-methylene blue agar) in a dose- and time-dependent manner: probiotics at a dose rate of 10(10) CFU inhibited MDR-EAEC isolates at 72 h post-inoculation (PI), whereas at lower concentrations (10(8) and 10(9) CFU) MDR-EAEC isolates were inhibited at 96 h PI. The synergistic antimicrobial effect of both probiotic strains (each at 10(10) CFU) was highly significant (P < 0.01) and inhibited the growth of MDR-EAEC isolates at 24 h PI. For in vivo evaluation, weaned mice were fed orally with 10(7) CFU of MDR-EAEC. At Day 3 post-infection, treated mice were fed orally with the probiotic strains (each at 10(10) CFU). Compared with the control, post-treatment a significant (P < 0.01) reduction in MDR-EAEC counts was observed in faeces by Day 2 and in intestinal tissues of treated mice by Days 3 and 4 as evidenced by plate count (mean 2.71 log and 2.27 log, respectively) and real-time PCR (mean 1.62 log and 1.57 log, respectively) methods. Histopathologically, comparatively mild changes were observed in the ileum and colon from Days 3 to 5 post-treatment with probiotics; however, from Day 6 the changes were regenerative or normal. These observations suggest that these probiotic strains can serve as alternative therapeutics against MDR-EAEC-associated infections in humans and animals.

Genetic diversity, virulence potential and antimicrobial susceptibility of Listeria monocytogenes recovered from different sources in India

Listeria monocytogenes isolates (n = 36) recovered from human and animal clinical cases and foods from different geographical regions of India were characterized using multiplex PCR-based serotyping, pulsed field gel electrophoresis (PFGE), in vitro and in vivo pathogenicity tests and antibiogram profiling. Multiplex PCR-based serotyping distributed L. monocytogenes isolates into 3 serogroups, of which 91.67% belonged to 4b, 4d, 4e serogroup, followed by 5.56% to 1/2a, 3a and 2.78% to 1/2b, 3b serogroups. PFGE analysis using ApaI and AscI restriction enzymes revealed 17 pulsotypes among 36 L. monocytogenes isolates with 6 major clusters having similar fingerprint profile within their cluster and 11 unique fingerprint profiles. Interestingly, PFGE analysis inferred that foods of animal origin could be a significant source of infection for spread of listeriosis among human populations. Furthermore, on comparison of in vitro and in vivo pathogenicity tests, an overall good correlation was observed between hemolytic titer assay and chick embryo inoculation test as most of the isolates with a hemolytic titer of ≥ 16 were found to be lethal to chick embryo. All the isolates were found to be susceptible to tested antimicrobials except for one animal isolate which showed resistance towards co-trimoxazole.

Genetic diversity and antibiogram profile of diarrhoeagenic Escherichia coli pathotypes isolated from human, animal, foods and associated environmental sources

Introduction Infectious diarrhoea particularly due to pathogenic bacteria is a major health problem in developing countries, including India. Despite significant reports of diarrhoeagenic Escherichia coli (DEC) pathotypes around the globe, studies which address genetic relatedness, antibiogram profile and their correlation with respect to their isolation from different sources are sparse. The present study determines isolation and identification of DEC pathotypes from different sources, their genetic characterisation, antibiogram profile and their correlation if any. Materials and methods A total of 336 samples comprising diarrhoeic stool samples from infants (n=103), young animal (n=106), foods (n=68) and associated environmental sources (n=59) were collected from Bareilly region of India. All the samples were screened by using standard microbiological methods for the detection of E. coli. The identified E. coli were then confirmed as DEC pathotypes using polymerase chain reaction–based assays. Those DEC pathotypes identified as Enteroaggregative E. coli (EAEC) were further confirmed using HEp-2 adherence assay. All the isolated DEC pathotypes were studied for their genetic diversity using pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing was performed by using disc diffusion method as per Clinical Laboratory Standards Institute guidelines. Results and discussion Of the four DEC pathotypes investigated, EAEC was found to be the predominant pathogen with an isolation rate of 16.5% from infants, 17.9% from young animals, 16.2% from foods and 3.4% from the associated environmental sources. These EAEC isolates, on further characterisation, revealed predominance of ‘atypical’ EAEC, with an isolation rate of 10.7% from infants, 15.1% from young animals, 16.2% from foods, and 3.4% from the associated environmental sources. On PFGE analysis, discrimination was evident within DEC pathotypes as 52 unique pulsotypes were observed for 59 recovered DEC pathotypes. However, a few EAEC isolates were found to be clonal (clusters A, B, C, D, F, G, and H) irrespective of their source of isolation, suggests sharing and/or circulation among different sources. Further, a high antibiotic resistance pattern was observed among isolated DEC pathotypes as almost 86.4% of isolates were found to be resistant against ≥3 tested drugs.

Seroprevalence and molecular detection of coxiellosis among cattle and their human contacts in an organized dairy farm

BACKGROUND The present investigation of Coxiella burnetii infection in cattle and farm workers on an organized cattle dairy farm, which appears to be the first of its kind in India, was undertaken to assess the status of this largely neglected and masked zoonosis. METHODS A total of 665 samples comprising of serum (n=224), milk (n=217) and vaginal swabs (n=224) collected from milch animals (n=224) with a history of reproductive disorders were screened. Besides these, ticks (n=114); animal feed (n=4) and environmental samples (n=13) as well as serum (n=19) of farm workers were also collected. The animal sera and milk samples as well as human sera were tested for antibodies against C. burnetii by commercial ELISA kit, whereas, all the collected samples were subjected to trans-PCR targeting the IS1111 gene of C. burnetii. RESULTS A high positivity for coxiellosis was detected in sera (29.91%) and milk (26.73%) samples of dairy cattle as well as sera from human contacts (84.21%) by ELISA. The trans-PCR detected the pathogen in 12.94% sera, 14.73% vaginal swabs and 5.53% milk samples of cattle, and in one soil sample, however, the sera of the farm workers and tick were tested negative. CONCLUSIONS The high positivity for coxiellosis among cattle and farm workers highlight the need to undertake extensive epidemiological studies to unravel the trends of C. burnetii infection in India.

Apparent prevalence and risk factors associated with occurrence of Coxiella burnetii infection in goats and humans in Chhattisgarh and Odisha, India

Coxiella burnetii is one of the most contagious pathogen associated with Q fever in humans, while, ruminants act as important source of infection for humans. In the present cross sectional study, a total of 464 samples were collected from 218 goats comprising of 218 sera, 218 blood and 28 milk from various parts of Chhattisgarh and Odisha region, India. Besides, environmental (33; soil- 4, faecal- 10, feed-6, drainage water- 6, drinking water- 7) and rodent (38) samples were also collected from the premises of the animals. Human sera samples (93) were collected from same sampling area comprised of workers at an organized dairy farm (43), and farmers (50). The samples were subjected to PCR targeting the trans and com1 genes and detection of antibodies using commercial ELISA kits. An overall 14.22% (95% CI: 10.2-19.47%) of the goat samples were positive using either PCR or ELISA. While, by using PCR and ELISA, 11.93% (26/218) and 9.63% (21/218) of the samples were positive for C. burnetii. A higher seropositivity (46.24%; 95% CI: 36.46-56.32%) was observed for antibodies against C. burnetii in samples collected from humans. None of the human, environmental and rodent samples were positive for C. burnetii using PCR. This seems to be the first cross sectional study to focus the hidden threat of coxiellosis among goat population and associated risk factors in India.

Seroscreening of lactating cattle for coxiellosis by TRANS-PCR and commercial ELISA in Kerala, India

The sero-epidemiology of Coxiella burnetii infection in domestic ruminants, which are considered as prime reservoirs, remains largely neglected and underreported in various states of India. The present study was aimed to assess coxiellosis among lactating cattle (n=134) based on their seroscreening at household level in Malappuram district of Kerala, India. On testing the cattle sera by trans-PCR targeting IS1111 gene of C. burnetii for the pathogen detection as well as by a commercial ELISA kit for the detection of antibodies against C. burnetii, the acute infection was noticed in 01 out of 134 animals (0.75%) that tested positive in trans-PCR assay while 06/134 (4.5%) animals indicated a persistent focalised infection based on their positivity in ELISA. Therefore, we conclude and recommend that the use of trans-PCR along with a serological assay like ELISA on serum samples is indispensable for early diagnosis of acute cases of coxiellosis in animals as well as to get a realistic assessment of the disease burden based on seroprevalence. This study seems to be the first epidemiological survey to highlight the hidden threat of coxiellosis among cattle population in Kerala, a southern state of India. However, other domestic and wild animals including cattle as well as their human contacts at high-risk need to be investigated through a large scale and multicentric epidemiological study for making a realistic assessment of the associated risk factors and to unmask the zoonotic potential of C. burnetii infection in the country. KEYWORDS

Pulsed-field gel electrophoresis of enterotoxic Clostridium perfringens type A isolates recovered from humans and animals in Kolkata, India

Abstract Clostridium perfringens is one of the most important globally recognised gastroenteric pathogen in humans as well as animals. The present study was aimed to know the similarities/divergence among C. perfringens type A isolates of human and animal origin using the pulsed-field gel electrophoresis (PFGE) as a molecular tool. The enterotoxic isolates obtained by screening of human diarrhoeal cases (n = 130), diarrhoeal cases of pig (n = 52) and goat (n = 50), meat samples viz., pork (n = 59) and chevon (n = 57) were characterized by standard cultural and biochemical methods followed by PCR Assays. Accordingly, a total of 11 C. perfringens type A characterized isolates (16S rRNA+, cpa+, cpb2+ and cpe+) recovered from human diarrhoeal cases (n = 3); diarrhoeal cases of pig (n = 2) and goat (n = 2); meat samples viz. pork (n = 2) and chevon (n = 2) were examined employing PFGE. The observed clustering pattern in PFGE analysis showed the relatedness between isolates from diarrhoeal goat and chevon (90–100%); diarrhoeal pig and pork (65–68%); moreover, isolates from human diarrhoeal cases were exhibiting lineage to cases from goat and pig diarrhoea as well pork and chevon by 62–68% relatedness. The outcome of the present study indicates the probable contamination of this pathogen to the human food chain through faeces from suspected food animals viz. goat and pig and their improperly cooked meat.

Isolation, Genotyping and Antibiogram Profile of Clostridium perfringens Isolates Recovered from Freshwater Fish and Fish Products from Kolkata Region

1Division of Veterinary Public Health, ICAR-Indian Veterinary Research Institute, Izatnagar-243122, India. 2Veterinary Public Health Laboratory, ICAR-Indian Veterinary Research Institute, Belgachia road, Kolkata 700 037, India. 3Department of Veterinary Public Health, College of Veterinary and Animal Sciences, Mannuthy, Kerala 680 651, India. 4Division of Livestock Products Technology, ICAR-Indian Veterinary Research Institute,Izatnagar-243122, India. 5Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar 243 122, India.