Methanolic extract of Hemidesmus indicus root augments the antibacterial and antibiofilm activity of amoxicillin and clindamycin against methicillin-resistant Staphylococcus aureus of bovine origin.

The present study evaluated the antibacterial and antibiofilm activity of methanolic extract of Hemidesmus indicus root (MHIR) in combination with amoxicillin and clindamycin against biofilm-forming methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk samples of mastitic cows. Microdilution susceptibility testing and microtitre plate assays were used to evaluate the in vitro efficacy of MHIR and antibiotic combinations against MRSA (n = 12). Furthermore, in vitro findings were validated in a murine model. Minimum inhibitory concentration and minimum biofilm inhibitory concentration of amoxicillin and clindamycin in combination with MHIR were significantly (P < 0·05) lower than when used alone against MRSA. In terms of antibacterial activity, MHIR showed additive interaction (fractional inhibitory concentrationindex >0·5-4) with amoxicillin and clindamycin against all the MRSA isolates, whereas MHIR synergizes (fractional biofilm inhibitory concentrationindex ≤0·5) the antibiofilm activity of amoxicillin and clindamycin against 58·33% and 83·33% of the MRSA isolates respectively. Amoxicillin/clindamycin in combination with MHIR significantly (P < 0·05) reduced disease activity score, and bacterial load and Gram-positive spots in kidney and liver of MRSA-infected mice. The combined efficacy of MHIR and amoxicillin/clindamycin was comparable to clindamycin alone but superior to amoxicillin alone. Hence, the combination of MHIR with amoxicillin/clindamycin is advocated in the treatment of MRSA-associated infections.

Occurrence of the genes encoding carbapenemases, ESBLs and class 1 integron-integrase among fermenting and non-fermenting bacteria from retail goat meat.

The present study was planned to detect the genes encoding carbapenemases, ESBLs and class 1 integron-integrase among bacteria obtained from retail goat meat. Fermenting and non-fermenting bacterial isolates (n = 57), recovered from 61 goat meat samples, were identified by 16S rRNA gene sequencing. Antimicrobial susceptibility of isolates was tested by the broth dilution method using ceftazidime, cefotaxime, meropenem and imipenem. Plasmids were isolated and tested for their physical characters. Plasmids were subjected to screening of carbapenemase, ESBL and intI1 gene. Conjugation assay was performed using blaNDM -positive isolates as the donor, and Escherichia coli HB101 as the recipient. Isolates showed the high rates of resistance to ceftazidime (77·2%), cefotaxime (70·2%), meropenem (22·8%) and imipenem (17·5%). They showed variability in number and size (~1 to >20 kb) of plasmids. Among all, 1, 4, 13 and 31 isolates showed the blaKPC , blaNDM , blaSHV and blaTEM genes, respectively. The blaKPC-2 gene was observed in one E. coli isolate. The blaNDM-1 gene was detected in Stenotrophomonas maltophilia (n = 2), Acinetobacter baumannii (n = 1) and Ochrobactrum anthropi (n = 1) isolates. These isolates co-harboured the blaTEM and blaSHV genes. The intI1 gene was detected in 22 (38·6%) isolates, and 16 of these isolates showed the carbapenemase and/or ESBL genes. The conjugative movement of blaNDM gene could not be proved after three repetitive mating experiments. The presence of genes encoding carbapenemases and ESBLs in bacteria from goat meat poses public health risks.

Carbapenemase and ESBL genes with class 1 integron among fermenting and nonfermenting bacteria isolated from water sources from India.

The present study was aimed to detect the carbapenemase, extended-spectrum ß-lactamase (ESBL), and intI1 gene of class 1 integron among fermenting (n = 61) and nonfermenting (n = 10) bacterial isolates recovered from water samples (n = 128). Isolates were identified by 16S rRNA sequencing. These isolates showed reduced-susceptibility to third-generation cephalosporins and carbapenems. The isolates varied in number and size of plasmids (2 kb to >20 kb). Plasmid DNA screening showed 5·6, 7, 11·2 and 26·7% prevalence of blaKPC , blaNDM , blaSHV and blaTEM genes respectively. Diverse blaNDM (blaNDM-1 and blaNDM-4 ) and blaSHV subtypes (blaSHV-2 and blaSHV-11 ) were recorded, unlike the single allelic blaKPC (blaKPC-2 ) and blaTEM (blaTEM-1 ) gene. Of the total 27 bla-gene-producing bacterial isolates, seven isolates co-harboured the carbapenemase genes (blaNDM or blaKPC or the both) along with the ESBL genes (blaSHV or blaTEM ). The intI1 gene of class 1 integron was detected among 12 (44·4%) of ESBL- and/or carbapenemase-harbouring isolates. Gene transferability was seen among four of the 10 Enterobacteriaceae donors. Carbapenemases and ESBLs with class 1 integron among aquatic environmental isolates raise the serious issue of the biosecurity and health of the ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: Anthropologically affected and polluted environment harbours the resistance threats, where a diverse bacterial species maintain, develop and exchange genetic determinants that constitute a risk to human and ecological health. The antimicrobial resistance (AMR) in Enterobacteriaceae and non-Enterobacteriaceae bacteria caused the failure of the therapy of last resort (carbapenems) and thus lead to life-threatening infections affecting public health. Surveillance and monitoring of AMR could be important for epidemiological, diagnostic testing and control of pathogens. This is a point-prevalence study reporting the comparative occurrence and co-occurrence of carbapenemase and extended-spectrum ß-lactamase genes among fermenting and nonfermenting bacteria isolated from the aquatic environment in India.

Isolation and characterization of bacteriophages with lytic activity against common bacterial pathogens.

AIM: Present investigation was conducted to isolate and characterize bacteriophages with lytic activity against common bacterial pathogens. MATERIALS AND METHODS: A total of 60 samples of animal waste disposal from cattle (42) and buffalo (18) farms were collected from three different strata, i.e., top, mid, and bottom of collection tank. Samples were primarily subjected to rapid detection methods, and then isolation of phage was done by double agar layer method using Bacillus subtilis (BsH) and Escherichia coli (EH) as host system. Phages were characterized on the basis of plaque morphology, temperature, pH susceptibility, and host range. RESULTS: Recovery of phages was higher from dairy cattle farm waste (78.57%) as compared to buffalo farm waste (72.22%) and bottom layer of tank showed maximum recovery. Bacillus subtilis (91%) supported the growth of more phages as compared to E. coli (9%). Three different phage morphotypes were observed each against Bacillus subtilis (BsHR1, BsHR2, and BsHR3) and E. coli (EHR1, EHR2, and EHR3). Mean phage titer of above six phage isolates ranged between 3×1010 and 5×1012 plaque forming units/ml. Viability of phages was by, and large unaffected at 70°C within 2-3 min, and phage isolates were completely inactivated below pH 3 and above 11. Coliphage EHR1 had widest host range followed by BsHR1 and BsHR2 while EHR2, EHR3, and BsHR3 had low lytic activity. CONCLUSION: It could be concluded from the present study that the Bacillus and Coli phage has wide host range and thus exhibits the potential to be used as drug substitute tool against common bacterial pathogens.

Characterization of Salmonella Gallinarum from an outbreak in Raigarh, Chhattisgarh.

AIM: The present investigation was conducted to isolate and characterize Salmonella Gallinarum from an outbreak of fowl typhoid in layer birds. MATERIALS AND METHODS: Clinically ill and dead layer birds from an outbreak were investigated. History, clinical signs, and postmortem lesions were suggestive of fowl typhoid. Postmortem samples including heart blood, intestinal contents, pieces of ovary, and liver were collected and processed immediately for bacterial culture, serotyping and antibiotic sensitivity tests. Isolates were further screened for the presence of extended spectrum beta lactamase (ESBL) (blaTEM) gene by polymerase chain reaction. RESULTS: On the basis of cultural, staining and biochemical characteristics; three bacterial isolates were confirmed as S. Gallinarum. On serotyping, somatic antigen O: 9 and 12 with nonflagellated antigen were detected in all three isolates. Isolates were intermediate sensitive to amoxycillin, amoxyclav, gentamicin and ciprofloxacin and resistant to most of the antibiotics including chloramphenicol, ampicillin, ceftazidime, cefexime, cefepime, azithromycin, nalidixin, tetracycline, oxytetracycline, and streptomycin. Two isolates were found to harbor ESBL (blaTEM) gene. CONCLUSION: Beta lactamase producer S. Gallinarum was confirmed as cause of increased mortality in layer birds during present investigation. Existence of multi drug resistant Salmonella poses serious threat to poultry industry in Chhattisgarh.

Comparative study on immunoglobulin Y transfer from breeding hens to egg yolk and progeny chicks in different breeds of poultry.

AIM: This study was undertaken to compare the immunoglobulin Y (IgY) level and its efficacy in laying hens of four different breeds of poultry (viz., Vanraja, Gramapriya, BlackRock, and KalingaBrown) and its relative transfer in egg yolk and chick. MATERIALS AND METHODS: This study was conducted in 48 apparently healthy laying hens vaccinated with Salmonella inactivated polyvalent vaccine, eggs and progeny chicks; 12 each from four different breeds of poultry, viz., Vanraja, Gramapriya, BlackRock, and KalingaBrown. The methodology included measurement of egg and yolk weight, total protein and IgY in egg yolk, total serum protein and IgY in breeding hens, and progeny chicks and extent of IgY transfer from hens to yolk then to chicks. Further, Salmonella-specific antibodies in breeding hens, egg yolk and progeny chicks were assessed using O and H antigen by tube agglutination test. RESULTS: The egg weight differed nonsignificantly (p>0.05) among breeds, however, breed wise significant variation (p<0.01) was reported in yolk weight. The weight of egg yolk significantly affects the total protein and IgY concentration although these levels per unit of volume did not differ. Total protein was significantly higher (p<0.01) in KalingaBrown and Gramapriya as compared to Vanraja and BlackRock. Non-significant (p>0.05) difference among breed was found in total protein of egg yolk and chick. The IgY concentration in hens, egg yolk and chick was found to be in the range of 5.35±0.63-5.83±0.65, 2.3±0.1-2.6±0.2, and 1.3±0.11-1.7±0.16 mg/ml, respectively which is uniform and independent of total protein concentration at all the three levels. Significant breed variations were not observed in maternal IgY transfer from breeding hens to chicks and were 25.62±1.42-36.06±4.34% of total IgY in parent flock. Moderate to higher rate of seroprevalence with peak titers of 1:640 against Salmonella-specific antibodies was observed in only 41.6% of breeding hens. CONCLUSION: No significant difference in the rate of transfer of IgY was observed in four breeds studied (viz., Vanraja, Gramapriya, BlackRock, and KalingaBrown) and moderate seropositivity was detected for Salmonella-specific antibodies in progeny chicks.

Pock forming ability of fowl pox virus isolated from layer chicken and its adaptation in chicken embryo fibroblast cell culture.

AIM: The objective of the present study was to examine pock forming ability of field strain and vaccine strain of fowl pox virus (FPV) in chorioallantoic membrane (CAM) of embryonated chicken eggs and its adaptation in chicken embryo fibroblast (CEF) cell culture. MATERIALS AND METHODS: Dry scabs were collected from 25 affected birds in glycerin-saline and preserved at 4°C until processed. Virus was isolated in 10-day-old embryonated chicken eggs by dropped CAM method. The identity of the virus is confirmed by clinical findings of affected birds, pock morphology and histopathology of infected CAM. In addition one field isolate and vaccine strain of FPV was adapted to CEF cell culture. CEF cell culture was prepared from 9-day-old embryonated chicken eggs. RESULT: Clinical symptoms observed in affected birds include pox lesion on comb, wattle, eyelids and legs, no internal lesions were observed. All field isolates produced similar findings in CAM. Pocks produced by field isolates ranged from 3 mm to 5 mm at the third passage while initial passages edematous thickening and necrosis of CAM was observed. Pocks formed by lyophilized strain were ranges from 0.5 mm to 2.5 mm in diameter scattered all over the membrane at the first passage. Intra-cytoplasmic inclusion bodies are found on histopathology of CAM. At third passage level, the CEF inoculated with FPV showed characteristic cytopathic effect (CPE) included aggregation of cells, syncytia and plaque formation. CONCLUSION: FPV field isolates and vaccine strain produced distinct pock lesions on CAMs. Infected CAM showed intracytoplasmic inclusion bodies. The CEF inoculated with FPV field isolate as well as a vaccine strain showed characteristic CPE at third passage level.