Prevalence and antibiotic resistance profile of Listeria spp. associated with seafoods from fish catchment areas in Kerala, India.

BACKGROUND AND AIM: Listeria monocytogenes is a ubiquitous, intracellular pathogen which has been implicated as a cause of several foodborne outbreaks. This study aimed to generate information on the prevalence and antibiotic resistance profile of Listeria species isolated from seafood. MATERIALS AND METHODS: A total of 400 samples of fresh fish, 100 samples of dry fish and 200 samples each of crustaceans and mollusks were collected from the fish catchment areas. All the samples were subjected to isolation and identification of Listeria spp. by two-step enrichment in UVM broth and plating on selective agar media (PALCAM) and then subjected to molecular characterization. L. monocytogenes isolates obtained during the study were subjected to serotyping by multiplex polymerase chain reaction. The isolates were also subjected to antibiotic sensitivity test. RESULTS: The prevalence of L. monocytogenes in seafoods in the present study was 0.55%. The isolates of L. monocytogenes were found to possess all virulence genes, namely, iap, hlyA, actA, prfA, plcA, and inlA. All the isolates belonged to serotype 4b. The occurrence of Listeria innocua was found to be more and was detected in 16.77% of seafood samples. Antibiotic sensitivity test revealed that all isolates were resistant to cefixime but were sensitive to almost all other commonly used antibiotics. CONCLUSION: The presence of Listeria spp. in raw seafood samples augments the need for implementation of good hygienic practices during the handling and processing of seafoods to safeguard the health of the consumers.

Occurrence of oxytetracycline residues in milk samples from Palakkad, Kerala, India.

BACKGROUND AND AIM: Food of animal origin such as milk is vital for nutritional security and should be free of any antibiotic residues due to its public health significance. We designed a study aiming to determine the occurrence of antibiotic residues and in further levels of oxytetracycline residues in pooled raw milk samples from Palakkad district, Kerala. MATERIALS AND METHODS: We collected pooled raw milk samples were collected from Alathur, Chittoor, and Palakkad blocks of Palakkad district, Kerala. A total of 215 samples were screened for antibiotic residues by microbial inhibition assay (MIA) and the positive samples were subjected to enzyme-linked immunosorbent assay (ELISA) to determine oxytetracycline residues, this was further confirmed using high-performance liquid chromatography (HPLC). RESULTS: We found that out of the 215 pooled raw milk samples screened for antibiotic residues using MIA, 22 samples (10.23%) were positive for antibiotic residues from Palakkad, Kerala. Out of these 22 samples, five (2.33%) were positive for oxytetracycline residues. We further calculated the mean concentration of oxytetracycline residues in these five samples and estimated it to be 201.00±41.25 ng/mL and 272.11±53.21 ng/mL using ELISA and HPLC, respectively. On analyzing these five samples, we found that four samples (1.86%) exceeded the maximum residue limits level of 100 ng/mL for oxytetracycline residues in milk as specified by Codex Alimentarius Commission/Food Safety and Standards Authority of India (FSSAI). CONCLUSION: This study revealed that the occurrence of oxytetracycline residues in pooled raw milk samples in the Palakkad district of Kerala. Hence, there is a need for surveillance and monitoring of antibiotic residues in milk due to its impact on public health to ensure consumer safety.

Exit-Site Infection: A Comparison of Classification Systems.

In general, in peritoneal dialysis (PD) practice in hospitals, Twardowski and Prowant's exit-site classification system is used, while the International Society for Peritoneal Dialysis (ISPD) exit-site scoring system is practical to use in community visits with less experienced healthcare personnel. Nevertheless, when exit-site scoring is 3 points under the ISPD exit-site score system and it falls in the category of equivocal under the Twardowski and Prowant's exit-site classification, the physician should be vigilant about the possibility of developing peritonitis, and hence, patients need to be kept under periodic monitoring.

An epidemiological investigation on occurrence of enterohemorrhagic Escherichia coli in raw milk.

AIM: The aim of the present investigation was to study the epidemiology of enterohemorrhagic Escherichia coli (EHEC) in raw milk and molecular characterization of isolates using multiplex polymerase chain reaction (PCR). MATERIALS AND METHODS: A total of 125 raw milk samples were subjected to isolation, identification, and confirmation of virulence-associated genes by multiplex PCR (mPCR). The samples were collected from a milk cooperative society of Thrissur district, Kerala. For further epidemiological investigation, samples such as dung (126), hair coat of cow (60), udder swab (60), udder wash (60), milking utensil wash (36), Milker's hand wash (36), water (36), soil (36), and feed (36) were collected from the households from which the raw milk tested positive for EHEC. RESULTS: The occurrence of EHEC in individual raw milk samples was found to be 8.8%. The major source of contamination to raw milk was found to be dung (19.84%) followed by udder swab (16.67%), hair coat of cow (15%), Milker's hand and milking utensils and water (11.11% each), and udder wash and soil (8.33% each). For identification of virulence genes, all the isolates were subjected to mPCR, of 75 isolates 73.33% of isolates harbored stx 2 gene while 53.33, 36, and 36% of isolates were encoded by stx 1, eae A, and hly A genes, respectively. On epidemiological survey, the multiple risk factors accountable for occurrence of EHEC in raw milk were found to be the quality of water used, improper and inadequate udder preparation, unhygienic hands of Milker's, use of insufficiently cleaned milking utensils, and using common utensil for washings of udder and milking purposes. CONCLUSION: The result of the present study signifies that raw milk was contaminated with EHEC and possesses a high public health threat. As dairy cattle and its environment serve as a potential niche for EHEC, hygienic milking practices should be adopted to curb the occurrence of EHEC in raw milk.

Occurrence and quantification of Shiga toxin-producing Escherichia coli from food matrices.

AIM: The objective of the study was to detect Shiga toxin-producing Escherichia coli (STEC) and develop a quantitative polymerase chain reaction (qPCR) assay to quantify the bacterial DNA present in different food matrices. MATERIALS AND METHODS: A total of 758 samples were collected during a period from January 2015 to December 2016 from Kozhikode, Thrissur, and Alappuzha districts of Kerala. The samples consisted of raw milk (135), pasteurized milk (100), beef (132), buffalo meat (130), chevon (104), beef kheema (115), and beef sausage (42). All the samples collected were subjected to isolation and identification of STEC by conventional culture technique. Confirmation of virulence genes was carried out using PCR. For the quantification of STEC in different food matrices, a qPCR was standardized against stx1 gene of STEC by the construction of standard curve using SYBR green chemistry. RESULTS: The overall occurrence of STEC in raw milk (n=135), beef (n=132), buffalo meat (n=130), chevon (n=104), and beef kheema (n=115) samples collected from Kozhikode, Thrissur, and Alappuzha districts of Kerala was 19.26%, 41.6%, 16.92%, 28.85%, and 41.74%, respectively. PCR revealed the presence of stx 1 and stx 2 genes in 88.46 and 83.64 and 30.77 and 40.00% of STEC isolates from raw milk and beef samples, respectively, while 100% of the STEC isolates from buffalo beef and beef kheema samples carried stx 1 gene. Real-time qPCR assay was used to quantify the bacterial cells present in different food matrices. The standard curve was developed, and the slopes, intercept, and R2 of linear regression curves were -3.10, 34.24, and 0.99, respectively. CONCLUSION: The considerably high occurrence of STEC in the study confirms the importance of foods of animal origin as a vehicle of infection to humans. In the present study, on comparing the overall occurrence of STEC, the highest percentage of occurrence was reported in beef kheema samples. The study shows the need for rigid food safety measures to combat the potential pathogenic effects of harmful bacteria throughout the production chain from production to consumption.

Prevalence of Listeria monocytogenes, Yersinia enterocolitica, Staphylococcusaureus, and Salmonella enterica Typhimurium in meat and meat products using multiplex polymerase chain reaction.

AIM: The objective of the study was to investigate the occurrence of Listeria monocytogenes, Yersinia enterocolitica, Staphylococcusaureus, and Salmonella enterica Typhimurium in meat and meat products using the multiplex polymerase chain reaction (PCR) method. MATERIALS AND METHODS: The assay combined an enrichment step in tryptic soy broth with yeast extract formulated for the simultaneous growth of target pathogens, DNA isolation and multiplex PCR. A total of 1134 samples including beef (n=349), chicken (n=325), pork (n=310), chevon (n=50), and meat products (n=100) were collected from different parts of Kerala, India. All the samples were subjected to multiplex PCR analysis and culture-based detection for the four pathogens in parallel. RESULTS: Overall occurrence of L. monocytogenes was 0.08 % by cultural method. However, no L. monocytogenes was obtained by multiplex PCR method. Yersinia enterocolitica was obtained from beef and pork samples. A high prevalence of S. aureus (46.7%) was found in all types of meat samples tested. None of the samples was positive for S. Typhimurium. CONCLUSION: Multiplex PCR assay used in this study can detect more than one pathogen simultaneously by amplifying more than one target gene in a single reaction, which can save time and labor cost.

Macroinvertebrate diversity of Veli and Kadinamkulam lakes, South Kerala, India.

The diversity and distribution pattern of benthic macroinvertebrates in two backwaters viz., Veli and Kadinamkulam of Kerala were assessed using diversity indices. The samples were collected once in three months for a period of two years from six sampling sites (K1, K2, K3, V1, V2 and V3) and community variations were analyzed. Overall, 24 families were identified represented by mollusca, annelida and arthropoda (crustaceans and insects). Among this, dominant taxon was Mytilidae of molluscan family and site-wise dominance was maximum in sites V1 and V2. Richness and abundance were highest in site V2 and lowest in site K2. Diversity index ranged from 0.27 (K2) to 2.33 (V1). The diversity and distribution patterns of certain species were clearly related to water quality as evident from the present study.

Antibacterial & antiplasmid activities of Helicteres isora L.

BACKGROUND & OBJECTIVES: The multiple drug resistance (MDR) is a serious health problem and major challenge to the global drug discovery programmes. Most of the genetic determinants that confer resistance to antibiotics are located on R-plasmids in bacteria. The present investigation was undertaken to investigate the ability of organic extract of the fruits of Helicteres isora to cure R-plasmids from certain clinical isolates. METHODS: Active fractions demonstrating antibacterial and antiplasmid activities were isolated from the acetone extracts of shade dried fruits of H. isora by bioassay guided fractionation. Minimal inhibitory concentration (MIC) of antibiotics and organic extracts was determined by agar dilution method. Plasmid curing activity of organic fractions was determined by evaluating the ability of bacterial colonies (pre treated with organic fraction for 18 h) to grow in the presence of antibiotics. The physical loss of plasmid DNA in the cured derivatives was further confirmed by agarose gel electrophoresis. RESULTS: The active fraction did not inhibit the growth of either the clinical isolates or the strains harbouring reference plasmids even at a concentration of 400 microg/ml. However, the same fraction could cure plasmids from Enterococcus faecalis, Escherichia coli, Bacillus cereus and E. coli (RP4) at curing efficiencies of 14, 26, 22 and 2 per cent respectively. The active fraction mediated plasmid curing resulted in the subsequent loss of antibiotic resistance encoded in the plasmids as revealed by antibiotic resistance profile of cured strains. The physical loss of plasmid was also confirmed by agarose gel electrophoresis. INTERPRETATION & CONCLUSIONS: The active fraction of acetone extract of H. isora fruits cured R-plasmids from Gram-positive and Gram-negative clinical isolates as well as reference strains. Such plasmid loss reversed the multiple antibiotic resistance in cured derivatives making them sensitive to low concentrations of antibiotics. Acetone fractions of H. isora may be a source to develop antiplasmid agents of natural origin to contain the development and spread of plasmid borne multiple antibiotic resistance.

Antiplasmid activity of 1'-acetoxychavicol acetate from Alpinia galanga against multi-drug resistant bacteria.

ETHNOPHARMACOLOGICAL RELEVANCE: Alpinia galanga (L.) Swartz is traditionally used in the treatment of various ailments across India, China, and Southeast Asian countries. In India it is a reputed drug in indigenous system of medicine and largely used as antibacterial and antiseptic. In southern India the rhizomes has been used as a domestic remedy against bacterial infections. AIM OF THE STUDY: To identify a potential antiplasmid compound from Alpinia galanga against multi-drug resistant bacteria. MATERIALS AND METHODS: The crude rhizome extract of Alpinia galanga was prepared in acetone. Antibacterial activity was checked by MIC and antiplasmid activity was checked by SIC. The principal compound responsible for the antiplasmid activity, in the crude extract, was identified by bioassay guided fractionation using hexane-acetone. Antibiotic resistance profile of plasmid harboring strains and plasmid cured strains was determined by disc diffusion method. RESULTS: The crude acetone extract of the rhizomes of Alpinia galanga exhibited antiplasmid activity against Salmonella typhi, Escherichia coli and vancomycin resistant Enterococcus faecalis with an efficiency of 92%, 82% and 8% respectively at 400 microg/ml SIC. The principal compound responsible for the activity was identified as 1'-acetoxychavicol acetate. 1'-Acetoxychavicol acetate demonstrated the ability to cure plasmid encoded antibiotic resistance in various multi-drug resistant bacterial strains of clinical isolates such as Enterococcus faecalis, Salmonella typhi, Pseudomonas aeruginosa, Escherichia coli and Bacillus cereus with curing efficiency of 66%, 75%, 70%, 32% and 6% respectively at SIC of 400-800 microg/ml. CONCLUSION: 1'-Acetoxychavicol acetate mediated R-plasmid curing significantly reduced the minimal inhibitory concentration of antibiotics required to inhibit growth of bacteria, thus making the antibiotic treatment more effective.

Sanitizing effect of salts on experimentally inoculated organisms on pork carcasses.

Pork forequarters procured from freshly slaughtered animals were decontaminated with hot water and inoculated with Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Listeria monocytogenes, Serratia marcescens, Pseudomonas aeruginosa and Proteusvulgaris. The forequarters were individually spray washed with 5% potassium sorbate and a combination of 5% sodium chloride and 2.5% each of sodium acetate, sodium citrate, sodium lactate and potassium sorbate solutions. The total viable count (TVC) of the treated meat samples was reduced by 0.96 and 1.31 log units by spraying with salt and salt combination respectively with marginal changes in colour and odour scores. Inoculated organisms were found to be highly sensitive to salt combination treatment as compared to potassium sorbate alone. Shelf-life of salt and salt combination treated samples was increased to 8 and 11days as against 4days in untreated samples. Carcass washing with salt and salt combination was found to be suitable for extension of shelf-life and improvement in the sensory and microbiological quality of meat.

A potential plasmid-curing agent, 8-epidiosbulbin E acetate, from Dioscorea bulbifera L. against multidrug-resistant bacteria.

Bioassay-guided fractionation of an aqueous methanolic extract of Dioscorea bulbifera L. bulbs was performed using organic solvents. A novel plasmid-curing compound was identified as 8-epidiosbulbin E acetate (EEA) (norditerpene) on the basis of modern spectroscopic analysis and X-ray crystallography. EEA exhibited broad-spectrum plasmid-curing activity against multidrug-resistant (MDR) bacteria, including vancomycin-resistant enterococci. EEA cured antibiotic resistance plasmids (R-plasmids) from clinical isolates of Enterococcus faecalis, Escherichia coli, Shigella sonnei and Pseudomonas aeruginosa with 12-48% curing efficiency. The reference plasmids of Bacillus subtilis (pUB110), E. coli (RP4), P. aeruginosa (RIP64) and Salmonella typhi (R136) were cured with efficiency ranging from 16% to 64%. EEA-mediated R-plasmid curing decreased the minimal inhibitory concentration of antibiotics against MDR bacteria, thus making antibiotic treatment more effective. The antibiotic resistance pattern revealed that the compound was effective in the reversal of bacterial resistance to various antibiotics. In addition, the compound did not show any cytotoxicity against a broad range of human cancer cell lines, namely MCF-7 (breast cancer), SiHa (cervical cancer) and A431 (epidermal carcinoma), and hence has the potential to be used as a lead compound for drug discovery programmes.

Microwave-assisted extraction of embelin from Embelia ribes.

A rapid and efficient microwave-assisted extraction (MAE) process for the selective extraction of embelin from Embelia ribes was developed. Solvent selection, microwave energy input and solid loading were optimized. The rate of extraction and purity of embelin depended upon the solvent used and exposure time to microwaves. Maximum MAE was achieved in acetone with total yield of 92% (w/w) embelin with 90% (w/w) purity with 1% (w/v) raw material loading at 150 W power level in 80 s. Non-polar solvents, such as hexane and dichloromethane, were not effective for the selective extraction of embelin.

Effect of food grade organic acids on inoculated S. aureus, L. monocytogenes, E. coli and S. Typhimurium in sheep/goat meat stored at refrigeration temperature.

Sheep/goat forequarters procured from freshly slaughtered animals were decontaminated with hot water and inoculated with Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella typhimurium. The forequarters were individually spray washed with 2% lactic acid and 1.5% acetic + 1.5% propionic acid combination. Total viable count (TVC) of the treated meat samples was reduced by about 0.52 and 1.16 log units with marginal changes in colour and odour scores. Inoculated organisms were found to be highly sensitive to acid combination treatment as compared to lactic acid alone. Shelf-life of acid and acid combination treated samples was increased to 8 and 11 days as against 3 days in untreated samples. Carcass washing with acid alone or acid combination was found to be suitable for extension of shelf-life and improvement in the sensory and microbiological quality of meat.

Chemical sprays as a method for improvement in microbiological quality and shelf-life of fresh sheep and goat meats during refrigeration storage (5-7°C).

The shelf-life of meats from freshly slaughtered sheep and goat carcasses at 5-7 °C was extended after spraying (pressure 3 kg/cm(2)) the carcasses with solution 'B' containing potassium sorbate, sodium acetate, sodium citrate, sodium lactate each at 2.5% and sodium chloride at 5% (prepared w/v in potable water), when compared with solution 'A' (without potassium sorbate). Solution B treatment inhibited Bacillus spp. to minimum and were not detected up to sixth day. It extended the lag phase of all organisms including psychrotrophes (pseudomonads) and reduction of differential counts in sheep and goat meat were noted throughout the refrigerated storage. On sixth day (control) and seventh day (solution "A treated") meat samples developed off odour and discoloration with total viable count (TVC)>10(7) cfu/g. Solution B treated meat samples showed no spoilage at seventh or eighth day, indicating an extended shelf-life of 3 and 2 days when compared with control and solution A treated meat, respectively.

Curcuma Raktakanda is a Potential Larvicide for Mosquito Control.

The leaves and tuber of Curcuma raktakanda were investigated as a mosquito larvicide against the early fourth instar larvae of four mosquito species, viz., Culex quinquefasciatus , Culex sitiens , Aedes aegypti and Anopheles stephensi . The petroleum ether extract of the leaves and tuber exhibited toxicity towards all the test species. The LC 90 values of leaf extract for Cx. quinquefasciatus , Cx. sitiens , Ae. aegypti and An. stephensi were 46.77, 27.45, 58.75 and 45.81 mg/l,respectively. The LC 90 values of crude extract of tuber were 44.88, 29.11, 48.08 and 37.49 mg/l for Cx. quinquefasciatus , Cx. sitiens , Ae. aegypti and An. stephensi , respectively. The crude extract of the tuber was fractionated using column chromatography and the activity of the fractions was studied against the test species. The LC 90 values of the biologically active fraction for Cx. quinquefasciatus , Cx. sitiens , Ae. aegypti and An. stephensi were 18.50, 12.82, 19.95 and 18.19 mg/l, respectively. The extracts were active after storage for one year.

Unnecessary injections given to children under five years.

Adults accompanying 64 children attending a hospital out-patient clinic were questioned about treatment and injections given for illnesses in the previous month. Half the children had received injections, almost all given by private doctors: we consider most of these injections to have been unnecessary. Three girls were paralysed by aggravation poliomyelitis after unnecessary injections. Adults approved of injections although they did not know what was injected.