Genomic Identity of Fluoroquinolone-Resistant blaCTX-M-15-Type ESBL and pMAmpC ß-Lactamase Producing Klebsiella pneumoniae from Buffalo Milk, India.

We investigated the occurrence of extended-spectrum ß-lactamase (ESBL) and AmpC-type ß-lactamase (ACBL) producing quinolone-resistant Klebsiella pneumoniae (KP) in milk samples of apparently healthy buffaloes (n = 348) and buffaloes (n = 19) with evidence of subclinical mastitis from seven districts of West Bengal, India. In total, 12 ESBL producing KP were isolated with blaCTX-M-15 gene and 7 of them were ACBL producers, as well. The blaCTX-M-15 genes were carried by transposable element ISEcp1. The plasmid-mediated quinolone resistance genes-qnrS, qnrA, qnrB, qepA, and aac(6')-Ib-cr were detected in five, one, three, four, and one isolate (s), respectively. In addition, eight isolates carried mutation in gyrase (gyrA) and six in topoisomerase IV (parC). Resistance markers/genes for sulfonamide (sul1), tetracycline [tet(A) and tet(B)], and aminoglycoside (aacC2) were also detected in eight, four, and one isolate(s), respectively. The class I integrons identified in five isolates carried aad2/aad5 and dfrA12/dfrA17 gene cassettes. The enterobacterial repetitive intergenic consensus-PCR revealed that all the isolates were genetically diverse and comprised a heterogeneous population. Isolation of multidrug-resistant KP, a typical nosocomial pathogen from buffalo milk, reiterates the need to monitor farm animals for ESBL producing Enterobacteriaceae and emphasizes on judicious use of antibiotics in animal husbandry sector.

First Report on Vancomycin-Resistant Staphylococcus aureus in Bovine and Caprine Milk.

The present investigation was carried out to study the vancomycin resistance pattern of Staphylococcus aureus isolates (n = 274) obtained from 352 milk samples of bovine (269) and caprine (63) clinical and subclinical mastitis from different districts of West Bengal, India. Of them, seven isolates (vancomycin-resistant S. aureus [VRSA] 1-7) exhibited resistance to vancomycin. Minimum inhibitory concentration of vancomycin (MICvan) for VRSA2 and VRSA3 was ≥16 µg/ml; thus categorized as VRSA. For rest of the isolates, MICvan was 8 µg/ml and they were grouped as vancomycin intermediate S. aureus (VISA). Even though all the isolates were resistant to cefoxitin and oxacillin and possessed mecA gene, none of them carried vancomycin resistance gene. Furthermore, all the seven isolates were subjected to Staphylococcal cassette chromosome mec (SCCmec) typing, Staphylococcal protein A (spa) typing, and enterobacterial repetitive intergenic consensus polymerase chain reaction. All the isolates except VRSA3 and VRSA4 from Kolkata district exhibited diverse genetic lineage, irrespective of their host and antibiotic resistance pattern. These two isolates showed clonal similarity (MRSA-SCCmec-V-spa t267) with methicillin-resistant S. aureus (MRSA) strains previously reported in human and animal infection. Isolation of VRSA and VISA could probably be due to intensive use of vancomycin in healthcare premises, which might have led to the development of glycopeptide-resistant strains and thereafter, further disseminated in the environment, including livestock farms. Detection of VRSA in milk is a serious concern as it may further cause health problems in the consumers. This is the first ever report of VRSA in food animals, even though the pathogen is otherwise prevalent in humans.

Co-infection of methicillin-resistant Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus and extended spectrum ß-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 ß-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.

Molecular and phylogenetic characterization of multidrug resistant extended spectrum beta-lactamase producing Escherichia coli isolated from poultry and cattle in Odisha, India.

The present study was undertaken to determine the occurrence and characterization of extended spectrum beta-lactamase (ESBL) producing Escherichia coli isolated from cattle and poultry in Odisha, India. Of 316 E. coli isolated from 305 samples (170 fecal samples from poultry and 135 milk samples from cattle), a total of 18 E. coli isolates were confirmed as ESBL producers by combination disc method and ESBL E-test. The isolates were resistant to oxyimino cephalosporins and monobactam as revealed by disc diffusion assay and determination of minimum inhibitory concentration. Resistance against other antibiotics was frequently noted as well. Further, beta-lactamase genes viz., blaSHV, blaCTXM, blaTEM and blaampC were detected in 17, 13, 9 and 2 isolates, respectively in PCR. Of the 18 ESBL strains, 16 were positive for class I integron (int1), nine of them carried sulphonamide resistance gene (sul1) and one harbored quinolone resistance gene (qnrB). Virulence markers for extraintestinal pathogenic E. coli like astA, tsh and iucD were also present in 4, 3 and 3 isolates, respectively. All the PCR amplified products were cloned and subjected to sequencing for homology analysis and data were submitted to gene bank. Sequence analysis of the amplified variable regions of class 1 integron of four representative isolates revealed the presence of aadA2 and dfrA12 gene cassettes conferring resistance to aminoglycosides and trimethoprim, respectively. Most of the ESBL producing strains emerged as single lineage through phylogenetic analysis by RAPD and ERIC PCR. This is the first ever systemic study on multidrug resistant ESBL producing E. coli in food producing animals from India.

Seasonal variations of EPG Levels in gastro-intestinal parasitic infection in a southeast asian controlled locale: a statistical analysis.

We present a data based statistical study on the effects of seasonal variations in the growth rates of the gastro-intestinal (GI) parasitic infection in livestock. The alluded growth rate is estimated through the variation in the number of eggs per gram (EPG) of faeces in animals. In accordance with earlier studies, our analysis too shows that rainfall is the dominant variable in determining EPG infection rates compared to other macro-parameters like temperature and humidity. Our statistical analysis clearly indicates an oscillatory dependence of EPG levels on rainfall fluctuations. Monsoon recorded the highest infection with a comparative increase of at least 2.5 times compared to the next most infected period (summer). A least square fit of the EPG versus rainfall data indicates an approach towards a super diffusive (i. e. root mean square displacement growing faster than the square root of the elapsed time as obtained for simple diffusion) infection growth pattern regime for low rainfall regimes (technically defined as zeroth level dependence) that gets remarkably augmented for large rainfall zones. Our analysis further indicates that for low fluctuations in temperature (true on the bulk data), EPG level saturates beyond a critical value of the rainfall, a threshold that is expected to indicate the onset of the nonlinear regime. The probability density functions (PDFs) of the EPG data show oscillatory behavior in the large rainfall regime (greater than 500 mm), the frequency of oscillation, once again, being determined by the ambient wetness (rainfall, and humidity). Data recorded over three pilot projects spanning three measures of rainfall and humidity bear testimony to the universality of this statistical argument.

Pleurotus florida lectin normalizes duration dependent hepatic oxidative stress responses caused by arsenic in rat.

Natural contamination of arsenic in ground water is a major health problem throughout the World. It is one of the most hazardous substances in the environment known to cause toxicity in multiple organs via oxidative stress. The molecular basis for arsenic toxicity involves direct or indirect damage to protein, lipid and DNA. Various studies have focused on the possible toxic effects of arsenic on membrane components and its correlation with oxidative damage. The present study was aimed to mitigation of arsenic induced hepatic oxidative stress by dietary modulation using of mushroom lectin in rats. Animals were divided into four groups; the first group was used as control. Groups 2, 3 and 4 were arsenic (20 ppm) exposed through drinking water, arsenic exposed plus oral ascorbic acid (25 mg/kg body weight) and arsenic exposed plus oral mushroom lectin (150 mg/kg body weight) respectively for a period of 12 weeks. We observed significant alterations in the antioxidant enzymes, oxidative stress intermediates and SOD(2) gene expression profile on arsenic exposure. These alterations were restored by co-administration of Pleurotus florida lectin which was as potent as standard antioxidant viz. ascorbic acid. The findings of the experiment suggested that P. florida lectin has capability of modulating arsenic mediated toxic effects and could be helpful in ameliorating them.

Mitigation of arsenic-mediated renal oxidative stress in rat by Pleurotus florida lectin.

Oyster mushroom, Pleurotus florida is regarded as one of the popular food with biopharmaceutical properties. Here, the study aimed to investigate the antioxidative effects of mushroom (Pleurotus florida) lectin against arsenic-induced nephrotoxicity in rats. Animals were divided into four groups; Group 1 was control. Groups 2, 3 and 4 were exposed to arsenic (20 parts per million [ppm] in drinking water), arsenic plus oral supplementation of ascorbic acid (25 mg/kg body weight) and arsenic plus oral supplementation of mushroom lectin (150 mg/kg body weight) respectively. Both ascorbic acid and mushroom lectin prevented the arsenic-mediated growth retardation and normalized the elevated kidney weight. Disrupted activities of superoxide dismutase (SOD) and catalase (CAT) and enhanced lipid peroxidation (LPO), protein carbonyl (PC) and nitric oxides (NO) production in kidney caused by arsenic could also be maintained towards normalcy by supplementation of mushroom lectin and ascorbic acid. These antioxidative effects were exhibited in a time-dependant manner. Further, arsenic-mediated down-regulation of messenger RNA (mRNA) expression of superoxide dismutase 2 (SOD(2)) gene was obstructed by these agents. Thus it was found that mushroom lectin reversed the effect of arsenic-mediated oxidative stress in a time-dependent manner.

Mushroom lectin protects arsenic induced apoptosis in hepatocytes of rodents.

Acute and chronic arsenic exposure result in toxicity both in human and animal beings and cause many hepatic and renal manifestations. The present study stated that mushroom lectin prevents arsenic-induced apoptosis. Apoptosis was measured by morphological alterations, cell proliferation index (CPI), phagocytic activity (nitro blue tetrazolium index; NBT), nitric oxide (NO) production, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, DNA fragmentation and caspase-3 activity. Arsenic exposure at 5 µM in the form of sodium arsenite resulted in significant elevation of deformed cells, NO production, TUNEL stained nuclei of hepatocytes, DNA fragmentation and caspase-3 activity. But the CPI and NBT index were significantly declined in arsenic-treated hepatocytes. The beneficial effect of mushroom lectin at 10 µg/mL, 20 µg/mL and 50 µg/mL) showed increased CPI and phagocytic activity. Mushroom lectin at those concentrations reduced deformed cells, NO production, DNA fragmentation and caspase-3 activity of hepatocytes. But significant better protection was observed in 50 µg/mL mushroom lectin-treated hepatocytes. This finding may be of therapeutic benefit in people suffering from chronic arsenic exposure.

Effect of chronic intake of arsenic-contaminated water on blood oxidative stress indices in cattle in an arsenic-affected zone.

This study aimed to determine the hemato-biochemical picture and blood oxidative stress in zebu cattle in an arsenic-contaminated zone. Significant decline in total erythrocyte count, packed cell volume, and total plasma protein was observed in cattle of that area in comparison to uncontaminated zone. There was significant elevation of plasma enzyme activities of both alanine aminotransaminase and aspertate aminotransaminase. Increased corpuscular osmotic fragility also proved to be a mechanism for deviation from normal functioning of erythrocytes. Cattle in the affected zone showed a significantly higher arsenic burden in blood. Those animals further showed decreased superoxide dismutase, catalase activities of erythrocytes, and plasma nitrite level, but increased lipid peroxidation and protein carbonyl level. Our finding concluded that cattle of the arsenic-contaminated zone is suffering from a subclinical form of arsenic toxicity, which is proved through altered hemato-biochemical indices and a certain extent of oxidative stress with higher arsenic concentration in blood.

Ground water arsenic contamination in West Bengal, India: a risk of sub-clinical toxicity in cattle as evident by correlation between arsenic exposure, excretion and deposition.

Arsenic contamination of ground water in West Bengal, India, is a great concern for both human and livestock populations. Our study investigated and correlated the arsenic concentration in the drinking water, urinary excretion and deposition of total arsenic in hair of cattle at an arsenic contaminated zone in West Bengal. The results of our study indicated that the average concentration of arsenic in tube well water in contaminated villages ranged from 0.042 to 0.251 ppm and a statistical significant (p < 0.01) difference was seen when compared to samples from a non-contaminated zone. The arsenic concentration in urine and hair of cattle ranged between 0.245-0.691 ppm and 0.461-0.984 ppm, respectively. A close relationship was found between the total arsenic in drinking water urinary excretion (r² = 0.03664, p < 0.05) and the arsenic concentration in hair (r² = 0.03668, p < 0.05). Our findings indicate that quantification of arsenic concentration in cattle urine and hair can serve as biomarkers for both present and past exposure in cattle population.

Supplementation of ascorbic acid prevents oxidative damages in arsenic-loaded hepatic tissue of rat: an ex vivo study.

Oxidative stress due to arsenic toxicity and ameliorative potentiality of L-ascorbic acid was evaluated in an ex vivo system of rat hepatic tissue. The study revealed that arsenic increased the activity of superoxide dismutase (SOD) and catalase (CAT) and the level of lipid peroxidation (LPO), protein carbonyl (PC) and nitric oxide (NO) at 1 hour, 1.5 hours and 2 hours of incubation. Co-treatment with L-ascorbic acid was found effective to normalize the activity of SOD and CAT and the production of LPO, PC and NO in hepatic tissue. This ex vivo study suggested that ascorbic acid is helpful to ameliorate arsenic-induced oxidative stress. This may be one of the alternative screening systems to study the efficacy of antioxidant and hepatoprotective agent.

Effect of ascorbic acid on blood oxidative stress in experimental chronic arsenicosis in rodents.

Ascorbic acid is a sugar acid and an essential vital food nutrient found mainly in fruits and vegetables. The purpose of this study was to investigate the effects of ascorbic acid against arsenic induced oxidative stress in blood of rat. In rat, treatment with ascorbic acid prevented the increased serum enzymatic activity of AST, ALT, ALP, ACP and LDH. In addition, treatment with ascorbic acid prevented elevated production of LPO, PC and NO and restored the depletion of reduced SOD and CAT activities. Interestingly, ascorbic acid markedly upregulated lymphocytes relative mRNA expression of lymphocytes SOD2 gene corresponding to GAPDH, house keeping candidate gene in arsenic-treated rat, which might provide anti-oxidative activity in the blood.

L-Ascorbate protects rat hepatocytes against sodium arsenite--induced cytotoxicity and oxidative damage.

Sodium arsenite-exposed hepatocytes of rat showed higher production of nitric oxide (NO) and increased lipid peroxidation (LPO) level vis-a-vis activity of superoxide dismutase (SOD) and catalase (CAT) were significantly lowered. Subsequently, the cell proliferation index (CPI) and cell viability were also reduced. Treatment with L-ascorbate was found effective in normalizing the arsenic-induced alteration of SOD and CAT activity and LPO level in rat hepatocytes. These observations indicated that L-ascorbate also has potent cytoprotective role as it could reduce the NO production and normalize the cell proliferation and viability of hepatocytes. Therefore, the in vitro study suggested that ascorbic acid is helpful to ameliorate the arsenic-induced cytotoxicity and oxidative stress of rat hepatocytes.