Extended-spectrum beta-lactamase (ESBL) producing and multidrug-resistant Escherichia coli in street foods: a public health concern.

Antimicrobial resistance (AMR) pattern and virulence genes of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli from foods of animal origin were evaluated. Based on combination disc method and ESBL E test, 42 of the 213 E. coli isolates were confirmed as ESBL producers where a high presence was observed in raw foods (60.62%), environmental samples (46.73%) and ready to eat foods (42.99%) of which 31(26.49%), 3(6.97%) and 7(15.21%) samples harbored ESBL E. coli, respectively. Higher contamination rates were observed in samples collected from meat vendors (54.36%), milk vendors (48.88%) and egg vendors (45.20%) of which 16.1%, 11.11% and 2.05%, respectively were ESBL E. coli. Among the 42 ESBL isolates, 85.71% (36/42) were multidrug-resistant. On polymerase chain reaction (PCR) analysis, expression of beta-lactamase genes viz., blaCTXM was noted in 69.04% (29/42) ESBL isolates, blaTEM in 66.66% (28/42) and blaOXA-1 in 19.04% (8/42) isolates, while blaSHV was not detected in any of the isolates. Other AMR genes viz., blaAmpC, sul1, sul2, tet(A), tet(B), catI, dhfrI, aac(3)-IIa(aacC2), aph(3')-Ia(aphA1), qnrB, qnrS were detected by PCR in 39, 28, 29, 3, 9, 5, 17, 11, 6, 6 and 33 isolates, respectively. None of the isolates harbored chloramphenicol (floR) and plasmid-mediated quinolone resistance (PMQR) (qnrA) genes. However, 21 isolates were positive for class I integron (int1), 5 for EPEC (eae) and 9 for ETEC (lt) while none were carrying bfp or stII genes. All ESBL producing isolates formed a single group when subjected to enterobacterial repetitive intergenic consensus (ERIC PCR) genotyping. The presence of multidrug-resistant ESBL E. coli in street foods of animal origin raises the issues of food safety and public health.

Evaluation of carboxyl beads based latex agglutination test for rapid sero-diagnosis of Japanese encephalitis.

Japanese encephalitis (JE) is a major public health problem in the South Asian countries including India. Pigs serve as a relevant sentinel model, the surveillance of which could predict a potential JE outbreak in human population nearby. However, existing serological detection methods like Enzyme-Linked Immuno Sorbent Assay (ELISA), virus neutralization test (VNT) and Haemagglutination Inhibition (HI) require elaborative laboratory facilities which are invariably not available in field conditions. Recognizing the lacunae, attempts were made to develop recombinant antigen (rNS1) based latex agglutination test (LAT) as a rapid on-site test using covalent coupling method. Four different formats were evaluated using different coupling buffers, blocking buffers and reaction conditions. The format in which borate buffer at alkaline pH (8.5) was used for coupling of antigen with carboxylated beads followed by blocking with skimmed milk powder was found to be the best amongst all. Developed latex based test was used for screening of 207 pig serum samples for JE which revealed relative diagnostic sensitivity and specificity of 80.2% and 95.2%, respectively in comparison with indirect IgG ELISA. Hence, the present study demonstrated that covalently coupled recombinant antigen based LAT could be used as a reliable screening test for surveillance of JE in pigs under field conditions.

Latex agglutination test for rapid on-site serodiagnosis of Japanese encephalitis in pigs using recombinant NS1 antigen.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) is a mosquitoe-borne viral zoonotic disease and globally around three billion people are at the risk of disease. The occurrence of JE cases has shown a rising trend during last decade in India. Pig is the amplifying host for JE virus and serves as a suitable sentinel model for the prediction of disease outbreak in humans. The development of a diagnostic test that is suitable for surveillance of JE in pigs is the need of the hour. The existing tests require elaborate laboratory facilities which make their application in rural settings difficult. Therefore, realizing the need for a rapid test, efforts were made to standardize a latex agglutination test (LAT) for serodiagnosis of JE in pigs. METHODS: Standardization of LAT by physical adsorption of recombinant NS1 (non-structural) protein of JE virus onto latex beads was done by altering six different variables, namely the antigen concentration, sensitization condition, surface blocking agent, blocking condition, particle concentration and reaction time. The standardized latex-protein complex was used for screening 246 pig serum samples under optimal conditions. RESULTS: The test was standardized with a diagnostic sensitivity and specificity of 82.24 and 87.83%, respectively. Screening of 246 field pig serum samples using standardized LAT showed a seropositivity of 50.4%. The results were available within 5 min after addition of test serum sample to the sensitized beads. INTERPRETATION & CONCLUSION: The findings of the study highlight the potential of LAT as a rapid on-site assay for JE diagnosis in pigs which would aid in predicting JE outbreaks in humans.

Virulent methicillin resistant Staphylococcus aureus (MRSA) in street vended foods.

Street foods are one of the important sources of foodborne infections and Staphylococcus aureus is an important infectious agent transmitted through various sources including street foods. The methicillin-resistant S. aureus (MRSA) are of public health significance, hence the study was taken to assess the street foods as a source of MRSA, for which 430 street vended foods of animal origin (meat, milk, eggs and their products) and associated environmental samples were processed for isolation and characterization. A total of 52 (12.1%) S. aureus were isolated and resistant was observed to oxacillin (36.5%), cefoxitin (25%) and penicillin G (82.7%) by disc diffusion test. On genotypic screening, mecA and blaZ have detected in 17.3% and 69.2% isolates, respectively. The virulence typing identified nuc, coa, clfA, spA, FnbA and enterotoxin A (sea) genes in 100%, 96.2%, 30.8%, 55.8, 50% and 7.7% isolates, respectively. Genetic diversity among the isolates was observed by enterobacterial repetitive intergenic consensus PCR with a D value of 0.77. The presence of virulent MRSA in street vended foods trigger the public health concern and emphasis to educate the consumers and street food vendors about quality and safety of such foods.

Lipoprotein particle concentration measured by nuclear magnetic resonance spectroscopy is associated with gestational age at delivery: a prospective cohort study.

OBJECTIVE: To estimate the association between lipoprotein particle concentrations in pregnancy and gestational age at delivery. DESIGN: Prospective cohort study. SETTING: The study was conducted in the USA at the University of North Carolina. POPULATION: We assessed 715 women enrolled in the Pregnancy, Infection, and Nutrition study from 2001 to 2005. METHODS: Fasting blood was collected at two time points (<20 and 24-29 weeks of gestation). Nuclear magnetic resonance (NMR) quantified lipoprotein particle concentrations [low-density lipoprotein (LDL), high-density lipoprotein (HDL), very-low density lipoprotein (VLDL)] and 10 subclasses of lipoproteins. Concentrations were assessed as continuous measures, with the exception of medium HDL which was classified as any or no detectable level, given its distribution. Cox proportional hazards models estimated hazard ratios (HR) for gestational age at delivery adjusting for covariates. MAIN OUTCOME MEASURES: Gestational age at delivery, preterm birth (<37 weeks of gestation), and spontaneous preterm birth. RESULTS: At <20 weeks of gestation, three lipoproteins were associated with later gestational ages at delivery [large LDLNMR (HR 0.78, 95% CI 0.64-0.96), total VLDLNMR (HR 0.77, 95% CI 0.61-0.98), and small VLDLNMR (HR 0.78, 95% CI 0.62-0.98], whereas large VLDLNMR (HR 1.19, 95% CI 1.01-1.41) was associated with a greater hazard of earlier delivery. At 24-28 weeks of gestation, average VLDLNMR (HR 1.25, 95% CI 1.03-1.51) and a detectable level of medium HDLNMR (HR 1.90, 95% CI 1.19-3.02) were associated with earlier gestational ages at delivery. CONCLUSION: In this sample of pregnant women, particle concentrations of VLDLNMR , LDLNMR , IDLNMR , and HDLNMR were each independently associated with gestational age at delivery for all deliveries or spontaneous deliveries <37 weeks of gestation. These findings may help formulate hypotheses for future studies of the complex relationship between maternal lipoproteins and preterm birth. TWEETABLE ABSTRACT: Nuclear magnetic resonance spectroscopy may identify lipoprotein particles associated with preterm delivery.

Hydraulic and treatment performance of pervious pavements under variable drying and wetting regimes.

Pervious pavements are an effective stormwater treatment technology. However, their performance under variable drying and wetting conditions have yet to be tested, particularly under a continuous time scale. This paper reports on the clogging behaviour and pollutant removal efficiency of three pervious pavement types over 26 accelerated years. These pavements were monolithic porous asphalt (PA), Permapave (PP) and modular Hydrapave (HP). Over a cycle of 13 days, the period of which was equivalent to the average annual Brisbane, Australia rainfall (1,200 mm), the pavements were randomly dosed with four different flows. Drying events of 3 h duration were simulated during each flow. Inflow and outflow samples were collected and analysed for Total Suspended Solids (TSS), Total Phosphorus (TP) and Total Nitrogen (TN). To evaluate the rate of clogging, a 1 in 5 year Brisbane storm event was simulated in the 6th, 8th, 12th, 16th, 20th and 24th week. Under normal dosing conditions, none of the pavements showed signs of clogging even after 15 years. However, under storm conditions, both PA and HP started to clog after 12 years, while PP showed no signs of clogging after 26 years. The drying and various flow events showed no effects in TSS removal, with all systems achieving a removal of approximately 100%. The average TP removal was 20% for all flows except for low flow, which had a significant amount of leaching over time. Leaching from TN was also observed during all flows except high flow. The TSS, TP and TN results observed during storm events were similar to that of high flow.

Evaluation of on-line preconcentration and flow-injection amperometry for phosphate determination in fresh and marine waters.

Dissolved reactive phosphorus (DRP) was determined as orthophosphate (PO(4)-P) in fresh and saline water samples by flow-injection (FI) amperometry, without and with in-valve column preconcentration. Detection is based on reduction of the product formed from the reaction of DRP with acidic molybdate at a glassy carbon working electrode (GCE) at 220 mV versus the Ag/AgCl reference electrode. A 0.1M potassium chloride solution was used as both supporting electrolyte and eluent in the preconcentration system. For the FI configuration without preconcentration, a detection limit of 3.4 microg Pl(-1) and sample throughput of 70 samples h(-1) were achieved. The relative standard deviations for 50 and 500 microg Pl(-1) orthophosphate standards were 5.2 and 5.9%, respectively. By incorporating an ion exchange preconcentration column, a detection limit of 0.18 microg Pl(-1) was obtained for a 2-min preconcentration time (R.S.D.s for 0.1 and 1 microg Pl(-1) standards were 22 and 1.0%, respectively). Potential interference from silicate, sulfide, organic phosphates and sodium chloride were investigated. Both the systems were applied to the analysis of certified reference materials and water samples.

Divalent ion effects and insights into the catalytic mechanism of protein tyrosine kinase Csk.

Csk (C-terminal Src kinase) is a protein tyrosine kinase which catalyzes the transfer of the gamma-phosphoryl group of ATP to the tyrosine hydroxyl of proteins in the presence of a divalent ion. Previous work with poly(Glu,Tyr) as the tyrosine-containing substrate and Mn as the divalent ion defined a ternary complex mechanism with ADP product release partially rate-determining [Cole, P. A., et al. (1994) J. Biol, Chem. 269, 30880-30887]. In this current study, ionic strength and divalent ion effects were probed. Increasing ionic strength led to a dramatic rise in the poly(Glu,Tyr) [4:l poly(glutamate:tyrosine)] K(m) and had little effect on the ATP K(m) or Kcat in Csk-mediated phosphoryl transfer. This finding allowed the dead-end peptide inhibitor EDNEFTA to be characterized as a linear competitive inhibitor of poly(Glu,Tyr) and a linear noncompetitive inhibitor of ATP. Taken together with previous data, the overall kinetic mechanism could now be assigned as random substrate binding, ternary complex. Compared to Mn, Mg was shown to sustain phosphoryl transfer with a 2.5-fold higher Kcat but K(m)'s for ATP and poly(Glu,Tyr) that were some 15-20-fold higher. An elevated ADP Ki and microviscosity effects were most suggestive of a kinetic mechanism with fast ADP release, and the chemical step fully rate-determining in the Mg-dependent reaction. Steady-state kinetic analyses of Csk reactions with Co and Ni in addition to Mg and Mn on wild-type and D314E Csk with ATP and ATP gamma S [adenosine 5'-O-(3-thiotriphosphate)] as substrates were performed. The Kcat thio effects [Kcat(ATP)/Kcat(ATP gamma S)] were inversely correlated with metal thiophilicity in both wild-type and D314E mutant Csk reactions, although the relationship was less pronounced in the latter. These results appear to underscore the role of gamma-phosphoryl hydrogen bonding/salt bridging in the wild-type Csk reaction transition state, which is somewhat perturbed in the D314E Csk reaction. In the case of the Ni reaction, the Kcat thio effect was reduced to about 2 in the wild-type and D314E mutant Csk reactions. Relevance with regard to the degree of nucleophilic attack in the transition state, i.e., associative vs dissociative character of phosphoryl transfer, is discussed.

NMR and stopped-flow studies of metal ion binding to alpha-lactalbumins.

1H-NMR spectroscopy and stopped-flow techniques have been used to investigate the binding of a host of metal ions to alpha-lactalbumins from bovine, goat, and human sources. We have identified two 1H-NMR markers diagnostic of metal ion binding to the high-affinity Ca2+-binding site of bovine alpha-lactalbumin, namely the signals corresponding to the delta-CH3 groups of Met-90, and a leucine, tentatively assigned to Leu-96. A number of metal ions other than Ca2+ bind to this site in either slow (La3+, Lu3+, Y3+, Sr2+, Sc3+) or fast (Cd2+, Ba2+, Pb2+) exchange. From competition experiments using this approach, we have determined an affinity series for metal ion binding at this site, in which lanthanides and Y3+ bind the strongest (Y3+>La3+, Lu3+>Ca2+>Sr2+>Cd2+, Pb2+, Ba2+>Sc3+). Several metal ions do not alter the 1H spectrum of bovine alpha-lactalbumin, retaining the protein in an 'apo-like' state. Evidence is given to support the notion that the paramagnetic divalent metal ions Co2+ and Cu2+ bind to a second distinct site, termed the 'zinc site', and that His-68 is involved in metal ion coordination. Finally, stopped-flow techniques using the indicator Xylenol orange were employed to obtain lanthanide off-rates for bovine, human, and goat alpha-lactalbumins (bovine and goat alpha-LA: k(off)(s-1) approximately 0.2 to 0.01 from La3+ to Lu3+; human alpha-LA: k(off)(s-1) approximately 0.02 to 0.001 from La3+ to Lu3+). In each case, we found that k(off) values decreased by an order of magnitude across the series, meaning that the dissociation constants for these metal ions are relatively constant. Data for the bovine and goat proteins are virtually identical, while the off-rates for human alpha-lactalbumin are appreciably slower. In addition, these rates are much slower than the Ca2+ off-rate for the bovine protein (k(off)(s-1) approximately 2 to 5), determined using the fluorescent indicator, BAPTA.

The role of the catalytic base in the protein tyrosine kinase Csk.

A potential distinguishing feature between protein tyrosine kinases and homologous serine/threonine kinases is the function of the catalytic base in these enzymes. In this study, we show that a peptide containing the unnatural amino acid trifluorotyrosine shows remarkably similar efficiency as a substrate of the tyrosine kinase Csk (C-terminal Src kinase) compared with the corresponding tyrosine-containing peptide despite a 4-unit change in the phenolic pKa. These results argue against the importance of early tyrosine deprotonation by a catalytic base in Csk. To further explore the role of the proposed catalytic base, the Csk mutant protein D314E was produced. This mutant displayed a significant reduction in kcat (approximately 10(4)) but relatively little effect on substrate Km values compared with wild-type Csk. Examination of the thio effect (kcat-ATP/kcat-adenosine 5'-O-(thiotriphosphate)) for D314E Csk led to the suggestion that a role of aspartate 314 may be to enhance the reactivity of the gamma-phosphate of ATP toward electrophilic attack. These results may have significant impact on protein tyrosine kinase inhibitor design.

Second-trimester abortion by dilatation and evacuation: an analysis of 11,747 cases.

The dilatation and evacuation procedure was explored in 1971 as an alternative method of second-trimester abortion. The results in 11,747 cases from 1972 through 1981 are presented. Although complications do occur--most notably hemorrhage, cervical laceration, fever, and perforation--the overall complication rate was lower than that reported for saline or prostaglandin in other large series. Further study and refinement of technique may help bring this shorter, safer, and more convenient procedure within the reach of larger numbers of women seeking second-trimester abortion.