Prevalence and molecular characterization of extended-spectrum ß-lactamase (ESBL) producing Escherichia coli isolated from dogs suffering from diarrhea in and around Kolkata.

Background: Dogs are the favorite companion animals among humans. The close interaction between dogs and people increases the risk of antibiotic resistance spreading. Surveillance for antimicrobial resistance and the identification of ESBL-producing Escherichia coli as an indicator bacterium is an important tool for managing antimicrobial drug therapy. Aims: The present study targeted to identify and characterize ESBL-producing E. coli among dogs suffering from diarrhea in and around Kolkata. Methods: Isolation and identification of E. coli from dogs suffering from diarrhea (n=70) along with screening for the production of both ESBL and AmpC. The isolates were further characterized through antimicrobial resistance profiling, resistance genes (bla CTX-M, bla TEM, and bla SHV) screening, and phylogenetic group study. Results: Among the 70 isolates, 21 (30%) were confirmed ESBL producers. An antibiogram typing of ESBL-producing E. coli revealed that the majority of them were resistant to norfloxacin (85.7%) followed by tetracycline (61.90%), doxycycline (57.14%), piperacillin/tazobactam (52.38%), cotrimoxazole (47.62%), gentamicin (42.62%), amikacin (23.81%), and chloramphenicol (19.05%). Major resistance genes included bla CTX-M (100%), bla TEM (28.57%), and bla SHV (9.50%). The predominant phylogenetic groups were phylogroup A (76%) followed by phylogroup D (24%). Conclusion: The current investigation reported a high prevalence of both ESBL and AmpC ß-lactamase (AmpC) producing E. coli, co-resistance to a distinct group of antibiotics, and co-existence of different ESBL genes in dogs. Our findings highlight the importance of diagnostic antimicrobial susceptibility testing for proper antimicrobial therapy and to prevent antimicrobial resistance from spreading to humans from dogs in Kolkata and the surrounding area.

Effect of vitamin E supplementation on mRNA expression of superoxide dismutase and interleukin-2 in arsenic exposed goat leukocytes.

The aim of this study was to quantify the expression level of genes involved in antioxidant defenses during inorganic arsenic (iAs) exposure in the blood of goats and to evaluate the regulative activity on these genes of antioxidant vitamin E in the diet. Twenty-four crossbred lactating goats (Alpine × Beetal) were distributed randomly into four equal groups (Control, T(1), T(2) and T(3)) of six in each, on the basis of average body weight (36.10 ± 0.11 kg) and milk yield (1.61 ± 0.004 kg/day). The animals in T(1), T(2) and T(3) were given 50 mg/kg dry matter arsenic daily, while in T(2) and T(3), vitamin E @100 IU and 150 IU/kg dry matter, respectively, was also supplemented additionally for the period of 12 months. Blood was sampled at 0 day then at 3 months interval and analyzed for the expression level of superoxide dismutase (Cu/Zn SOD) and interleukin-2 (IL-2) using real-time PCR technique. Initially there was no difference (p > 0.05) in relative expression of the two genes. But, at 3 months, relative expression of Cu/Zn SOD increased (p < 0.05) in T(1) groups then, at 6 and 9 months expression was decreased (p < 0.05) in all the iAs treated groups whereas at 12 months, vitamin E supplementation increased (p < 0.05) the expression which is comparable to control groups. IL-2 mRNA expression was decreased (p < 0.05) at 6 months in all iAs treated groups, at 9 months there was decline trend but not significantly different whereas at 12 months decline trend was less (p < 0.05) in vitamin E supplemented groups. The result suggests that vitamin E may have a controlling effect on oxidative stress through modulation of SOD and IL-2 expression.

Analysis of breeding and pathology helps refine management practices of a large-scale N'-ethyl-N'-nitrosourea mouse mutagenesis programme.

N'-ethyl-N'-nitrosourea (ENU) is a powerful germline mutagen used in conjunction with phenotype-driven screens to generate novel mouse mutants. ENU also induces genetic lesions in somatic cells and dosage requires optimization between maximum germline mutation rate versus induced sterility and tumourigenesis that compromise the welfare and fecundity of the ENU-treated males. Here, we present our experience with BALB/cAnNCrl and C57BL/6J mice in terms of the pathology induced by ENU and its impact on breeding. In both mouse strains, morbidity and mortality rises with ENU dose. In more than 75% of C57BL/6J males, morbidity and mortality were attributable to the development of malignant T-lymphoblastic lymphoma. Approximately 50% of ENU-treated BALB/cAnNCrl males develop early malignant T-lymphoblastic lymphoma, but the cohort that survives develops late-onset lung carcinoma. Within strains, the latency of these clinically important tumour(s) was not dosage-dependent, but the proportion of mice developing tumours and consequently removed from the breeding programme increased with ENU dosage. The median number of offspring per ENU-treated C57BL/6J male in standard matings with C3H/HeH females decreased with increasing dosage. The two most important underlying causes for lower male fecundity were increased infertility in the highest dosage group and reduced numbers of litters born to the remaining fertile C57BL/6J males due to a higher incidence of morbidity. These findings have allowed us to refine breeding strategy. To maximize the number of offspring from each ENU-treated male, we now rotate productive males between two cages to expose them to more females. This optimizes the number of mutation carrying offspring while reducing the number of ENU-treated males that must be generated.

Ambiguous abbreviations: an audit of abbreviations in paediatric note keeping.

OBJECTIVE: To assess the frequency, nature and understanding of abbreviations in medical records. DESIGN: Audit of abbreviation use and meaning in paediatric handover sheets and medical notes compared to two standards, the Trust Intranet Medical Dictionary (TID) and Mosby's Medical Dictionary (MMD). A selection of abbreviations was shown to healthcare professionals to examine interpretation of abbreviations. SETTING: Large inner-city district general hospital, Birmingham, UK. MAIN OUTCOME MEASURES: Frequency, nature and understanding of abbreviations in paediatric medical records. RESULTS: On 25 handover sheets a total of 2286 abbreviations were used, with 221 different abbreviations; the standards recognised 14% (TID) and 20% (MMD) of these abbreviations. In 168 sets of medical notes a total of 3668 abbreviations were used, with 479 different abbreviations; the standards recognised 15% (TID) and 17% (MMD). Some words were shortened in different forms, for example, normal (N, Nl, NAD) and some abbreviations had multiple interpretations that differed from those intended, for example, TOF (tetralogy of Fallot, tracheo-oesophageal fistula). When presented with a selection of abbreviations, paediatric doctors recognized 56-94% and other healthcare professionals recognised 31-63%. CONCLUSION: Abbreviation use was widespread in paediatric note keeping. There was no systematic approach to this and difficulties in interpretation were demonstrated. The use of standardised abbreviations to avoid confusion is suggested.

Naturally acquired antibodies to polymorphic and conserved epitopes of Plasmodium falciparum merozoite surface protein 3.

Many studies on the role of merozoite surface protein 3 (MSP3) in immunity against malaria have focused on a conserved section of MSP3. New evidence suggests that polymorphic sequences within MSP3 are under immune selection. We report a detailed analysis of naturally-acquired antibodies to allele-specific and conserved parts of MSP3 in a Kenyan cohort. Indirect and competition ELISA to heterologous recombinant MSP3 proteins were used for antibody assays, and parasites were genotyped for msp3 alleles. Antibody reactivity to allele-specific and conserved epitopes of MSP3 was heterogeneous between individuals. Overall, the prevalence of allele-specific antibody reactivity was significantly higher (3D7-specific 54%, K1-specific 41%) than that to a recombinant protein representing a conserved portion of C-terminal MSP3 (24%, P < 0.01). The most abundant IgG subclass was IgG3, followed by IgG1. Allele-specific reactivity to the K1-type of MSP3 was associated with a lower risk of clinical malaria episodes during a 6-month follow-up in individuals who were parasitized at the start of the malaria transmission season (Relative risk 0.41 with 95% confidence interval 0.20-0.81, P = 0.011). The potential importance of allele-specific immunity to MSP3 should be considered in addition to immunity to conserved epitopes, in the development of an MSP3 malaria vaccine.

Linkage of human cytomegalovirus glycoprotein gO variant groups identified from worldwide clinical isolates with gN genotypes, implications for disease associations and evidence for N-terminal sites of positive selection.

Previously, we identified the glycoprotein gO gene, UL74, as a hypervariable locus in the human cytomegalovirus (HCMV) genome [Virology 293 (2002) 281]. Here, we analyze gO from 50 isolates from congenitally infected newborns, transplant recipients, and HIV/AIDS patients from Italy, Australia, and UK. These are compared to four gO groups described from USA transplantation patients [J. Virol. 76 (2002) 10841]. Phylogenetic analyses identified seven genotypes. Divergence between genotypes was up to 55% and within 3%. Discrete linkage was shown between seven hypervariable gO and gN genotypes, but not with gB. This suggests interactions, while gN and gO are known to form complexes with distinct conserved glycoproteins gM, gH/gL, respectively, both are involved in fusogenic entry and exit. Codon-based maximum likelihood models showed evidence for sites of positive selection. Further analyses of disease relationships should take into account these newly defined gO/gN groups.

Differential expression of cold- and diet-specific genes encoding two carp liver delta 9-acyl-CoA desaturase isoforms.

Carp respond to cold by the upregulated expression of Delta9-acyl-CoA desaturase. Here we report the cloning and characterization of Cds2, a second Delta9-acyl CoA-desaturase expressed in carp liver. Both Cds1 and Cds2 complemented the ole1 mutation in Saccharomyces cerevisiae, permitting the synthesis of delta9-monounsaturates, confirming their identity as delta9-desaturases. We demonstrate that under a standard feeding regime it is the Cds2, and not Cds1, transcript that is transiently upregulated during the first few days of cooling from 30 degrees C to 10 degrees C, the period when cold-induced membrane restructuring occurs. Cds2 exists as two differentially spliced transcripts, differing by a small segment from the 3'-untranslated region, the ratio of which varies with temperature. Feeding a diet enriched in saturated fats produced a fourfold increase in Cds1 transcript levels, which was blocked by cooling to 15 degrees C. Cds2 transcript levels, however, showed no substantial response to the saturated diet. Thus carp liver uniquely expresses two isoforms of delta9-acyl CoA desaturase, possibly formed by a recent duplication event, that are differentially regulated by cooling and dietary treatment.

The role of desaturases in cold-induced lipid restructuring.

All organisms respond to environmental challenge by adaptive responses, although, in many cases, the underlying molecular mechanisms are not understood. In the case of membranes, the physical structure of membrane phospholipids is conserved in the face of cold, rigidifying conditions by the elevated proportions of unsaturated fatty acids. We have observed a clear positional specificity in this substitution and head group preferences in carp liver membranes. We have also demonstrated changes in the activity of lipid desaturases that mediate the unsaturation response, caused by both transcriptional and post-translational mechanisms. Another hepatic isoform has recently been discovered with sensitivity, not to cooling, but to dietary variations. Finally, we are testing the importance of desaturase inductions in the inducible cold tolerance of the whole animal.

Advantages of glutamate dehydrogenase as a blood biomarker of acute hepatic injury in rats.

In a recent study in rats, alanine aminotransferase (ALT), the preferred plasma biomarker of hepatocellular injury in rats, was ineffective at detecting marked hepatic necrosis produced by acetaminophen (Human and Experimental Toxicology 19, 277-83, 2000). In contrast, glutamate dehydrogenase (GLDH) was markedly elevated. Accordingly, these enzymes were comprehensively evaluated as plasma biomarkers of hepatocellular injury in rats using several other models of hepatic injury, including partial hepatectomy and exposure to methapyrilene, dexamethasone, cyproterone, isoniazid, lead nitrate, and Wyeth-14643. Other enzymes also evaluated were aspartate aminotransferase (AST), sorbitol dehydrogenase (SDH), and the hepatobiliary marker alkaline phosphatase (ALP). Compared to plasma ALT increases, plasma GLDH increases were up to 10-fold greater, up to 3-fold more persistent, and occurred at times following hepatocellular injury when plasma ALT was not increased. Plasma GLDH activity was not inhibited by the test compounds, whereas ALT was substantially inhibited by both isoniazid and lead nitrate. While plasma GLDH activity was unaffected by induction, ALT was induced by cyproterone and dexamethasone, and ALP was induced by Wyeth-14643 and partial hepatectomy. GLDH was concluded to be a more effective biomarker of acute hepatic injury than ALT, AST, SDH or ALP in the rat, based primarily on the large increase following hepatocellular injury, prolonged persistence in the blood following injury, high sensitivity for detection of injury (including pre-necrotic injury), high tissue specificity, and lower susceptibility to inhibition or induction.

Measuring immune selection.

Immune responses that kill pathogens or reduce their reproductive rate are generally important in protecting hosts from infection and disease. Pathogens that escape the full impact of such responses will survive, and any heritable genetic basis of this evasion will be selected. Due to the memory component of vertebrate immune responses, pathogens with rare alleles of a target antigen can have an advantage over those with common alleles, leading to the maintenance of a polymorphism. At the genetic level, there ought to be detectable signatures of balancing selection in the genes encoding these antigens. Here, methods for identifying these selective signatures are reviewed. Their practical utility for identifying which antigens are targets of protective immune responses is discussed.

Strong diversifying selection on domains of the Plasmodium falciparum apical membrane antigen 1 gene.

The surface-accessible ectodomain region of the Plasmodium falciparum apical membrane antigen 1 (AMA1) is a malaria vaccine candidate. The amino acid sequence may be under selection from naturally acquired immune responses, and previous analyses with a small number of allele sequences indicate a non-neutral pattern of nucleotide variation. To investigate whether there is selection to maintain polymorphism within a population, and to identify the parts of the ectodomain under strongest selection, a sample of 51 alleles from a single endemic population was studied. Analyses using Fu and Li's D and F tests, Tajima's D test, and the McDonald-Kreitman test (with the chimpanzee parasite P. reichenowi as outgroup) show significant departure from neutrality and indicate the selective maintenance of alleles within the population. There is also evidence of a very high recombination rate throughout the sequence, as estimated by the recombination parameter, C, and by the rapid decline in linkage disequilibrium with increasing nucleotide distance. Of the three domains (I-III) encoding structures determined by disulfide bonds, the evidence of selection is strongest for Domains I and III. We predict that these domains in particular are targets of naturally acquired protective immune responses in humans.

An integrated proteomic approach to studying glomerular nephrotoxicity.

A single dose of puromycin aminonucleoside (PAN) given parenterally to rats induces ultrastructural glomerular changes and a nephrotic syndrome similar in many respects to human minimal change nephropathy. The exact aetiologies of both the human and the experimental syndromes are unknown, and are probably multifactorial. However, among the observed consequences in humans and rats is increased plasma protein excretion in urine, beginning in the latter typically 3-6 days after PAN administration. In view of this, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to profile urinary proteins during PAN-induced nephrotoxicity and subsequent recovery in the rat. In addition, urinary high performance liquid chromatography (HPLC) profiles and high resolution proton nuclear magnetic resonance (NMR) spectroscopy has been utilised to simultaneously detect toxin-induced changes in the relative concentrations of a number of metabolites. The proteomic approach, in conjunction with these other techniques, has the potential to provide significantly more mechanistic information than is provided readily by traditional clinical chemistry.

High-resolution magic angle spinning 1H NMR spectroscopic studies on intact rat renal cortex and medulla.

High-resolution magic angle spinning 1H NMR (MAS-NMR) spectroscopy was used to investigate the biochemical composition of normal renal cortex and renal papilla samples from rats, and results were compared with those from conventional 1H NMR analysis of protein-free tissue extracts. 1H MAS NMR spectra of samples obtained from inner and outer cortex were found to be broadly similar in terms of metabolite profile, and intra- and inter-animal variability was small. However, the MAS NMR spectra from renal papilla samples were qualitatively and quantitatively different from those obtained from cortex. High levels of free amino acids and several organic acids were detected in the cortex, together with choline, glucose, and trimethylamine-N-oxide. The dominant metabolite resonances observed in papillary tissue were from glycerophosphocholine (GPC), betaine, myo-inositol, and sorbitol. On increasing the magic angle spinning rate from 4,200 to 12,000 Hz, the lipid MAS 1H NMR signal profile remained largely unchanged in papillary tissue, whereas "new" resonances from triglycerides appeared in the spectra of cortical tissue, this effect being reversible on returning the spinning rate to 4,200 Hz. Further investigation into the behavior of the lipid components under different spinning rates suggested that the lipids in the cortex were present in more motionally constrained environments than those in the papilla. 1H MAS NMR spectra of tissues are of value both in interrogation of the biochemical composition of whole tissue, and in obtaining information on the mobility and compartmentalization of certain metabolites.

Molecular characterisation of meaB, a novel gene affecting nitrogen metabolite repression in Aspergillus nidulans.

Mutations within the meaB gene elicit the inappropriate expression of several activities subject to nitrogen metabolite repression in Aspergillus nidulans and also have some unrelated phenotypic effects. We have cloned meaB and isolated a full length cDNA clone. Northern analysis has shown that meaB expression is not subject to nitrogen metabolite repression. meaB encodes a novel protein of 418 amino acids and contains a significantly high number of S/TPXX motifs, a motif common in transcriptional regulatory proteins. We have sequenced three mutations within meaB, two of which have an identical phenotype to that produced by gene disruption.

Predictive value of hCG level 14 days after embryo transfer.

OBJECTIVE: It has been reported that the quantitative serum hCG level 14 days after embryo transfer (ET) correlated with pregnancy outcome as well as a likelihood of a multiple gestation pregnancy. This prospective study was designed to assess the predictive value of a 14-day post-ET hCG level with pregnancy outcome and multiple gestation pregnancies. METHODS: Patients undergoing in vitro fertilization (IVF) and ET were monitored by serum quantitative hCG levels 14 days after ET. If positive, serial values of hCG were obtained and transvaginal ultrasound was performed 3 weeks after ET and weekly until fetal cardiac activity was seen. Ongoing pregnancies were defined as greater than 20 weeks. RESULTS: One hundred eleven patients had positive serum quantitative hCG levels 14 days post-ET; 89/111, or 80.2%, had ongoing pregnancies. The spontaneous miscarriage rate was, therefore, 19.8% (22/111). If the level was less than 300, the ongoing multiple pregnancy rate was 9% (5/57). If the level was between 300 and 600, the ongoing pregnancy rate was 40% (10/25). If the hCG level was greater than 600, the multiple pregnancy rate was 100% (7/7). CONCLUSIONS: These data support the hypothesis that hCG levels greater than 200 mIU/ml on 14 days post-ET are more likely to have ongoing pregnancies; hCG levels greater than 600 have a high likelihood of a multiple gestation pregnancy.