Molecular characterization of Escherichia coli strains that cause symptomatic and asymptomatic urinary tract infections.

The differences between Escherichia coli strains associated with symptomatic and asymptomatic urinary tract infections (UTIs) remain to be properly determined. Here we examined the prevalence of plasmid types and bacteriocins, as well as genetic relatedness, in a defined collection of E. coli strains that cause UTIs. Comparative analysis identified a subgroup of strains with a high number of virulence genes (VGs) and microcins M/H47. We also identified associations between microcin genes, VGs, and specific plasmid types.

Molecular characterization of commensal Escherichia coli adapted to different compartments of the porcine gastrointestinal tract.

The role of Escherichia coli as a pathogen has been the focus of considerable study, while much less is known about it as a commensal and how it adapts to and colonizes different environmental niches within the mammalian gut. In this study, we characterize Escherichia coli organisms (n = 146) isolated from different regions of the intestinal tracts of eight pigs (dueodenum, ileum, colon, and feces). The isolates were typed using the method of random amplified polymorphic DNA (RAPD) and screened for the presence of bacteriocin genes and plasmid replicon types. Molecular analysis of variance using the RAPD data showed that E. coli isolates are nonrandomly distributed among different gut regions, and that gut region accounted for 25% (P < 0.001) of the observed variation among strains. Bacteriocin screening revealed that a bacteriocin gene was detected in 45% of the isolates, with 43% carrying colicin genes and 3% carrying microcin genes. Of the bacteriocins observed (H47, E3, E1, E2, E7, Ia/Ib, and B/M), the frequency with which they were detected varied with respect to gut region for the colicins E2, E7, Ia/Ib, and B/M. The plasmid replicon typing gave rise to 25 profiles from the 13 Inc types detected. Inc F types were detected most frequently, followed by Inc HI1 and N types. Of the Inc types detected, 7 were nonrandomly distributed among isolates from the different regions of the gut. The results of this study indicate that not only may the different regions of the gastrointestinal tract harbor different strains of E. coli but also that strains from different regions have different characteristics.

Green fluorescent protein-based biosensor to detect and quantify stress responses induced by DNA-degrading colicins.

Here we report the development of a whole-cell biosensor to detect and quantify the induction of the SOS response activated by DNA-degrading colicins. This biosensor utilizes the SOS-responsive cda promoter to regulate the expression of green fluorescent protein. The biosensor assay revealed induction of stress for all DNA-degrading reference colicins (E2, E7, and E8).

Virulence characteristics of translocating Escherichia coli and the interleukin-8 response to infection.

Four efficiently translocating Escherichia coli (TEC) strains isolated from the blood of humans (HMLN-1), pigs (PC-1) and rats (KIC-1 and KIC-2) were tested for their ability to adhere and translocate across human gut epithelial Caco-2 and HT-29 cells, to elicit a proinflammatory response and for the presence of 47 pathogenic E. coli virulence genes. HMLN-1 and PC-1 were more efficient in adhesion and translocation than rat strains, had identical biochemical phenotype (BPT) and serotype (O77:H18) and phylogenetic group (D). KIC-2 adhered more than KIC-1, belonged to different BPT and serotype but the same phylogenetic group as KIC-1. TEC strains elicited significantly higher IL-8 response in both cell lines (P < 0.05) and monocytic THP-1 (P < 0.0001) cells than non-TEC strains. KIC-2 induced the highest IL-8 response which may be associated with its immunostimulatory flagellin. Apart from adhesin genes fimH and bmaE that were carried by all strains, HMLN-1 and PC-1 carried capsule synthesis gene kpsMT III and KIC-2 carried the EAST1 toxin gene. The lack of known virulence genes and the ability of TEC to efficiently adhere and translocate whilst causing proinflammatory response suggests that these strains may carry as yet unidentified genes that enable their translocating ability.

No change in impedance upon intravascular injection of D5W.

PURPOSE: Electrical impedance increases following test injections of non-conducting solutions around nerves; however, this increase should diminish rapidly with intravascular needle placement, wherein the systemic circulation will dissipate the solution. For this observational study, we hypothesized that the impedance increases significantly at the perineural space after an injection of 5% dextrose in water (D5W), but that it does not increase correspondingly at the intravascular location METHODS: After Ethics Research Board approval, electrical impedance was measured by a nerve stimulator displaying resistance, Stimuplex HNS 12, before and during (30 sec) an injection of D5W 3 mL: 1) during intravenous cannula placement using an insulated stimulating needle sheathed in its plastic cannula, MultiSet NanoLine with 18G needle; and 2) during needle placement (Pajunk 22G insulated) for an ultrasound-guided supraclavicular block in patients undergoing hand surgery. The impedance changes at each location were analyzed and compared. RESULTS: Data were collected from 16 patients. Baseline impedance was lower intravascularly (mean 16.5 +/- standard deviation 7.2 kOmega) compared with perineurally (23.5 +/- 8.3 kOmega) (P = 0.037). Peak impedance after intravascular D5W injection was 20.1 +/- 6.8 kOmega, which was not a significant change (P = 0.15). In contrast, peak impedance after perineural D5W injection was 58.6 +/- 29.1 kOmega, an increase of 35.1 +/- 26.4 kOmega (155 +/- 117%), and then it reached a plateau of 36.7 +/- 19.6 kOmega. The increase in impedance was significantly greater at the perineural location (P < 0.0001). CONCLUSION: The absence of a significant increase in impedance upon injection of D5W prior to injection of local anesthetic may provide useful information to warn of intravascular injection.

Colonisation dynamics and virulence of two clonal groups of multidrug-resistant Escherichia coli isolated from dogs.

The study established the virulence potential of multidrug-resistant Escherichia coli (MDREC) isolates from nosocomial infections in hospitalised dogs. The isolates were resistant to fluoroquinolones, belonged to two distinct clonal groups (CG1 and CG2) and contained a plasmid-mediated AmpC (CMY-7) beta-lactamase. CG1 isolates (n=14) possessed two of 36 assayed extraintestinal virulence genes (iutA and traT) and belonged to phylogenetic group A, whereas CG2 isolates (n=19) contained four such genes (iutA, ibeA, fimH and kpsMT K5) and belonged to group D. In a mouse gastrointestinal tract colonisation model, colonisation by index CG1 strain C1 was transient, in contrast to the index CG2 strain C2b, which persisted up to 40days post-inoculation. In a mouse subcutaneous challenge model, both strains were less virulent than archetypal group B2 extraintestinal pathogenic E. coli (ExPEC) strain CFT073; strain C1 caused no systemic signs and strain C2b was lethal to only one of six mice. In a mouse urinary tract infection model, strain C2b colonised the mouse bladder over 2 logs higher compared to strain C1. Whilst both groups of canine MDREC appear less virulent than a reference human ExPEC strain, CG2 strains have greater capacity for colonisation and virulence.

Amyloid beta protein modulates glutamate-mediated neurotransmission in the rat basal forebrain: involvement of presynaptic neuronal nicotinic acetylcholine and metabotropic glutamate receptors.

Amyloid beta (Abeta) protein, a 39-43 amino acid peptide deposited in brains of individuals with Alzheimer's disease (AD), has been shown to interact directly with a number of receptor targets including neuronal nicotinic acetylcholine receptors (nAChRs) and glutamate receptors. In this study, we investigated the synaptic effects of Abeta(1-42) on glutamate-mediated neurotransmission in the diagonal band of Broca (DBB), a cholinergic basal forebrain nucleus. Glutamatergic miniature EPSCs (mEPSCs) were recorded using whole-cell patch-clamp recordings from identified cholinergic DBB neurons in rat forebrain slices. In 54% of DBB neurons, bath application of Abeta(1-42) (100 nM), but not Abeta(42-1) (inverse fragment), significantly increased the frequency of mEPSCs without affecting amplitude or kinetic parameters (rise or decay time). In 32% of DBB neurons, bath application of Abeta(1-42) significantly decreased only the frequency but not amplitude of mEPSCs. Application of dihydro-beta-erythroidine (DHbetaE) (an antagonist for the alpha4beta2 subtype of nAChRs) but not alpha-bungarotoxin (an antagonist for the alpha7 subtype of nAChRs) blocked Abeta(1-42)-mediated increases in mEPSC frequency. The Abeta(1-42)-mediated increase in glutamatergic transmission is thus presynaptic and mediated via non-alpha7 AChRs. In contrast, Abeta(1-42)-mediated decreases in mEPSC frequency could not be antagonized by either DHbetaE or alpha-bungarotoxin. However, the Abeta(1-42)-evoked depression in mEPSC frequency was antagonized by (RS)-alpha-methyl-4-carboxyphenyglycine, a nonselective group I/II metabotropic glutamate receptor antagonist. These observations provide further insight into the mechanisms whereby Abeta affects synaptic function in the brain and may be relevant in the context of synaptic failure observed in AD.

Safety of raw meat and shellfish in Vietnam: an analysis of Escherichia coli isolations for antibiotic resistance and virulence genes.

This study was conducted to examine a current baseline profile of antimicrobial resistance and virulence of Escherichia coli isolated from foods commonly sold in the market place in Vietnam. E. coli were isolated from 180 samples of raw meat, poultry and shellfish and also isolated from 43 chicken faeces samples. Ninety-nine E. coli isolates recovered from all sources were selected for the investigation of their susceptibility to 15 antimicrobial agents by the disk diffusion method. Eighty-four percent of the isolates were resistant to one or more antibiotics, and multi-resistance, defined as resistance to at least 3 different classes of antibiotics, was detected in all sources. The rates of multi-resistance were up to 89.5% in chicken, 95% in chicken faeces and 75% in pork isolates. Resistance was most frequently observed to tetracycline (77.8%), sulfafurazole (60.6%), ampicillin (50.5%), amoxicillin (50.5%), trimethoprim (51.5%), chloramphenicol (43.4%), streptomycin (39.4%), nalidixic acid (34.3%) and gentamicin (24.2%). In addition, the isolates also displayed resistance to fluoroquinolones (ciprofloxacin 16.2%, norfloxacin 17.2%, and enrofloxacin 21.2%), with chicken isolates showing the highest rates of resistance to these antibiotics (52.6-63.2%). Thirty-eight multi-resistant isolates were selected for further the examination of antibiotic resistance genes and were also evaluated for virulence gene profiles by multiplex and uniplex polymerase chain reaction. The beta-lactam TEM gene and tetracycline resistance tetA, tetB genes were frequently detected in the tested isolates (84.2% and 89.5% respectively). Genes which are responsible for resistance to streptomycin (aadA) (68.4%), chloramphenicol (cmlA) (42.1%), sulfonamides (sulI) (39.5%), trimethoprim (dhfrV) (26.3%) and kanamycin (aphA-1) (23.7%) were also widely distributed. Plasmid-mediated ampC genes were detected in E. coli isolates from chicken and pork. The isolates were tested for the presence of 58 virulence genes for adhesins, toxins, capsule synthesis, siderophores, invasins and others from different E. coli pathotypes. All of the tested isolates contained at least one virulence gene and there were 16 genes detected. Virulence genes detected were fimH (92.1%), bmaE (84.2%), TSPE4.C2 (42.1%), aidA AIDA-I (orfB) (31.6%), east1 (26.3%), traT (23.7%), and others including fyuA, iutA, chuA, yjaA, iss, iroN(E. coli), ibeA, aah (orfA), iha and papG allele III (10.5-2.6%). Typical toxin genes produced by enterohemorrhagic and enterotoxigenic E. coli pathotypes (a heat-stable toxin (ST), heat-labile toxin (LT) and Shiga toxin stx1, stx2) were not detected in any of these 38 isolates. The study has revealed that E. coli in raw foods is a significant reservoir of resistance and virulence genes.

Heterosexual HIV transmission dynamics: implications for prevention and control.

Understanding the epidemiologic definition of epidemic versus non-epidemic spread of an infectious disease agent and the different patterns of heterosexual HIV transmission are needed to fully understand the low potential for heterosexual HIV epidemics in most heterosexual populations. Epidemic sexual HIV transmission can occur only in populations where there are large numbers of persons who have unprotected sex with multiple and concurrent sex partners. How high HIV prevalence may reach in these populations depends on the size and overlap of sex networks, and the prevalence of facilitating and protective factors that can greatly increase or limit the amount of infected blood and sexual fluids exchanged during intercourse. The wide difference in potentials for heterosexual HIV epidemics that exists within and between countries must be recognized, accepted and monitored in order to design and focus prevention strategies where they are most needed and most effective.

Pathotypes and serogroups of enterotoxigenic Escherichia coli isolated from pre-weaning pigs in north Vietnam.

The contribution of enterotoxigenic Escherichia coli (ETEC) to pre-weaning diarrhoea was investigated over a 6 month period at five selected commercial piggeries (CPs) in north Vietnam with at least 100 sows each. Diarrhoea was found to affect 71.5% of the litters born during the period of study. Of 406 faecal specimens submitted for bacteriological culture, 200 (49.3%) yielded a heavy pure culture of E. coli and 126 (31%) were confirmed by PCR to carry at least one of eight porcine ETEC virulence genes. ETEC was responsible for 43% of cases of diarrhoea in neonatal pigs during the first 4 days of life and 23.9% of the remaining cases up until the age of weaning. Pathotypes were determined by PCR for the 126 ETEC isolates together with 44 ETEC isolates obtained from village pigs (VPs) raised by smallholder farmers. The CP isolates belonged to five pathotypes, four of which were also identified in VP isolates. Haemolytic serogroup O149 : K91 isolates that belonged to F4/STa/STb/LT were most commonly identified in both CPs (33% of isolates) and VPs (45.5%). Other combinations identified in both production systems included O64 (F5/STa), O101 (F4/STa/STb) and O-nontypable (F-/STb). A high proportion of CP isolates (22.3%) possessed all three enterotoxins (STa/STb/LT), lacked the genes for all five tested fimbriae (F4, F5, F6, F41 and F18) and belonged to serogroup O8. These unusual O8 F- isolates were haemolytic and were isolated from all ages of diarrhoeic piglets at each CP, suggesting that they have pathogenic potential.

Emergence and spread of two distinct clonal groups of multidrug-resistant Escherichia coli in a veterinary teaching hospital in Australia.

Multidrug-resistant Escherichia coli (MDREC) expressing AmpC beta-lactamases have emerged as a cause of opportunistic infections in dogs. Following a cluster of extraintestinal infections caused by two distinct clonal groups (CGs) of bla(CMY)-producing MDREC, a 12-month infection control study was undertaken at a veterinary teaching hospital in Brisbane, Australia. Swabs from the rectum of hospitalized dogs (n=780), hospital staff (n=16) and the hospital environment (n=220) were plated onto selective agar to obtain multidrug-resistant (MDR) coliforms. These were then tested by multiplex PCR for E. coli uspA, bla(CMY) and the class 1 integron-associated dfrA17-aadA5 gene cassette for rapid identification of MDREC CG 1 (positive for all three genes) and CG 2 (positive for uspA and bla(CMY) only). A total of 16.5 % of the dog rectal swabs and 4.1% of the hospital environmental swabs yielded MDREC, and on the basis of multiplex PCR, PFGE and plasmid profiling, these were confirmed to belong to either CG 1 or CG 2. Both CG 1 and CG 2 isolates were obtained from clinical cases of extraintestinal infection and rectal swabs from hospitalized dogs over the same period of time, whereas only CG 1 isolates were obtained from the hospital environment. Both CGs were prevalent during the first 6 months, but only CG 2 was isolated during the second 6 months of the study. Two isolates obtained from rectal swabs of staff working in the hospital belonged to CG 2, with one of the isolates possessing the same REDP as nine isolates from dogs, including six isolates associated with cases of extraintestinal infection. CG 1 isolates belonged to E. coli serotypes O162 : H-, OR : H- or Ont : H-, whereas CG 2 isolates belonged to O153 : HR, OR : HR or OR : H34. These results confirm that in this particular outbreak, canine MDREC were highly clonal and CG 2 MDREC may colonize both humans and dogs.

Development of a group-specific PCR combined with ARDRA for the identification of Bacillus species of environmental significance.

A group-specific primer pair was designed to amplify the 16S rRNA gene of representative reference strains from environmentally sourced, mesophilic aerobic spore-forming Bacillus taxa. The PCR generated a 1114 bp amplicon but did not do so with DNA extracted from 16 other Eubacterial species. When amplicons were digested with restriction enzymes AluI or TaqI, different profiles containing between 2 and 5 fragments ranging in size from 76 to 804 base pairs were seen with different Bacillus species. This procedure, known otherwise as amplified ribosomal DNA restriction analysis or ARDRA, produced unique and distinguishable patterns to differentiate between 15 ATCC reference strains (10 Bacillus, 3 Paenibacillus and 2 Brevibacillus member species) as well as 3 misidentified Bacillus probiotic strains in a commercial collection. Our simplified PCR-ARDRA protocol provides a facile method for the identification of most environmentally important species of Bacillus.

An aromatic amino acid auxotrophic mutant of Bordetella bronchiseptica is attenuated and immunogenic in a mouse model of infection.

We have constructed an aromatic amino acid auxotrophic mutant of Bordetella bronchiseptica, harbouring mutations in aroA and trpE to investigate the use of such a strain as a live-attenuated vaccine. B. bronchiseptica aroA trpE was unable to grow in minimal medium without aromatic supplementation. Compared to the parental wild-type strain, the mutant displayed significantly reduced abilities to invade and survive within the mouse macrophage-like cell line J774A.1 in vitro and in the murine respiratory tract following experimental intranasal infection. Mice vaccinated with B. bronchiseptica aroA trpE displayed significant dose-dependent increases in B. bronchiseptica-specific antibody responses, and exhibited increases in the number of B. bronchiseptica-reactive spleen cells in lymphoproliferation assays. Immunised animals were protected against lung colonisation after challenge with the wild-type parental strain. With such a broad host range displayed by B. bronchiseptica, the attenuated strain constructed in this study may not only be used for the prevention of B. bronchiseptica-associated disease, but also for the potential delivery of heterologous antigen.

Canine model for investigating the impact of oral enrofloxacin on commensal coliforms and colonization with multidrug-resistant Escherichia coli.

A model was developed in dogs to determine the impact of oral enrofloxacin administration on the indigenous coliform population in the gastrointestinal tract and subsequent disposition to colonization by a strain of multidrug-resistant Escherichia coli (MDREC). Dogs given a daily oral dose of 5 mg enrofloxacin kg(-1) for 21 consecutive days showed a significant decline in faecal coliforms to levels below detectable limits by 72 h of administration. Subsequently, faecal coliforms remained suppressed throughout the period of enrofloxacin dosing. Upon termination of antibiotic administration, the number of excreted faecal coliforms slowly returned over an 8-day period, to levels comparable to those seen prior to antibiotic treatment. Enrofloxacin-treated dogs were more effectively colonized by MDREC, evidenced by a significantly increased count of MDREC in the faeces (7.1 +/- 1.5 log(10) g(-1)) compared with non-antibiotic-treated dogs (5.2 +/- 1.2; P = 0.003). Furthermore, antibiotic treatment also sustained a significantly longer period of MDREC excretion in the faeces (26.8 +/- 10.5 days) compared with animals not treated with enrofloxacin (8.5 +/- 5.4 days; P = 0.0215). These results confirm the importance of sustained delivery of an antimicrobial agent to maintain and expand the colonization potential of drug-resistant bacteria in vivo, achieved in part by reducing the competing commensal coliforms in the gastrointestinal tract to below detectable levels in the faeces. Without in vivo antimicrobial selection pressure, commensal coliforms dominated the gastrointestinal tract at the expense of the MDREC population. Conceivably, the model developed could be used to test the efficacy of novel non-antibiotic strategies aimed at monitoring and controlling gastrointestinal colonization by multidrug-resistant members of the Enterobacteriaceae that cause nosocomial infections.

The evolving role of the pediatric nurse practitioner in hospital medicine.

BACKGROUND: This program evaluation sought to compare cost and pediatric patient outcomes among a pediatric nurse practitioner (PNP) hospitalist team, a combined PNP/doctor of medicine (MD) team, and 2 resident teams without PNPs. METHODS: Administrative and electronic medical record data from July 1, 2009 to June 30, 2010 was retrospectively reviewed from Children's Hospital Colorado inpatient medical unit and inpatient satellite sites in the Children's Hospital network of care (NOC). The top 3 All Patient Refined Diagnosis Related Groups (APR-DRG) admission codes bronchiolitis and respiratory syncytial virus (RSV) pneumonia, pneumonia not elsewhere classified (NEC), and asthma were selected for this analysis. Inpatient records representing these APR-DRG admission codes were reviewed (N = 1664). Measures included adherence with relevant clinical care guidelines (CCGs), length of stay (LOS), and cost of care. Chi square, t tests, and analysis of variance were used to analyze between-group differences. RESULTS: Approximately 20% of these admissions were on the PNP team, 45% were on the resident teams, and 35% were on the PNP/MD team in the NOC. PNP adherence to CCGs was comparable to resident teams for selected measures. There was no significant difference in LOS among the PNP team, the PNP/MD team, and the resident teams. The direct cost of patient care per encounter provided by the PNP team was significantly less than the PNP/MD team and the resident teams. CONCLUSIONS: There is evidence to suggest that PNP hospitalists provide inpatient care comparable to resident teams at a lower cost for patients with uncomplicated bronchiolitis, pneumonia, and asthma.

Lovastatin in Aspergillus terreus: fermented rice straw extracts interferes with methane production and gene expression in Methanobrevibacter smithii.

Lovastatin, a natural byproduct of some fungi, is able to inhibit HMG-CoA (3-hydroxy-3 methyl glutaryl CoA) reductase. This is a key enzyme involved in isoprenoid synthesis and essential for cell membrane formation in methanogenic Archaea. In this paper, experiments were designed to test the hypothesis that lovastatin secreted by Aspergillus terreus in fermented rice straw extracts (FRSE) can inhibit growth and CH4 production in Methanobrevibacter smithii (a test methanogen). By HPLC analysis, 75% of the total lovastatin in FRSE was in the active hydroxyacid form, and in vitro studies confirmed that this had a stronger effect in reducing both growth and CH4 production in M. smithii compared to commercial lovastatin. Transmission electron micrographs revealed distorted morphological divisions of lovastatin- and FRSE-treated M. smithii cells, supporting its role in blocking normal cell membrane synthesis. Real-time PCR confirmed that both commercial lovastatin and FRSE increased (P < 0.01) the expression of HMG-CoA reductase gene (hmg). In addition, expressions of other gene transcripts in M. smithii. with a key involvement in methanogenesis were also affected. Experimental confirmation that CH4 production is inhibited by lovastatin in A. terreus-fermented rice straw paves the way for its evaluation as a feed additive for mitigating CH4 production in ruminants.

Optimization of benzoxazole-based inhibitors of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase.

Cryptosporidium parvum is an enteric protozoan parasite that has emerged as a major cause of diarrhea, malnutrition, and gastroenteritis and poses a potential bioterrorism threat. C. parvum synthesizes guanine nucleotides from host adenosine in a streamlined pathway that relies on inosine 5'-monophosphate dehydrogenase (IMPDH). We have previously identified several parasite-selective C. parvum IMPDH (CpIMPDH) inhibitors by high-throughput screening. In this paper, we report the structure-activity relationship (SAR) for a series of benzoxazole derivatives with many compounds demonstrating CpIMPDH IC50 values in the nanomolar range and >500-fold selectivity over human IMPDH (hIMPDH). Unlike previously reported CpIMPDH inhibitors, these compounds are competitive inhibitors versus NAD(+). The SAR study reveals that pyridine and other small heteroaromatic substituents are required at the 2-position of the benzoxazole for potent inhibitory activity. In addition, several other SAR conclusions are highlighted with regard to the benzoxazole and the amide portion of the inhibitor, including preferred stereochemistry. An X-ray crystal structure of a representative E·IMP·inhibitor complex is also presented. Overall, the secondary amine derivative 15a demonstrated excellent CpIMPDH inhibitory activity (IC50 = 0.5 ± 0.1 nM) and moderate stability (t1/2 = 44 min) in mouse liver microsomes. Compound 73, the racemic version of 15a, also displayed superb antiparasitic activity in a Toxoplasma gondii strain that relies on CpIMPDH (EC50 = 20 ± 20 nM), and selectivity versus a wild-type T. gondii strain (200-fold). No toxicity was observed (LD50 > 50 µM) against a panel of four mammalian cells lines.

Molecular serogrouping of porcine enterotoxigenic Escherichia coli from Australia.

Enterotoxigenic Escherichia coli (ETEC) is a common etiological agent of neonatal, pre and post weaning diarrhoea in piglets. One of the most important steps in the diagnosis and epidemiological understanding of this organism is accurate serogrouping. In many instances, however, conventional serogrouping fails to produce accurate identification of serogroups. In this communication we report a modified and simplified molecular serogrouping method (rfb-RFLP) for the accurate identification of the most common porcine ETEC strains that cause neonatal, pre and post weaning diarrhoea in Australia.

Comparative analysis of virulence genes, genetic diversity, and phylogeny of commensal and enterotoxigenic Escherichia coli isolates from weaned pigs.

If the acquisition of virulence genes (VGs) for pathogenicity were not solely acquired through horizontal gene transfers of pathogenicity islands, transposons, and phages, then clonal clusters of enterotoxigenic Escherichia coli (ETEC) would contain few or even none of the VGs found in strains responsible for extraintestinal infections. To evaluate this possibility, 47 postweaning diarrhea (PWD) ETEC strains from different geographical origins and 158 commensal E. coli isolates from the gastrointestinal tracts of eight group-housed healthy pigs were screened for 36 extraintestinal and 18 enteric VGs using multiplex PCR assays. Of 36 extraintestinal VGs, only 8 were detected (fimH, traT, fyuA, hlyA, kpsMtII, k5, iha, and ompT) in the ETEC collection. Among these, hlyA (alpha-hemolysin) and iha (nonhemagglutinating adhesin) occurred significantly more frequently among the ETEC isolates than in the commensal isolates. Clustering analysis based on the VG profiles separated commensal and ETEC isolates and even differentiated serogroup O141 from O149. On the other hand, pulsed-field gel electrophoresis (PFGE) successfully clustered ETEC isolates according to both serotype and geographical origin. In contrast, the commensal isolates were heterogeneous with respect to both serotype and DNA fingerprint. This study has validated the use of VG profiling to examine pathogenic relationships between porcine ETEC isolates. The clonal relationships of these isolates can be further clarified by PFGE fingerprinting. The presence of extraintestinal VGs in porcine ETEC confirmed the hypothesis that individual virulence gene acquisitions can occur concurrently against a background of horizontal gene transfers of pathogenicity islands. Over time, this could enable specific clonotypes to respond to host selection pressure and to evolve into new strains with increased virulence.

Beta-amyloid enhances intracellular calcium rises mediated by repeated activation of intracellular calcium stores and nicotinic receptors in acutely dissociated rat basal forebrain neurons.

Beta-amyloid, a 39-43 amino acid peptide, may exert its biological effects via neuronal nicotinic acetylcholine receptors. Using the ratiometric dye, fura-2, we examined the effect of soluble beta-amyloid(1-42) on the concentration of intracellular Ca(2+) ([Ca(2+)](i)) in acutely dissociated rat basal forebrain neurons. Focal applications of nicotine (0.5-20 mM), evoked dose-dependent increases in intracellular [Ca(2+)](i) that were mediated by the entry of extracellular Ca(2+) via nicotinic acetylcholine receptors, and the release of intracellular Ca(2+) from stores. With repeated nicotine challenges, the nicotinic responses were potentiated by 98 +/- 12% (P < 0.05) while beta-amyloid(1-42)(100 nM) was present for approximately 5 min. This potentiation became larger during the subsequent washout of beta-amyloid(1-42), which was associated with a gradual rise in baseline [Ca(2+)](i). Application of beta-amyloid(1-42)by itself did not alter [Ca(2+)](i), and beta-amyloid(1-42)also had no significant effect on the response to repeated KCl challenges. Therefore, beta-amyloid(1-42) caused neither gross disturbance of cellular Ca(2+) homeostasis nor enhancement of voltage-gated Ca(2+) channels. Interestingly, beta-amyloid(1-42) transiently potentiated the response to repeated caffeine challenges, which was also associated with a transient rise in baseline [Ca(2+)](i). beta-amyloid(1-42) potentiation of nicotine-evoked rises in [Ca(2+)](i) was reversed by the SERCA pump inhibitor, thapsigargin, and the mitochondrial Na(+)/Ca(2+) exchanger inhibitor, CGP-37157. These results suggest that the dysregulation of [Ca(2+)](i) by beta-amyloid(1-42) during multiple challenges with nicotine or caffeine involved the sensitization or overfilling of intracellular stores that are maintained by SERCA pump and Ca(2+) efflux from the mitochondria.