Considerations when using discriminant function analysis of antimicrobial resistance profiles to identify sources of fecal contamination of surface water in Michigan

The goals of this study were to (i) identify issues that affect the ability of discriminant function analysis (DA) of antimicrobial resistance profiles to differentiate sources of fecal contamination, (ii) test the accuracy of DA from a known-source library of fecal Escherichia coli isolates with isolates from environmental samples, and (iii) apply this DA to classify E. coli from surface water. A repeated cross-sectional study was used to collect fecal and environmental samples from Michigan livestock, wild geese, and surface water for bacterial isolation, identification, and antimicrobial susceptibility testing using disk diffusion for 12 agents chosen for their importance in treating E. coli infections or for their use as animal feed additives. Nonparametric DA was used to classify E. coli by source species individually and by groups according to antimicrobial exposure. A modified backwards model-building approach was applied to create the best decision rules for isolate differentiation with the smallest number of antimicrobial agents. Decision rules were generated from fecal isolates and applied to environmental isolates to determine the effectiveness of DA for identifying sources of contamination. Principal component analysis was applied to describe differences in resistance patterns between species groups. The average rate of correct classification by DA was improved by reducing the numbers of species classifications and antimicrobial agents. DA was able to correctly classify environmental isolates when fewer than four classifications were used. Water sample isolates were classified by livestock type. An evaluation of the performance of DA must take into consideration relative contributions of random chance and the true discriminatory power of the decision rules.     

A chromatographic tool for preparing combinatorial quinone-thiol conjugate libraries

Quinones are well established as key players in the production of reactive oxygen species within cellular environments. Many factors govern their cytotoxicity but most studies have been restricted to a few, core, derivatives. A new strategy for the in situ production of quinone derivatives has been developed such that libraries of diverse functionality can be rapidly created without recourse to extensive synthetic procedures. The approach relies upon nucleophilic addition by reduced thiol derivatives to the quinone core within a pre-culture assay mixture and provides a generic strategy that exploits the large reservoir of commercial thiols currently available. A readily accessible chromatographic method has been developed that allows the derivatisation process to be easily monitored and the purity of the resulting one pot preparation to be assessed. The viability of the combinatorial approach has been fully validated through comparison with a range of quinone-S-conjugates prepared using conventional bench synthesis. The latter have been fully characterised.     

Siderophore-mediated cooperation and virulence in Pseudomonas aeruginosa

Why should organisms cooperate with each other? Helping close relatives that are likely to share the same genes (kin selection) is one important explanation that is likely to apply across taxa. The production of metabolically costly extracellular iron-scavenging molecules (siderophores) by microorganisms is a cooperative behaviour because it benefits nearby conspecifics. We review experiments focusing on the production of the primary siderophore (pyoverdin) of the opportunistic bacterial pathogen, Pseudomonas aeruginosa, which test kin selection theories that seek to explain the evolution of cooperation. First, cooperation is indeed favoured when individuals interact with their close relatives and when there is competition between groups of cooperators and noncooperators, such that the benefit of cooperation can be realized. Second, the relative success of cheats and cooperators is a function of their frequencies within populations. Third, elevated mutation rates can confer a selective disadvantage under conditions when cooperation is beneficial, because high mutation rates reduce how closely bacteria are related to each other. Fourth, cooperative pyoverdin production is also shown to be favoured by kin selection in vivo (caterpillars), and results in more virulent infections. Finally, we briefly outline ongoing and future work using this experimental system.     

The antimutagenicity of 2-substituted selenazolidine-4-(R)-carboxylic acids

Selenium can have cancer chemopreventive activity, although the mechanism of action has not been well defined. Selenazolidine-4-(R)-carboxylic acids (SCAs) were devised as prodrugs of L-selenocysteine, to provide selenium in a form and at a concentration commensurate with cancer chemopreventive activity. In the present study, a series of selenazolidines has been evaluated in the Salmonella typhimurium TA98 tester strain and all were found to possess antimutagenic activity. There was little difference between the seven selenazolidines in their effectiveness against either benzo[a]pyrene (B[a]P) or 3,6-bis(dimethylamino)acridine (acridine orange), agents which differ in their requirement for mammalian enzyme bioactivation for mutagenicity. Antimutagenic activity against acridine orange was dependent on selenazolidine concentration, and EC50 values were in the 5-10 microM range. At 25 microM, the concentration tested in common for the two mutagens, the selenazolidines were more effective antimutagens against acridine orange than against B[a]P, with reductions in mutant frequency ranging from 54 to 71% for B[a]P and 79 to 93% for acridine orange. Efficacy against B[a]P was not enhanced when the concentration was increased to 50 microM. The similarity in efficacy among the selenazolidines against B[a]P mutagenicity, contrasted with inter-compound differences in their ability to inhibit S9 CYP1A activity. The CYP1A Ki values ranged from a low of 63 microM (2-[2'-hydroxyphenyl]SCA) to a high of 1.1mM (2-cyclohexylSCA), but all were above the concentration required to inhibit mutagenicity by 50%. Thus, all the SCAs possess antimutagenic activity against both B[a]P and acridine orange, the efficacy varies little between the individual selenazolidines, and for B[a]P, the efficacy is not proportional to the inhibitory effect on the mutagen bioactivating enzyme.     

HKT1;5-like cation transporters linked to Na+ exclusion loci in wheat, Nax2 and Kna1

Bread wheat (Triticum aestivum) has a greater ability to exclude Na⁺ from its leaves and is more salt tolerant than durum wheat (Triticum turgidum L. subsp. durum [Desf.]). A novel durum wheat, Line 149, was found to contain a major gene for Na⁺ exclusion, Nax2, which removes Na⁺ from the xylem in the roots and leads to a high K⁺-to-Na⁺ ratio in the leaves. Nax2 was mapped to the distal region on chromosome 5AL based on linkage to microsatellite markers. The Nax2 locus on 5AL coincides with the locus for a putative Na⁺ transporter, HKT1;5 (HKT8). The Nax2 region on 5AL is homoeologous to the region on chromosome 4DL containing the major Na⁺ exclusion locus in bread wheat, Kna1. A gene member of the HKT1;5 family colocates to the deletion bin containing Kna1 on chromosome 4DL. This work provides evidence that Nax2 and Kna1 are strongly associated with HKT1;5 genes.     

Isolation of a field strain of Babesia bigemina (Piroplasma: Babesiidae) and establishment of in vitro culture for antigen production

Isolation of a field strain of Babesia bigemina (Piroplasma: Babesiidae) and establishment of in vitro culture for antigen production. Bovine b abesiosis, caused by Babesia bigemina, is a barrier for livestock development; it results in high economic loss to Mexican livestock. Control requires adequate antigens for diagnosis and vaccination programs. However, because of antigenic variation among Babesia strains, it is necessary to use antigens prepared from local strains. The purpose of the present study was to isolate a local field strain and to establish the in vitro culture of B. bigemina by the evaluation of the constituent's concentration of culture media. Thirty engorged female Boophilus microplus were collected from cattle suffering clinical babesiosis (B. bigemina) in Yucatan state, Mexico. These ticks were sent to the laboratory for detection of Babesia sp. vermicules. Eggs were kept at 83-85 % humidity and 27 degrees C until hatching. Larvae were transferred to an esplenectomized calf (B-1). The resulting nymphs were transferred to an esplenectomized calf (B-2). Twelve days later, B. bigemina (local strain) was detected in calf B-2 and its infected blood was frozen in liquid nitrogen to initiate the in vitro culture. The Microaerophilus Stationary Phase (MASP) in vitro culture method was used to reactivate the parasite. Three different concentrations of culture media (70, 60 and 50%), serum (30, 40 and 50%) and uninfected red blood cells (5, 10 and 15 %) were used in order to know the convenient concentrations to obtain the highest percentage of infected red blood cells (PEI). The cultured strain was used to prepare antigens for the Immunofluorescence Antibody Test (IFAT) and several concentrations of serum and conjugate were tested. Strain isolation was successful; 30 days were needed to obtain a PEI of 1.5%. The isolated strain was frozen in liquid nitrogen and the parasites were reactivated with the in vitro culture MASP method. The concentration of culture media that produced the highest PEI (14%) (p < 0.05) was 30% serum, 70% M199 and 5%. Uninfected Red Blood cells antigens were successfully used in the IFAT and the best dilutions to differentiate between positive and negative controls were serum 1:80 and conjugate 1:80. The isolated B. bigemina local strain requires particular conditions of in vitro culture by the MASP method to reach high numbers of infected red blood cells, needed to prepare and provide high quality antigens for serological diagnosis of B. bigemina.     

Release method and anatomical hook location: effects on short-term mortality of angler-caught Acanthopagrus australis and Argyrosomus japonicus

One field and 3 aquaria experiments were done to quantify the short-term mortality of yellowfin bream Acanthopagrus australis and mulloway Argyrosomus japonicus after being angled and subjected to 3 general handling treatments. Anglers were supplied with identical J-type hooks and asked to handle hooked fish by either (1) physically removing the hook or (2) cutting the line (5 cm from the mouth of the fish) and leaving the hook in. Some hooked A. japonicus were subjected to a third handling treatment where the line was cut underwater without exposing the fish to air. Technical and biological data were collected before all fish were released into sea cages and monitored for 5 d. Control fish were seined and similarly caged and monitored. Concentrations of plasma glucose and cortisol were collected from a sample of fish on the first and last day of the experiments. Significant predictors of mortality for both species involved the presence of blood at the mouth and an interaction between anatomical hook location and hook removal. A. australis and A. japonicus that had their ingested hooks removed experienced the greatest mortalities (87.5 and 72.7%, respectively). Typically, these fish suffered damage to their oesophagus, stomach wall and vital organs. Mortality rates of A. australis and A. japonicus were significantly decreased to 1.7 and 16%, respectively, when they were released with their lines cut, with some of these fish free of hooks after 5 d. In contrast, few mortalities occurred in either species when the hooks were removed or the lines cut on mouth-hooked fish or in A. japonicus when it was released with no air exposure. For A. australis, the field- and aquaria-based experiments provided comparable results in terms of identifying treatment-specific effects, but there were potential biases in rates of hook ingestion. Irrespective of the treatment of fish, all experiments caused physiological changes measured as elevations in either plasma cortisol or glucose. We concluded that anglers should cut the line from hook-ingested A. australis and A. japonicus, but remove the hook from mouth-hooked individuals to prevent subsequent ingestion. Further research is required to examine the longer-term consequences of these handling practices on fish health.