Prevalence of multiple antibiotic resistance in 443 Campylobacter spp. isolated from humans and animals

Aims: In view of recent findings that a multidrug efflux pump CmeABC exists in Campylobacter jejuni, 391 C. jejuni and 52 Campylobacter coli of human and animal origin were examined for a multidrug resistance phenotype. Materials and methods: The MICs of ampicillin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, tetracycline, cetrimide, triclosan, acridine orange, paraquat and ethidium bromide were determined. Resistance to organic solvents and the effect of salicylate (known inducer of the marRAB operon in Escherichia coli and Salmonella) were also examined. Results: Two C. coli and 13 C. jejuni isolates, mainly from pigs or poultry, were resistant to three or more antibiotics and 12 of these strains had reduced susceptibility to acridine orange and/or ethidium bromide. Strains (n=20) that were less susceptible to acridine orange, ethidium bromide and triclosan were significantly more resistant (P<0.05) to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid and tetracycline, with two- to four-fold increases in MIC values compared with strains (n=20) most susceptible to acridine orange, ethidium bromide and triclosan. Growth of strains with 1 mM salicylate caused a small (up to two-fold) but statistically significant (Pless than or equal to0.005) increase in the MICs of chloramphenicol, ciprofloxacin, erythromycin and tetracycline. Conclusions: These data indicate that multiple antibiotic resistant (MAR)-like Campylobacter strains occur and it may be postulated that these may overexpress cmeABC or another efflux system.

Antibiotic resistance genes, integrons and multiple antibiotic resistance in thirty-five serotypes of Salmonella enterica isolated from humans and animals in the UK

Objectives: To examine 397 strains of Salmonella enterica of human and animal origin comprising 35 serotypes for the presence of aadB, aphAI-IAB, aadA1, aadA2, bla(Carb(2)) or pse1, bla(Tem), cat1, cat2, dhfr1, floR, strA, sul1, sul2, tetA(A), tetA(B) and tetA(G) genes, the presence of class 1 integrons and the relationship of resistance genes to integrons and antibiotic resistance. Results: Some strains were resistant to ampicillin (91), chloramphenicol (85), gentamicin (2), kanamycin (14), spectinomycin (81), streptomycin (119), sulfadiazine (127), tetracycline (108) and trimethoprim (45); 219 strains were susceptible to all antibiotics. bla(Carb(2)), floR and tetA(G) genes were found in S. Typhimurium isolates and one strain of S. Emek only. Class 1 integrons were found in S. Emek, Haifa, Heidelberg, Mbandaka, Newport, Ohio, Stanley, Virchow and in Typhimurium, mainly phage types DT104 and U302. These strains were generally multi-resistant to up to seven antibiotics. Resistance to between three and six antibiotics was also associated with class 1 integron-negative strains of S. Binza, Dublin, Enteritidis, Hadar, Manhattan, Mbandaka, Montevideo, Newport, Typhimurium DT193 and Virchow. Conclusion: The results illustrate specificity of some resistance genes to S. Typhimurium or non- S. Typhimurium serotypes and the involvement of both class 1 integron and non-class 1 integron associated multi-resistance in several serotypes. These data also indicate that the bla(Carb(2)), floR and tetA(G) genes reported in the SG1 region of S. Typhimurium DT104, U302 and some other serotypes are still predominantly limited to S. Typhimurium strains.

Prolonged treatment of Salmonella enterica serovar Typhimurium with commercial disinfectants selects for multiple antibiotic resistance, increased efflux and reduced invasiveness

Objectives: To study how disinfectants affect antimicrobial susceptibility and phenotype of Salmonella enterica serovar Typhimurium SL1344. Methods: Wild-type strain SL1344 and its isogenic gyrA mutant were passaged daily for 7 days in subinhibitory concentrations, and separately for 16 days in gradually increasing concentrations of a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde (QACFG), an oxidizing compound blend (OXC), a phenolic tar acids-based disinfectant (TOP) and triclosan. The MICs of antimicrobials and antibiotics for populations and representative isolates and the proportion of cells resistant to the MICs for the wild-type were determined. Expression of acrB gene, growth at 37 degrees C and invasiveness of populations in Caco-2 intestinal epithelial cells were assessed. Results: QACFG and triclosan showed the highest selectivity for variants with reduced susceptibility to chloramphenicol, tetracycline, ampicillin, acriflavine and triclosan. Populations treated with the above biocides had reduced invasiveness in Caco-2 cells, and altered growth kinetics. Resistance to disinfectants was observed only after exposure to gradually increasing concentrations of triclosan, accompanied with a 2000-fold increase in its MIC. Growth in OXC and TOP did not affect the MICs of antibiotics, but resulted in the appearance of a proportion of cells resistant to the MIC of acriflavine and triclosan for the wild-type. Randomly selected stable variants from all populations, except the one treated with TOP, over-expressed acrB. Conclusions: In vitro exposure to QACFG and triclosan selects for Salmonella Typhimurium cells with reduced susceptibility to several antibiotics. This is associated with overexpression of AcrAB efflux pump, but accompanied with reduced invasiveness.

Commonly used farm disinfectants can select for mutant Salmonella enterica serovar Typhimurium with decreased susceptibility to biocides and antibiotics without compromising virulence

Objectives: To determine if one passage of Salmonella enterica serovar Typhimurium in the presence of farm disinfectants selected for mutants with decreased susceptibility to disinfectants and/or antibiotics. Methods: Eight Salmonella Typhimurium strains including field isolates and laboratory mutants were exposed to either a tar oil phenol (PFD) disinfectant, an oxidizing compound disinfectant (OXC), an aldehyde based disinfectant (ABD) or a dairy sterilizer disinfectant (based on quaternary ammonium biocide) in agar. The susceptibility of mutants obtained after disinfectant exposure to antibiotics and disinfectants was determined as was the accumulation of norfloxacin. The proteome of SL1344 after exposure to PFD and OXC was analysed using two-dimensional liquid chromatography mass spectrometry. Results: Strains with either acrB or tolC inactivated were more susceptible to most disinfectants than other strains. The majority (3/5) of mutants recovered after disinfectant exposure required statistically significantly longer exposure times to disinfectants than their parent strains to generate a 5 log kill. Small decreases in antibiotic susceptibility were observed but no mutants were multiply antibiotic-resistant (MAR). Notably exposure to ABD decreased susceptibility to ciprofloxacin in some strains. Mutants with increased disinfectant tolerance were able to survive and persist in chicks as well as in parent strains. Analysis of proteomes revealed significantly increased expression of the AcrAB-TolC efflux system after PFD exposure. Conclusions: Data presented demonstrate that efflux pumps are required for intrinsic resistance to some disinfectants and that exposure to disinfectants can induce expression of the AcrAB-TolC efflux system, but that single exposure was insufficient to select for MAR strains.

Plant–pathogen interactions: disease resistance in modern agriculture

The growing human population will require a significant increase in agricultural production. This challenge is made more difficult by the fact that changes in the climatic and environmental conditions under which crops are grown have resulted in the appearance of new diseases, whereas genetic changes within the pathogen have resulted in the loss of previously effective sources of resistance. To help meet this challenge, advanced genetic and statistical methods of analysis have been used to identify new resistance genes through global screens, and studies of plant-pathogen interactions have been undertaken to uncover the mechanisms by which disease resistance is achieved. The informed deployment of major, race-specific and partial, race-nonspecific resistance, either by conventional breeding or transgenic approaches, will enable the production of crop varieties with effective resistance without impacting on other agronomically important crop traits. Here, we review these recent advances and progress towards the ultimate goal of developing disease-resistant crops.

Random Prism: a noise-tolerant alternative to Random Forests

Ensemble learning can be used to increase the overall classification accuracy of a classifier by generating multiple base classifiers and combining their classification results. A frequently used family of base classifiers for ensemble learning are decision trees. However, alternative approaches can potentially be used, such as the Prism family of algorithms that also induces classification rules. Compared with decision trees, Prism algorithms generate modular classification rules that cannot necessarily be represented in the form of a decision tree. Prism algorithms produce a similar classification accuracy compared with decision trees. However, in some cases, for example, if there is noise in the training and test data, Prism algorithms can outperform decision trees by achieving a higher classification accuracy. However, Prism still tends to overfit on noisy data; hence, ensemble learners have been adopted in this work to reduce the overfitting. This paper describes the development of an ensemble learner using a member of the Prism family as the base classifier to reduce the overfitting of Prism algorithms on noisy datasets. The developed ensemble classifier is compared with a stand-alone Prism classifier in terms of classification accuracy and resistance to noise.

Effect of Rht alleles on the tolerance of wheat grain set to high temperature and drought stress during booting and anthesis

Factorial pot experiments were conducted to compare the responses of GA-sensitive and GA-insensitive reduced height (Rht) alleles in wheat for susceptibility to heat and drought stress during booting and anthesis. Grain set (grains/spikelet) of near isogenic lines (NILs) was assessed following three day transfers to controlled environments imposing day temperatures (t) from 20 to 40°C. Transfers were during booting and/or anthesis and pots maintained at field capacity (FC) or had water withheld. Logistic responses (y = c/1+e-b(t -m)) described declining grain set with increasing t, and t5 was that fitted to give a 5% reduction in grain set. Averaged over NIL, t5 for anthesis at FC was 31.7±0.47°C (S.E.M, 26 d.f.). Drought at anthesis reduced t5 by <2°C. Maintaining FC at booting conferred considerable resistance to high temperatures (t5=33.9°C) but booting was particularly heat susceptible without water (t5 =26.5°C). In one background (cv. Mercia), for NILs varying at the Rht-D1 locus, there was progressive reduction in t5 with dwarfing and reduced gibberellic acid (GA) sensitivity (Rht-D1a, tall, 32.7±0.72; Rht-D1b, semi-dwarf, 29.5±0.85; Rht-D1c, severe dwarf, 24.2±0.72). This trend was not evident for the Rht-B1 locus, or for Rht-D1b in an alternative background (Maris Widgeon). The GA-sensitive severe dwarf Rht12 was more heat tolerant (t5=29.4±0.72) than the similarly statured GA-insensitive Rht-D1c. The GA-sensitive, semi-dwarfing Rht8 conferred greater drought tolerance in one experiment. Despite the effects of Rht-D1 alleles in Mercia on stress tolerance, the inconsistency of the effects over background and locus led to the conclusion that semi-dwarfing with GA-insensitivity did not necessarily increase sensitivity to stress at booting and flowering. In comparison to effects of semi-dwarfing alleles, responses to heat stress are much more dramatically affected by water availability and the precise growth stage at which the stress is experienced by the plants.

Investigation of milk proteins binding to the oral mucosa

High protein dairy beverages are considered to be mouth drying. The drying sensation may be due to the product protein content; however the mechanism of this mouth drying is uncertain. This study investigated the potential adhesion of milk proteins to porcine oral mucosal tissues and their resistance to wash out with simulated saliva was monitored using fluorescence microscopy. Cadein was found to be more adhesive to porcine mucosa then lactogloubulin. Some investigation into the reason for this difference in mucoadhesion was conducted by thiol-content analysis, rheology and zeta-potential measurements. The higher viscosity of casein solution and smaller zeta-potential is believed to be responsible for its better retention on mucosal surfaces. These findings suggest that casein and whey protein are both capable of binding and eliciting mouth drying in high protein dairy beverages.

Detection and molecular characterisation of Pyrenopeziza brassicae isolates resistant to methyl benzimidazole carbamates

BACKGROUND Methyl benzimidazole carbamate (MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in β-tubulin, will enable resistance diagnostics and thereby inform resistance management strategies. RESULTS P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. The MBC-target encoding gene β-tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with β-tubulin amino acid substitutions L240F and E198A. The highest level of MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. The substitutions E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. The frequencies of L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. There were no differences in the frequencies of these alleles between P. brassicae populations sampled from different locations or after fungicide treatment regimes. CONCLUSIONS The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations.

Inflammatory modulation of exercise salience: using hormesis to return to a healthy lifestyle

Most of the human population in the western world has access to unlimited calories and leads an increasingly sedentary lifestyle. The propensity to undertake voluntary exercise or indulge in spontaneous physical exercise, which might be termed "exercise salience", is drawing increased scientific attention. Despite its genetic aspects, this complex behaviour is clearly modulated by the environment and influenced by physiological states. Inflammation is often overlooked as one of these conditions even though it is known to induce a state of reduced mobility. Chronic subclinical inflammation is associated with the metabolic syndrome; a largely lifestyle-induced disease which can lead to decreased exercise salience. The result is a vicious cycle that increases oxidative stress and reduces metabolic flexibility and perpetuates the disease state. In contrast, hormetic stimuli can induce an anti-inflammatory phenotype, thereby enhancing exercise salience, leading to greater biological fitness and improved functional longevity. One general consequence of hormesis is upregulation of mitochondrial function and resistance to oxidative stress. Examples of hormetic factors include calorie restriction, extreme environmental temperatures, physical activity and polyphenols. The hormetic modulation of inflammation, and thus, exercise salience, may help to explain the highly heterogeneous expression of voluntary exercise behaviour and therefore body composition phenotypes of humans living in similar obesogenic environments.

Lifestyle-induced metabolic inflexibility and accelerated ageing syndrome: insulin resistance, friend or foe?

The metabolic syndrome may have its origins in thriftiness, insulin resistance and one of the most ancient of all signalling systems, redox. Thriftiness results from an evolutionarily-driven propensity to minimise energy expenditure. This has to be balanced with the need to resist the oxidative stress from cellular signalling and pathogen resistance, giving rise to something we call 'redox-thriftiness'. This is based on the notion that mitochondria may be able to both amplify membrane-derived redox growth signals as well as negatively regulate them, resulting in an increased ATP/ROS ratio. We suggest that 'redox-thriftiness' leads to insulin resistance, which has the effect of both protecting the individual cell from excessive growth/inflammatory stress, while ensuring energy is channelled to the brain, the immune system, and for storage. We also suggest that fine tuning of redox-thriftiness is achieved by hormetic (mild stress) signals that stimulate mitochondrial biogenesis and resistance to oxidative stress, which improves metabolic flexibility. However, in a non-hormetic environment with excessive calories, the protective nature of this system may lead to escalating insulin resistance and rising oxidative stress due to metabolic inflexibility and mitochondrial overload. Thus, the mitochondrially-associated resistance to oxidative stress (and metabolic flexibility) may determine insulin resistance. Genetically and environmentally determined mitochondrial function may define a 'tipping point' where protective insulin resistance tips over to inflammatory insulin resistance. Many hormetic factors may induce mild mitochondrial stress and biogenesis, including exercise, fasting, temperature extremes, unsaturated fats, polyphenols, alcohol, and even metformin and statins. Without hormesis, a proposed redox-thriftiness tipping point might lead to a feed forward insulin resistance cycle in the presence of excess calories. We therefore suggest that as oxidative stress determines functional longevity, a rather more descriptive term for the metabolic syndrome is the 'lifestyle-induced metabolic inflexibility and accelerated ageing syndrome'. Ultimately, thriftiness is good for us as long as we have hormetic stimuli; unfortunately, mankind is attempting to remove all hormetic (stressful) stimuli from his environment.

Paralog re-emergence: a novel, historically contingent mechanism in the evolution of antimicrobial resistance

Evolution of resistance to drugs and pesticides poses a serious threat to human health and agricultural production. CYP51 encodes the target site of azole fungicides, widely used clinically and in agriculture. Azole resistance can evolve due to point mutations or overexpression of CYP51, and previous studies have shown that fungicide-resistant alleles have arisen by de novo mutation. Paralogs CYP51A and CYP51B are found in filamentous ascomycetes, but CYP51A has been lost from multiple lineages. Here, we show that in the barley pathogen Rhynchosporium commune, re-emergence of CYP51A constitutes a novel mechanism for the evolution of resistance to azoles. Pyrosequencing analysis of historical barley leaf samples from a unique long-term experiment from 1892 to 2008 indicates that the majority of the R. commune population lacked CYP51A until 1985, after which the frequency of CYP51A rapidly increased. Functional analysis demonstrates that CYP51A retains the same substrate as CYP51B, but with different transcriptional regulation. Phylogenetic analyses show that the origin of CYP51A far predates azole use, and newly sequenced Rhynchosporium genomes show CYP51A persisting in the R. commune lineage rather than being regained by horizontal gene transfer; therefore, CYP51A re-emergence provides an example of adaptation to novel compounds by selection from standing genetic variation.

The current status of anticoagulant resistance in rats and mice in the UK

Introduction: Resistance to anticoagulants in Norway rats (Rattus norvegicus) and house mice (Mus domesticus) has been studied in the UK since the early 1960s. In no other country in the world is our understanding of resistance phenomena so extensive and profound. Almost every aspect of resistance in the key rodent target species has been examined in laboratory and field trials and results obtained by independent researchers have been published. It is the principal purpose of this document to present a short synopsis of this information. More recently, however, the development of genetical techniques has provided a definitive means of detection of resistant genotypes among pest rodent populations. Preliminary information from a number of such surveys will also be presented. Resistance in Norway rats: A total of nine different anticoagulant resistance mutations (single nucleotide polymorphisms or SNPs) are found among Norway rats in the UK. In no other country worldwide are present so many different forms of Norway rat resistance. Among these nine SNPs, five are known to confer on rats that carry them a significant degree of resistance to anticoagulant rodenticides. These mutations are: L128Q, Y139S, L120Q, Y139C and Y139F. The latter three mutations confer, to varying degrees, practical resistance to bromadiolone and difenacoum, the two second-generation anticoagulants in predominant use in the UK. It is the recommendation of RRAG that bromadiolone and difenacoum should not be used against rats carrying the L120Q, Y139C and Y139F mutations because this will promote the spread of resistance and jeopardise the long-term efficacy of anticoagulants. Brodifacoum, flocoumafen and difethialone are effective against these three genotypes but cannot presently be used because of the regulatory restriction that they can only be applied against rats that are living and feeding predominantly indoors. Our understanding of the geographical distribution of Norway rat resistance in incomplete but is rapidly increasing. In particular, the mapping of the focus of L120Q Norway rat resistance in central-southern England by DNA sequencing is well advanced. We now know that rats carrying this resistance mutation are present across a large part of the counties of Hampshire, Berkshire and Wiltshire, and the resistance spreads into Avon, Oxfordshire and Surrey. It is also found, perhaps as outlier foci, in south-west Scotland and East Sussex. L120Q is currently the most severe form of anticoagulant resistance found in Norway rats and is prevalent over a considerable part of central-southern England. A second form of advanced Norway rat resistance is conferred by the Y139C mutation. This is noteworthy because it occurs in at least four different foci that are widely geographically dispersed, namely in Dumfries and Galloway, Gloucestershire, Yorkshire and Norfolk. Once again, bromadiolone and difenacoum are not recommended for use against rats carrying this genotype and a concern of RRAG is that continued applications of resisted active substances may result in Y139C becoming more or less ubiquitous across much of the UK. Another type of advanced resistance, the Y139F mutation, is present in Kent and Sussex. This means that Norway rats, carrying some degree of resistance to bromadiolone and difenacoum, are now found from the south coast of Kent, west into the city of Bristol, to Yorkshire in the north-east and to the south-west of Scotland. This difficult situation can only deteriorate further where these three genotypes exist and resisted anticoagulants are predominantly used against them. Resistance in house mice: House mouse is not so well understood but the presence in the UK of two resistant genotypes, L128S and Y139C, is confirmed. House mice are naturally tolerant to anticoagulants and such is the nature of this tolerance, and the presence of genetical resistance, that house mice resistant to the first-generation anticoagulants are considered to be widespread in the UK. Consequently, baits containing warfarin, sodium warfarin, chlorophacinone and coumatetralyl are not approved for use against mice. This regulatory position is endorsed by RRAG. Baits containing brodifacoum, flocoumafen and difethialone are effective against house mice and may be applied in practice because house mouse infestations are predominantly indoors. There are some reports of resistance among mice in some areas to the second-generation anticoagulant bromadiolone, while difenacoum remains largely efficacious. Alternatives to anticoagulants: The use of habitat manipulation, that is the removal of harbourage, denial of the availability of food and the prevention of ingress to structures, is an essential component of sustainable rodent pest management. All are of importance in the management of resistant rodents and have the advantage of not selecting for resistant genotypes. The use of these techniques may be particularly valuable in preventing the build-up of rat infestations. However, none can be used to remove any sizeable extant rat infestation and for practical reasons their use against house mice is problematic. Few alternative chemical interventions are available in the European Union because of the removal from the market of zinc phosphide, calciferol and bromethalin. Our virtual complete reliance on the use of anticoagulants for the chemical control of rodents in the UK, and more widely in the EU, calls for improved schemes for resistance management. Of course, these might involve the use of alternatives to anticoagulant rodenticides. Also important is an increasing knowledge of the distribution of resistance mutations in rats and mice and the use of only fully effective anticoagulants against them.

Tannin containing legumes as a model for nutraceuticals against digestive parasites in livestock

Parasitic infections with gastrointestinal nematodes (GINs) still represent a worldwide major pathological threat associated with the outdoor production of various livestock species. Because of the widespread resistance to synthetic chemical anthelmintics, there is a strong impetus to explore novel approaches for a more integrated management of the infections. The use of nutraceuticals in the control of GINs is one of the alternatives which has been widely studied for since 20 years. The objectives of this review are: i) to define and illustrate the concept of ‘nutraceutical’ in the context of veterinary parasitology based on data obtained on the most studied GIN models in small ruminants, the tannin-containing legumes (Fabaceae); ii) to illustrate how the ‘nutraceutical concept’ could be expanded to other plants, other livestock production systems and other GI parasitic diseases, and iii) to explain how this concept is opening up new research fields for better understanding the interactions between the host, the digestive parasites and the environment.

Contrasting responses to drought stress in herbaceous perennial legumes

Background and aims Medicago sativa L. is widely grown in southern Australia, but is poorly adapted to dry, hot summers. This study aimed to identify perennial herbaceous legumes with greater resistance to drought stress and explore their adaptive strategies. Methods Ten herbaceous perennial legume species/accessions were grown in deep pots in a sandy, low-phosphorus field soil in a glasshouse. Drought stress was imposed by ceasing to water. A companion M. sativa plant in each pot minimised differences in leaf area and water consumption among species. Plants were harvested when stomatal conductance of stressed plants decreased to around 10% of well watered plants. Results A range of responses to drought stress were identified, including: reduced shoot growth; leaf curling; thicker pubescence on leaves and stems; an increased root:shoot ratio; an increase, decrease or no change in root distribution with depth; reductions in specific leaf area or leaf water potential; and osmotic adjustment. The suite of changes differed substantially among species and, less so, among accessions. Conclusions The inter- and intra-specific variability of responses to drought-stress in the plants examined suggests a wide range of strategies are available in perennial legumes to cope with drying conditions, and these could be harnessed in breeding/selection programs.