Antimicrobial resistance in Escherichia coli strains isolated from Swiss weaned pigs and sows

Based on Directive (EC) No 99/2003, monitoring programs on the development of antimicrobial resistance in bacteria from livestock are implemented in many European countries. The aim of the present study was (i) to establish comprehensive baseline data on the antimicrobial resistance situation in Escherichia coli isolates obtained from healthy pigs (pooled fecal samples) originating from 60 Swiss pig-breeding farms, and (ii) to analyze differences in the resistance frequency between Escherichia coli isolates from weaned pigs and sows. Susceptibility testing (disc diffusion method) was performed on 429 isolates from weaned pigs and 431 isolates from sows. Overall, 17.7% of the isolates from weaned pigs and 22.5% of the Escherichia coli isolates from sows were susceptible to all antibiotics tested. Low resistance prevalence was found for amoxicillin, amoxicillin/clavulanic acid, ampicillin, cefquinome, ciprofloxacin, colistin, florfenicol, and gentamicin. The most frequently found resistances were against streptomycin (60.6% of the isolates from weaners and 64.3% of the isolates from sows), sulfonamide (51.5% and 26.9%), tetracycline (35.2% and 22.0%), and trimethoprim (27.5% and 11.1%). With exception of colistin, most resistances were found for those antibiotics commonly used on the farms. Except for ciprofloxacin and streptomycin, isolates from weaned pigs showed higher resistance prevalence than those from sows. This difference was significant for cefquinome, florfenicol, sulfonamide, tetracycline, and trimethoprim (p<0.05).

Harmonised monitoring of antimicrobial resistance in Salmonella and Campylobacter isolates from food animals in the European Union

Many Member States of the European Union (EU) currently monitor antimicrobial resistance in zoonotic agents, including Salmonella and Campylobacter. According to Directive 2003/99/EC, Member States shall ensure that the monitoring provides comparable data on the occurrence of antimicrobial resistance. The European Commission asked the European Food Safety Authority to prepare detailed specifications for harmonised schemes for monitoring antimicrobial resistance. The objective of these specifications is to lay down provisions for a monitoring and reporting scheme for Salmonella in fowl (Gallus gallus), turkeys and pigs, and for Campylobacter jejuni and Campylobacter coli in broiler chickens. The current specifications are considered to be a first step towards a gradual implementation of comprehensive antimicrobial resistance monitoring at the EU level. These specifications propose to test a common set of antimicrobial agents against available cut-off values and a specified concentration range to determine the susceptibility of Salmonella and Campylobacter. Using isolates collected through programmes in which the sampling frame covers all epidemiological units of the national production, the target number of Salmonella isolates to be included in the antimicrobial resistance monitoring per Member State per year is 170 for each study population (i.e., laying hens, broilers, turkeys and slaughter pigs). The target number of Campylobacter isolates to be included in the antimicrobial resistance monitoring per Member State per year is 170 for each study population (i.e., broilers). The results of the antimicrobial resistance monitoring are assessed and reported in the yearly national report on trends and sources of zoonoses, zoonotic agents and antimicrobial resistance.

Non-phenotypic tests to detect and characterize antibiotic resistance mechanisms in Enterobacteriaceae

In the past 2 decades, we have observed a rapid increase of infections due to multidrug-resistant Enterobacteriaceae. Regrettably, these isolates possess genes encoding for extended-spectrum β-lactamases (e.g., blaCTX-M, blaTEM, blaSHV) or plasmid-mediated AmpCs (e.g., blaCMY) that confer resistance to last-generation cephalosporins. Furthermore, other resistance traits against quinolones (e.g., mutations in gyrA and parC, qnr elements) and aminoglycosides (e.g., aminoglycosides modifying enzymes and 16S rRNA methylases) are also frequently co-associated. Even more concerning is the rapid increase of Enterobacteriaceae carrying genes conferring resistance to carbapenems (e.g., blaKPC, blaNDM). Therefore, the spread of these pathogens puts in peril our antibiotic options. Unfortunately, standard microbiological procedures require several days to isolate the responsible pathogen and to provide correct antimicrobial susceptibility test results. This delay impacts the rapid implementation of adequate antimicrobial treatment and infection control countermeasures. Thus, there is emerging interest in the early and more sensitive detection of resistance mechanisms. Modern non-phenotypic tests are promising in this respect, and hence, can influence both clinical outcome and healthcare costs. In this review, we present a summary of the most advanced methods (e.g., next-generation DNA sequencing, multiplex PCRs, real-time PCRs, microarrays, MALDI-TOF MS, and PCR/ESI MS) presently available for the rapid detection of antibiotic resistance genes in Enterobacteriaceae. Taking into account speed, manageability, accuracy, versatility, and costs, the possible settings of application (research, clinic, and epidemiology) of these methods and their superiority against standard phenotypic methods are discussed.

Thiazolides, a Novel Class of Anti-Infective Drugs, Effective Against Viruses, Bacteria, Intracellular and Extracellular Protozoan Parasites and Proliferating Mammalian Cells

The thiazolide nitazoxanide (2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide; NTZ) is composed of a nitrothiazole- ring and a salicylic acid moiety, which are linked together through an amide bond. NTZ exhibits a broad spectrum of activities against a wide range of helminths, protozoa, enteric bacteria, and viruses infecting animals and humans. Since the first synthesis of the drug, a number of derivatives of NTZ have been produced, which are collectively named thiazolides. These are modified versions of NTZ, which include the replacement of the nitro group with bromo-, chloro-, or other functional groups, and the differential positioning of methyl- and methoxy-groups on the salicylate ring. The presence of a nitro group seems to be the prerequisite for activities against anaerobic or microaerophilic parasites and bacteria. Intracellular parasites and viruses, however, are susceptible to non-nitro-thiazolides with equal or higher effectiveness. Moreover, nitro- and bromo-thiazolides are effective against proliferating mammalian cells. Biochemical and genetic approaches have allowed the identification of respective targets and the molecular basis of resistance formation. Collectively, these studies strongly suggest that NTZ and other thiazolides exhibit multiple mechanisms of action. In microaerophilic bacteria and parasites, the reduction of the nitro group into a toxic intermediate turns out to be the key factor. In proliferating mammalian cells, however, bromo- and nitro-thiazolides trigger apoptosis, which may also explain their activities against intracellular pathogens. The mode of action against helminths may be similar to mammalian cells but has still not been elucidated.

P2X7 receptors mediate resistance to toxin-induced cell lysis

In the majority of cells, the integrity of the plasmalemma is recurrently compromised by mechanical or chemical stress. Serum complement or bacterial pore-forming toxins can perforate the plasma membrane provoking uncontrolled Ca(2+) influx, loss of cytoplasmic constituents and cell lysis. Plasmalemmal blebbing has previously been shown to protect cells against bacterial pore-forming toxins. The activation of the P2X7 receptor (P2X7R), an ATP-gated trimeric membrane cation channel, triggers Ca(2+) influx and induces blebbing. We have investigated the role of the P2X7R as a regulator of plasmalemmal protection after toxin-induced membrane perforation caused by bacterial streptolysin O (SLO). Our results show that the expression and activation of the P2X7R furnishes cells with an increased chance of surviving attacks by SLO. This protective effect can be demonstrated not only in human embryonic kidney 293 (HEK) cells transfected with the P2X7R, but also in human mast cells (HMC-1), which express the receptor endogenously. In addition, this effect is abolished by treatment with blebbistatin or A-438079, a selective P2X7R antagonist. Thus blebbing, which is elicited by the ATP-mediated, paracrine activation of the P2X7R, is part of a cellular non-immune defense mechanism. It pre-empts plasmalemmal damage and promotes cellular survival. This mechanism is of considerable importance for cells of the immune system which carry the P2X7R and which are specifically exposed to toxin attacks.

VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart

Angiogenic growth factors have recently been linked to tissue metabolism. We have used genetic gain- and loss-of function models to elucidate the effects and mechanisms of action of vascular endothelial growth factor-B (VEGF-B) in the heart. A cardiomyocyte-specific VEGF-B transgene induced an expanded coronary arterial tree and reprogramming of cardiomyocyte metabolism. This was associated with protection against myocardial infarction and preservation of mitochondrial complex I function upon ischemia-reperfusion. VEGF-B increased VEGF signals via VEGF receptor-2 to activate Erk1/2, which resulted in vascular growth. Akt and mTORC1 pathways were upregulated and AMPK downregulated, readjusting cardiomyocyte metabolic pathways to favor glucose oxidation and macromolecular biosynthesis. However, contrasting with a previous theory, there was no difference in fatty acid uptake by the heart between the VEGF-B transgenic, gene-targeted or wildtype rats. Importantly, we also show that VEGF-B expression is reduced in human heart disease. Our data indicate that VEGF-B could be used to increase the coronary vasculature and to reprogram myocardial metabolism to improve cardiac function in ischemic heart disease.

In vitro activity of clinically implemented β-lactams against Aerococcus urinae: presence of non-susceptible isolates in Switzerland.

We analyzed the in vitro susceptibility to several ?-lactams and vancomycin of 80 Aerococcus urinae isolates col- lected during 2011-2012 in Switzerland. MICs were determined by Etest (bioMérieux) on Müller-Hinton agar with 5% sheep blood and interpreted according to the CLSI and EUCAST criteria set for viridans streptococci. MIC50/90 for penicillin, amoxicillin, ceftriaxone and vancomycin were 0.016/0.064 mg/l, 0.032/0.064 mg/l, 0.125/0.5 mg/l and 0.38/0.5 mg/l, respectively. Three (3.8%) isolates were resistant to ceftriaxone regardless of the criteria used (MICs ?2 mg/l); one of them was also non-susceptible to penicillin (MIC of 0.25 mg/l) according to CLSI. ?-lactam resis- tance in A. urinae is a concern and suggests that more studies are needed to determine the molecular mechanisms of such resistance.

In vitro Activity of Fosfomycin Alone and in Combination with Ceftriaxone or Azithromycin Against Clinical Neisseria gonorrhoeae Isolates.

New therapeutic strategies are needed to combat the emergence of infections due to multidrug-resistant Neisseria gonorrhoeae (Ng). In this study, fosfomycin (FOS) was tested against 89 Ng using the Etest method and showing MIC50/90s of only 8/16 μg/ml (range ≤ 1-32 μg/ml). FOS in combination with ceftriaxone (CRO) or azithromycin (AZT) was then evaluated using the checkerboard method for eight strains, including F89 (CRO-resistant) and AZT-HLR (high-level AZT-resistant). All combinations including FOS gave indifferent effects (fractional inhibitory concentration [FIC] index values between 1.2-2.3 for FOS plus CRO and between 1.8-3.2 for FOS plus AZT). Time-kill experiments for FOS, CRO, AZT and their combinations (at concentrations of 0.5×, 1×, 2× and 4× MIC) were performed against ATCC 49226, one Ng of NG-MAST ST1407, F89 and AZT-HLR. For all strains, at 24 hours results indicated that: i) FOS was bactericidal at 2× MIC concentrations but after >24 hours there was re-growth of bacteria; ii) CRO was bactericidal at 0.5× MIC; iii) AZT was bactericidal at 4× MIC; iv) CRO plus AZT was less bactericidal than CRO alone; v) FOS plus AZT was bactericidal at 2× MIC; vi) CRO plus AZT and FOS plus CRO were both bactericidal at 0.5× MIC, but the latter had more rapid effects. FOS is appealing for the management of Ng infections because of its single and oral formulation. However, our results suggest its use in combination with CRO. This strategy could, after appropriate clinical trials, be implemented for the treatment of infections due to isolates possessing resistance to CRO and/or AZT.

Assessing the paradox between transmitted and acquired HIV-1 drug resistance in the Swiss HIV Cohort Study from 1998 to 2012.

BACKGROUND  Transmitted HIV-1 drug-resistance mutations(TDR) are transmitted from treatment-failing or treatment-naïve patients. Although prevalence of drug-resistance in treatment-failing patients has declined in developed countries, TDR prevalence has not. Mechanisms causing this paradox are poorly explored. METHODS  We included recently-infected, treatment-naïve patients with genotypic-resistance-tests performed ≤1year post-infection and <2013. Potential risk factors for TDR were analyzed using logistic regression. Association of TDR prevalences with population viral load(PVL) from treatment-patients during 1997-2011 was estimated with Poisson regression for all TDR and individually for most frequent resistance-mutations against each drug class(M184V/L90M/K103N). RESULTS  We included 2421 recently-infected, treatment-naïve patients and 5399 treatment-failing patients. TDR prevalence fluctuated considerably over time. Two opposing developments could explain these fluctuations: generally continuous increases in TDR(Odds Ratio[OR]=1.13,p=0.010), punctuated by sharp decreases when new drug-classes were introduced. Overall, TDR prevalence increased with decreasing PVL(Rate Ratio[RR]=0.91/1000Log10-PVL,p=0.033). Additionally, we observed that the transmitted high-fitness-cost mutation M184V was positively associated with PVL of treatment-failing patients carrying M184V(RR=1.50/100Log10-PVL,p<0.001). Such association was absent and negative for K103N(RR-K103N=1.00/100Log10-PVL,p=0.99) and L90M(RR-L90M=0.75/100Log10-PVL,p=0.022), respectively. CONCLUSIONS  Transmission of antiretroviral drug-resistance is temporarily reduced by the introduction of new drug classes and driven by treatment-failing and treatment-naïve patients. These findings suggest a continuous need for new drugs, early detection/treatment of HIV-1-infection.

A copper-induced quinone degradation pathway provides protection against combined copper/quinone stress in Lactococcus lactis IL1403.

Quinones are ubiquitous in the environment. They occur naturally but are also in widespread use in human and industrial activities. Quinones alone are relatively benign to bacteria, but in combination with copper, they become toxic by a mechanism that leads to intracellular thiol depletion. Here, it was shown that the yahCD-yaiAB operon of Lactococcus lactis IL1403 provides resistance to combined copper/quinone stress. The operon is under the control of CopR, which also regulates expression of the copRZA copper resistance operon as well as other L. lactis genes. Expression of the yahCD-yaiAB operon is induced by copper but not by quinones. Two of the proteins encoded by the operon appear to play key roles in alleviating quinone/copper stress: YaiB is a flavoprotein that converts p-benzoquinones to less toxic hydroquinones, using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as reductant; YaiA is a hydroquinone dioxygenase that converts hydroquinone putatively to 4-hydroxymuconic semialdehyde in an oxygen-consuming reaction. Hydroquinone and methylhydroquinone are both substrates of YaiA. Deletion of yaiB causes increased sensitivity of L. lactis to quinones and complete growth arrest under combined quinone and copper stress. Copper induction of the yahCD-yaiAB operon offers protection to copper/quinone toxicity and could provide a growth advantage to L. lactis in some environments.

The maize lipoxygenase, ZmLOX10 , mediates green leaf volatile, jasmonate and herbivore-induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, majorregulatory genes and the signals that modulate these defense metabolites are vastly understudied, especiallyin important agro-economic monocot species. Here we show that products and signals derived from a singleZea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbiv-ory. We provide genetic evidence that two 13-LOXs, ZmLOX10 and ZmLOX8, specialize in providing substratefor the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the spe-cialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indi-cating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression ofJA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10-derived signaling isrequired for LOX8-mediated JA. The possible role of GLVs in JA signaling is supported by their ability to par-tially restore wound-induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produceGLVs and JA led to dramatic reductions in herbivore-induced plant volatiles (HIPVs) and attractiveness toparasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic linkto the diurnal regulation of GLVs and HIPVs. GLV-, JA- and HIPV-deficient lox10 mutants display compro-mised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence thatLOX10-dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene toagro-ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.

Volatiles produced by soil-borne endophytic bacteria increase plant pathogen resistance and affect tritrophic interactions

Volatile organic compounds (VOCs) released by soil microorganisms influence plant growth and pathogen resistance. Yet, very little is known about their influence on herbivores and higher trophic levels. We studied the origin and role of a major bacterial VOC, 2,3-butanediol (2,3-BD), on plant growth, pathogen and herbivore resistance, and the attraction of natural enemies in maize. One of the major contributors to 2,3-BD in the headspace of soil-grown maize seedlings was identified as Enterobacter aerogenes, an endophytic bacterium that colonizes the plants. The production of 2,3-BD by E. aerogenes rendered maize plants more resistant against the Northern corn leaf blight fungus Setosphaeria turcica. On the contrary, E. aerogenes-inoculated plants were less resistant against the caterpillar Spodoptera littoralis. The effect of 2,3-BD on the attraction of the parasitoid Cotesia marginiventris was more variable: 2,3-BD application to the headspace of the plants had no effect on the parasitoids, but application to the soil increased parasitoid attraction. Furthermore, inoculation of seeds with E. aerogenes decreased plant attractiveness, whereas inoculation of soil with a total extract of soil microbes increased parasitoid attraction, suggesting that the effect of 2,3-BD on the parasitoid is indirect and depends on the composition of the microbial community.

Induced Immunity Against Belowground Insect Herbivores- Activation of Defenses in the Absence of a Jasmonate Burst

Roots respond dynamically to belowground herbivore attack. Yet, little is known about the mechanisms and ecological consequences of these responses. Do roots behave the same way as leaves, or do the paradigms derived from aboveground research need to be rewritten? This is the central question that we tackle in this article. To this end, we review the current literature on induced root defenses and present a number of experiments on the interaction between the root herbivore Diabrotica virgifera and its natural host, maize. Currently, the literature provides no clear evidence that plants can recognize root herbivores specifically. In maize, mild mechanical damage is sufficient to trigger a root volatile response comparable to D. virgifera induction. Interestingly, the jasmonate (JA) burst, a highly conserved signaling event following leaf attack, is consistently attenuated in the roots across plant species, from wild tobacco to Arabidopsis. In accordance, we found only a weak JA response in D. virgifera attacked maize roots. Despite this reduction in JA-signaling, roots of many plants start producing a distinct suite of secondary metabolites upon attack and reconfigure their primary metabolism. We, therefore, postulate the existence of additional, unknown signals that govern induced root responses in the absence of a jasmonate burst. Surprisingly, despite the high phenotypic plasticity of plant roots, evidence for herbivore-induced resistance below ground is virtually absent from the literature. We propose that other defensive mechanisms, including resource reallocation and compensatory growth, may be more important to improve plant immunity below ground.

Biodiversity increases the resistance of ecosystem productivity to climate extremes

It remains unclear whether biodiversity buffers ecosystems against climate extremes, which are becoming increasingly frequent worldwide. Early results suggested that the ecosystem productivity of diverse grassland plant communities was more resistant, changing less during drought, and more resilient, recovering more quickly after drought, than that of depauperate communities. However, subsequent experimental tests produced mixed results. Here we use data from 46 experiments that manipulated grassland plant diversity to test whether biodiversity provides resistance during and resilience after climate events. We show that biodiversity increased ecosystem resistance for a broad range of climate events, including wet or dry, moderate or extreme, and brief or prolonged events. Across all studies and climate events, the productivity of low-diversity communities with one or two species changed by approximately 50% during climate events, whereas that of high-diversity communities with 16–32 species was more resistant, changing by only approximately 25%. By a year after each climate event, ecosystem productivity had often fully recovered, or overshot, normal levels of productivity in both high- and low-diversity communities, leading to no detectable dependence of ecosystem resilience on biodiversity. Our results suggest that biodiversity mainly stabilizes ecosystem productivity, and productivity-dependent ecosystem services, by increasing resistance to climate events. Anthropogenic environmental changes that drive biodiversity loss thus seem likely to decrease ecosystem stability, and restoration of biodiversity to increase it, mainly by changing the resistance of ecosystem productivity to climate events.

Silencing OsHI-LOX makes rice more susceptible to chewing herbivores, but enhances resistance to a phloem feeder

The jasmonic acid (JA) pathway plays a central role in plant defense responses against insects. Some phloem-feeding insects also induce the salicylic acid (SA) pathway, thereby suppressing the plant’s JA response. These phenomena have been well studied in dicotyledonous plants, but little is known about them in monocotyledons. We cloned a chloroplast-localized type 2 13-lipoxygenase gene of rice, OsHI-LOX, whose transcripts were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis and the rice brown planthopper (BPH) Niaparvata lugens, as well as by mechanical wounding and treatment with JA. Antisense expression of OsHI-LOX (as-lox) reduced SSB- or BPH-induced JA and trypsin protease inhibitor (TrypPI) levels, improved the larval performance of SBB as well as that of the rice leaf folder (LF) Cnaphalocrocis medinalis, and increased the damage caused by SSB and LF larvae. In contrast, BPH, a phloem-feeding herbivore, showed a preference for settling and ovipositing on WT plants, on which they consumed more and survived better than on as-lox plants. The enhanced resistance of as-lox plants to BPH infestation correlated with higher levels of BPH-induced H2O2 and SA, as well as with increased hypersensitive response-like cell death. These results imply that OsHI-LOX is involved in herbivore-induced JA biosynthesis, and plays contrasting roles in controlling rice resistance to chewing and phloem-feeding herbivores. The observation that suppression of JA activity results in increased resistance to an insect indicates that revision of the generalized plant defense models in monocotyledons is required, and may help develop novel strategies to protect rice against insect pests.