SummaryPlants both produce and utilize carbohydrates and have developed mechanisms to regulate their sugar status and co-ordinate carbohydrate partitioning. High sugar levels result in a feedback inhibition of photosynthesis and an induction of storage processes. We used a genetic approach to isolate components of the signalling pathway regulating the induction of starch biosynthesis. The regulatory sequences of the sugar inducible ADP-glucose pyrophosphorylase subunit ApL3 were fused to a negative selection marker. Of the four impaired sucrose induction (isi) mutants described here, two (isi1 and isi2) were speci®c to this screen. The other two mutants (isi3 and isi4) showed additional phenotypes associated with sugarsensing screens that select for seedling establishment on high-sugar media. The isi3 and isi4 mutants were found to be involved in the abscisic acid signalling pathway. isi3 is allelic to abscisic acid insensitive4 (abi4), a gene encoding an Apetala2-type transcription factor; isi4 was found to be allelic to glucose insensitive1 (gin1) previously reported to reveal cross-talk between ethylene and glucose signalling. Here we present an alternative interpretation of gin1 as an allele of the ABA-de®cient mutant aba2. Expression analysis showed that ABA is unable to induce ApL3 gene expression by itself, but greatly enhances ApL3 induction by sugar. Our data suggest a major role for ABA in relation to sugar-signalling pathways, in that it enhances the ability of tissues to respond to subsequent sugar signals.

Brachyspira hyodysenteriae is the aetiological agent of swine dysentery, a globally distributed disease that causes profound economic loss, impedes the free trade and movement of animals, and has significant impact on pig health. Infection is generally treated with antibiotics of which pleuromutilins, such as tiamulin, are widely used for this purpose, but reports of resistance worldwide threaten continued effective control. In Brachyspira hyodysenteriae pleuromutilin resistance has been associated with mutations in chromosomal genes encoding ribosome-associated functions, however the dynamics of resistance acquisition are poorly understood, compromising stewardship efforts to preserve pleuromutilin effectiveness. In this study we undertook whole genome sequencing (WGS) and phenotypic susceptibility testing of 34 UK field isolates and 3 control strains to investigate pleuromutilin resistance in Brachyspira hyodysenteriae. Genome-wide association studies identified a new pleuromutilin resistance gene, tva(A) (tiamulin valnemulin antibiotic resistance), encoding a predicted ABC-F transporter. In vitro culture of isolates in the presence of inhibitory or sub-inhibitory concentrations of tiamulin showed that tva(A) confers reduced pleuromutilin susceptibility that does not lead to clinical resistance but facilitates the development of higher-level resistance via mutations in genes encoding ribosome-associated functions. Genome sequencing of antibiotic-exposed isolates identified both new and previously described mutations in chromosomal genes associated with reduced pleuromutilin susceptibility, including the 23S rRNA gene and rplC, which encodes the L3 ribosomal protein. Interesting three antibiotic-exposed isolates harboured mutations in fusA, encoding Elongation Factor G, a gene not previously associated with pleuromutilin resistance. A longitudinal molecular epidemiological examination of two episodes of swine dysentery at the same farm indicated that tva(A) contributed to development of tiamulin resistance in vivo in a manner consistent with that seen experimentally in vitro. The in vitro studies further showed that tva(A) broadened the mutant selection window and raised the mutant prevention concentration above reported in vivo antibiotic concentrations obtained when administered at certain doses. We show how the identification and characterisation of tva(A), a new marker for pleuromutilin resistance, provides evidence to inform treatment regimes and reduce the development of resistance to this class of highly important antimicrobial agents.

T he extensive use of antimicrobials has selected resistance in many bacterial species, and this has become a major public health issue worldwide (1). The problem of multidrug resistance is currently most acute in Gram-negative bacteria, where treatment options can be severely limited, and there are few promising new antibiotics with activity against Gram-negative bacteria under advanced development (1). Clonal spread of resistant strains and horizontal transfer of resistance genes both contribute to the rising global prevalence of multiresistant Gram-negative bacteria, with horizontal transfer enabling the acquisition of multidrug resistance by previously susceptible bacteria (2).Most methods currently employed by clinical diagnostic laboratories, e.g., the Vitek (bioMérieux, Marcy l'Etoile, France) and MicroScan (Siemens, Camberley, United Kingdom) (3) systems, provide insight only into the resistance phenotypes, require susceptibility testing following bacterial culture, and can take from approximately 9 to 20 h to obtain results. Molecular methods are faster but present their own challenges. In particular, conventional or real-time PCR, single-gene sequencing, or in situ hybridization usually detects only a few resistance genes simultaneously. Microarrays, however, offer the potential for simultaneous detection of large numbers of resistance genes, allowing prediction of an isolate's repertoire of resistances to multiple antibiotic classes (4-7).We previously developed a microarray that detected 51 resistance genes in Escherichia coli and Salmonella and facilitated epidemiological studies (4, 8). Its coverage included genes that conferred acquired resistance to a range of antimicrobials of clinical importance as well as two integrase genes associated with class 1 and class 2 integrons and not just those genes encoding ␤-lactamases (6). This work sought to extend the microarray to make it relevant to the broader range of Gram-negative bacterial genera commonly encountered in clinical diagnostic or reference laboratories and to include in its coverage newly critical gene groups, including genes encoding carbapenemases. MATERIALS AND METHODS Bacterial strains used and determination of antibiotic susceptibilities.A panel of 132 bacterial isolates from our laboratory collections and clinical isolates representing a diverse range of Enterobacteriaceae and nonfermenter genera, including Pseudomonas and Acinetobacter, was assembled. The antimicrobial susceptibilities (MICs) of these isolates to 19 antibiotics (tobramycin, amikacin, gentamicin, streptomycin, ampicillin, amoxicillin-clavulanic acid [co-amoxiclav], aztreonam, cefotaxime, ceftazidime, cefpirome, cefoxitin, piperacillin, imipenem, meropenem, chloramphenicol, ciprofloxacin, sulfonamide, tetracycline, and trimethoprim) were determined using the British Society for Antimicrobial Chemotherapy (BSAC) agar dilution methodology or disk diffusion (9). Susceptibility was defined using BSAC/European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinic...

The chicken gastrointestinal tract is richly populated by commensal bacteria that fulfill various beneficial roles for the host, including helping to resist colonization by pathogens. It can also facilitate the conjugative transfer of multidrug resistance (MDR) plasmids between commensal and pathogenic bacteria which is a significant public and animal health concern as it may affect our ability to treat bacterial infections. We used an in vitro chemostat system to approximate the chicken cecal microbiota, simulate colonization by an MDR Salmonella pathogen, and examine the dynamics of transfer of its MDR plasmid harboring several genes, including the extended-spectrum beta-lactamase blaCTX-M1. We also evaluated the impact of cefotaxime administration on plasmid transfer and microbial diversity. Bacterial community profiles obtained by culture-independent methods showed that Salmonella inoculation resulted in no significant changes to bacterial community alpha diversity and beta diversity, whereas administration of cefotaxime caused significant alterations to both measures of diversity, which largely recovered. MDR plasmid transfer from Salmonella to commensal Escherichia coli was demonstrated by PCR and whole-genome sequencing of isolates purified from agar plates containing cefotaxime. Transfer occurred to seven E. coli sequence types at high rates, even in the absence of cefotaxime, with resistant strains isolated within 3 days. Our chemostat system provides a good representation of bacterial interactions, including antibiotic resistance transfer in vivo. It can be used as an ethical and relatively inexpensive approach to model dissemination of antibiotic resistance within the gut of any animal or human and refine interventions that mitigate its spread before employing in vivo studies.

The aim of this study was to screen for the presence of antimicrobial resistance genes within the saliva and faecal microbiomes of healthy adult human volunteers from five European countries. Two non-culture based approaches were employed to obviate potential bias associated with difficult to culture members of the microbiota. In a gene target-based approach, a microarray was employed to screen for the presence of over 70 clinically important resistance genes in the saliva and faecal microbiomes. A total of 14 different resistance genes were detected encoding resistances to six antibiotic classes (aminoglycosides, β-lactams, macrolides, sulphonamides, tetracyclines and trimethoprim). The most commonly detected genes were erm(B), bla TEM, and sul2. In a functional-based approach, DNA prepared from pooled saliva samples was cloned into Escherichia coli and screened for expression of resistance to ampicillin or sulphonamide, two of the most common resistances found by array. The functional ampicillin resistance screen recovered genes encoding components of a predicted AcrRAB efflux pump. In the functional sulphonamide resistance screen, folP genes were recovered encoding mutant dihydropteroate synthase, the target of sulphonamide action. The genes recovered from the functional screens were from the chromosomes of commensal species that are opportunistically pathogenic and capable of exchanging DNA with related pathogenic species. Genes identified by microarray were not recovered in the activity-based screen, indicating that these two methods can be complementary in facilitating the identification of a range of resistance mechanisms present within the human microbiome. It also provides further evidence of the diverse reservoir of resistance mechanisms present in bacterial populations in the human gut and saliva. In future the methods described in this study can be used to monitor changes in the resistome in response to antibiotic therapy.