A link between the newly described colistin resistance gene mcr-9 and clinical Enterobacteriaceae isolates carrying blaSHV-12 from horses in Sweden.

OBJECTIVES: The aim of this study was to investigate the occurrence of the newly described transferable colistin resistance gene mcr-9 in extended-spectrum ß-lactamase (ESBL)-producing clinical Enterobacteriaceae isolates from horses in Sweden. METHODS: A total of 56 whole-genome sequenced ESBL-producing Enterobacteriaceae isolates from horses were subjected to in silico detection of antimicrobial resistance genes and identification of plasmid replicons types. The colistin minimum inhibitory concentration (MIC) for mcr-positive isolates was determined by broth microdilution. Relatedness between Enterobacteriaceae carrying mcr genes was determined by multilocus sequence typing (MLST) and core genome MLST. RESULTS: Thirty ESBL-producing Enterobacteriaceae isolates from horses were positive for the colistin resistance gene mcr-9. These isolates included Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca and Citrobacter freundii and belonged to diverse MLST sequence types within each species. Two of the mcr-9-containing isolates originated from the same horse. All mcr-9-positive isolates had colistin MICs below or equal to the EUCAST epidemiological cut-off value of 2 mg/L and were negative for the two potential regulatory genes qseB-like and qseC-like for mcr-9. Except for one isolate carrying only blaTEM-1B, all of the isolates carried blaSHV-12 and blaTEM-1B, and were all considered multidrug-resistant as they harboured genes encoding resistance to aminoglycosides, chloramphenicol, fosfomycin, macrolides, quinolones, sulfonamides, trimethoprim and tetracyclines. Plasmid replicon types IncHI2 and IncHI2A were detected in all mcr-9-positive isolates. CONCLUSION: The occurrence of mcr-9 was common among clinical ESBL-producing Enterobacteriaceae isolates from horses in Sweden and was linked to the ESBL-encoding gene blaSHV-12 and plasmid replicon types IncHI2 and IncHI2A.

Clonal spread of Escherichia coli resistant to cephalosporins and quinolones in the Nordic broiler production.

The intestinal flora of healthy broilers can contain Escherichia coli resistant to extended spectrum cephalosporins (ESC) and fluoroquinolones (FQ), representing a possible public health problem. We investigated the clonal epidemiology of E. coli with reduced susceptibility to ESC or FQ in broilers in three Nordic countries interconnected by a common source of breeding animals. Isolates (n = 319 and n = 132 non-wild type for ESC and FQ, respectively) from Norwegian, Swedish and Icelandic production originated mainly from the intestinal flora of broilers at the age of 20-35 days. Genetic relationships were investigated by ten loci multilocus variable number tandem repeat analyses (MLVA) and representative isolates of inter-Nordic clusters were subjected to multilocus sequence typing (MLST). Antimicrobial susceptibility data based on minimum inhibitory concentrations was compiled. Approximately one third of the ESC non-wild type isolates, including isolates from all three countries, clustered together. These isolates belonged to sequence type (ST) 38 and contained blaCMY-2. The FQ non-wild type isolates were more genetically diverse, but related isolates occurred in more than one country. MLST typing showed clusters belonging to ST10, ST355, ST349, ST665 and ST93. Our study demonstrated inter-Nordic distribution of E. coli ST38 with blaCMY-2, suggesting clonal proliferation as a contributing factor for spread of ESC resistance in the broiler production. The international trade in breeding material may explain introduction of resistant E. coli. The reason for their success and the success of certain clonal lineages in broiler production not exposed to antimicrobial selection pressure is currently unknown.

Evidence of household transfer of ESBL-/pAmpC-producing Enterobacteriaceae between humans and dogs - a pilot study.

BACKGROUND: Extended-spectrum cephalosporin-resistant Enterobacteriaceae (ESCRE) are an increasing healthcare problem in both human and veterinary medicine. The spread of ESCRE is complex with multiple reservoirs and different transmission routes. The aim of this study was to investigate if ESCRE carriage in dogs is more prevalent in households with a known human carrier, compared to households where humans are known to be negative for ESCRE. Identical ESCRE strains in humans and dogs of the same household would suggest a possible spread between humans and dogs. METHODS: Twenty-two dog owners with a positive rectal culture for ESCRE each collected a rectal sample from their dog. In addition, a control group of 29 healthy dog owners with a documented negative rectal culture for ESCRE each sampled their household dog. Samples were cultivated for ESCRE using selective methods. In households where both humans and dogs carried ESCRE, isolates were further analysed for antimicrobial susceptibility by disc diffusion or microdilution and for genotype and genetic relatedness using molecular methods. RESULTS: In 2 of 22 households studied, identical ESCRE strains with respect to bacterial species, antibiogram, genotype, and MLVA type were found in humans and dogs. The ESCRE found in the two households were ESBL-producing E. coli with the resistance gene blaCTX-M-27 and AmpC-producing E. coli with blaCMY-2, blaTEM-1. ESCRE were not found in dogs in the control group. CONCLUSIONS: In households where humans are carrying ESCRE, identical strains were to a limited extent found also in household dogs, indicating a transfer between humans and dogs. In contrast, ESCRE were not found in dogs in households without human carriers.

Transferable genes putatively conferring elevated minimum inhibitory concentrations of narasin in Enterococcus faecium from Swedish broilers.

The minimum inhibitory concentration (MIC) of the polyether ionophore antibiotic narasin is elevated in a large proportion of Enterococcus faecium from Swedish broilers. The aim of this study was to identify gene(s) responsible for these elevated MICs. Six plasmids, four conferring vancomycin resistance and elevated MIC of narasin and two only conferring resistance to vancomycin, were sequenced. The genes for a putative mechanism for elevated MIC of narasin was used to design a PCR assay which in turn was used to screen 100 isolates of E. faecium from Swedish broilers. A 5.9 kb area was only found in the plasmids transferring elevated MIC of narasin. This area included two genes coding for an ABC-type transporter; an 'ABC transporter permease protein' and an 'ABC-type multidrug transport system, ATPase component'. These genes are known to confer resistance to the ionophore tetronasin. PCR investigation confirmed a correlation between the presence of the genes and a MIC of narasin ≥ 2 mg/L. The results of this study indicate that the ABC permease together with the ABC ATPase are responsible for the elevated MIC of narasin present among E. faecium in Swedish broilers. To our knowledge, this is the first report of a putative transferable mechanism for elevated MIC of narasin.

The Arabidopsis thaliana transcriptional activator STYLISH1 regulates genes affecting stamen development, cell expansion and timing of flowering.

SHORT-INTERNODES/STYLISH (SHI/STY)-family proteins redundantly regulate development of lateral organs in Arabidopsis thaliana. We have previously shown that STY1 interacts with the promoter of the auxin biosynthesis gene YUCCA (YUC)4 and activates transcription of the genes YUC4, YUC8 and OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF (ORA)59 independently of protein translation. STY1 also affects auxin levels and auxin biosynthesis rates. Here we show that STY1 induces the transcription of 16 additional genes independently of protein translation. Several of these genes are tightly co-expressed with SHI/STY-family genes and/or down-regulated in SHI/STY-family multiple mutant lines, suggesting them to be regulated by SHI/STY proteins during plant development. The majority of the identified genes encode transcription factors or cell expansion-related enzymes and functional studies suggest their involvement in stamen and leaf development or flowering time regulation.

ABA is required for Leptosphaeria maculans resistance via ABI1- and ABI4-dependent signaling.

Abscisic acid (ABA) is a defense hormone with influence on callose-dependent and -independent resistance against Leptosphaeria maculans acting in the RLMcol pathway. ABA-deficient and -insensitive mutants in Ler-0 background (abal-3 and abil-1) displayed susceptibility to L. maculans, along with a significantly decreased level of callose depositions, whereas abi2-1 and abi3-1 remained resistant, together with the abi5-1 mutant of Ws-0 background. Suppressor mutants of abil-1 confirmed that the L. maculans-susceptible response was due to the dominant negative nature of the abil-1 mutant. Highly induced camalexin levels made ABA mutants in Col-0 background (aba2-1, aba3-1, and abi4-1) appear resistant, but displayed enhanced susceptibility as double mutants with pad3-1, impaired in camalexin biosynthesis. beta-Aminobutyric acid (BABA) pretreatment of Ler-0 contributed to an elevated level of endogenous ABA after L. maculans inoculation. Comparisons between (RLM1co1)pad3 and rlmlLerpad3 showed that ABA and BABA enhancement of callose deposition requires induction from RLM1col. ABII, but not ABI2, was found to be involved in a feedback mechanism that modulates RLM1co, expression. Genetic analysis showed further that this feedback occurs upstream of ABI4 and that components downstream of ABI4 modulate ABIJ activity. ABA and BABA treatments of the L. maculans-susceptible callose synthase mutant pmr4 showed that ABA also induces a callose-independent resistance. Similar treatments enhanced callose depositions and induced resistance to L. maculans in oilseed rape, and BABA-induced resistance was found to be independent of salicylic acid.

STY1 regulates auxin homeostasis and affects apical-basal patterning of the Arabidopsis gynoecium.

Gynoecia of the Arabidopsis mutant sty1-1 display abnormal style morphology and altered vascular patterning. These phenotypes, which are enhanced in the sty1-1 sty2-1 double mutant, suggest that auxin homeostasis or signalling might be affected by mutations in STY1 and STY2, both members of the SHI gene family. Chemical inhibition of polar auxin transport (PAT) severely affects the apical-basal patterning of the gynoecium, as do mutations in the auxin transport/signalling genes PIN1, PID and ETT. Here we show that the apical-basal patterning of sty1-1 and sty1-1 sty2-1 gynoecia is hypersensitive to reductions in PAT, and that sty1-1 enhances the PAT inhibition-like phenotypes of pin1-5, pid-8 and ett-1 gynoecia. Furthermore, we show that STY1 activates transcription of the flavin monooxygenase-encoding gene THREAD/YUCCA4, involved in auxin biosynthesis, and that changes in expression of STY1 and related genes lead to altered auxin homeostasis. Our results suggest that STY1 and related genes promote normal development of the style and affect apical-basal patterning of the gynoecium through regulation of auxin homeostasis.