Clonal Spread of Salmonella enterica Serovar Infantis in Serbia: Acquisition of Mutations in the Topoisomerase Genes gyrA and parC Leads to Increased Resistance to Fluoroquinolones

Quinolone‐resistant Salmonella Infantis (n = 64) isolated from human stool samples, food and poultry during the years 2006–2011 were analysed for their resistance phenotypes, macrorestriction patterns and molecular mechanisms of decreased susceptibility to fluoroquinolones. Minimum inhibitory concentrations (MICs) of nalidixic acid (NAL) and ciprofloxacin (CIP) were determined by the agar dilution procedure, and the susceptibility to additional antimicrobial agents was determined by the disc diffusion method. To assess the influence of enhanced efflux activity, MICs were determined in the presence and absence of the inhibitor PAβN. The results of pulsed‐field gel electrophoresis (PFGE) typing revealed that quinolone‐resistant S. Infantis in Serbia had similar or indistinguishable PFGE profiles, suggesting a clonal spread. All S. Infantis showed combined resistance to NAL and tetracycline, whereas multiple drug resistance to three or more antibiotic classes was rare (2 isolates of human origin). The MICs ranged between 512 and 1024 μg/mL for NAL and 0.125–2 μg/mL for CIP. A single‐point mutation in the gene gyrA leading to a Ser83→Tyr exchange was detected in all isolates, and a second exchange (Ser80→Arg) in the gene parC was only present in eight S. Infantis isolates exhibiting slightly higher MICs of CIP (2 μg/mL). The inhibitor PAβN decreased the MIC values of CIP by two dilution steps and of NAL by at minimum 3–6 dilution steps, indicating that enhanced efflux plays an important role in quinolone resistance in these isolates. The plasmid‐mediated genes qnr, aac(6′)‐lb‐cr and qepA were not detected by PCR assays.     

Occurrence,Virulence Genes and Antibiotic Resistance of Enteropathogenic Escherichia coli (EPEC) from Twelve Bovine Farms in the North‐East of Ireland

Cattle faecal samples (n = 480) were collected from a cluster of 12 farms, and PCR screened for the presence of the intimin gene (eae). Positive samples were cultured, and colonies were examined for the presence of eae and verocytotoxin (vtx) genes. Colonies which were positive for the intimin gene and negative for the verocytotoxin genes were further screened using PCR for a range of virulence factors including bfpA, espA, espB, tir ehxA, toxB, etpD, katP, saa, iha, lpfA_{O157/OI‐141} and lpfA_{O157/OI‐154.} Of the 480 faecal samples, 5.8% (28/480) were PCR positive, and one isolate was obtained from each. All 28 isolates obtained were bfpA negative and therefore atypical EPEC (aEPEC). The serotypes detected included O2:H27, O8:H36, O15:H2, O49:H+, O84:H28, O105:H7 and O132:H34 but half of the isolates could not be serogrouped using currently available antisera. Twenty‐two (79%) of the isolates carried the tir gene but only 25% were espB positive, and all other virulence genes tested for were scarce or absent. Several isolates showed intermediate resistance to ciprofloxacin, kanamycin, nalidixic acid, minocycline and tetracycline; full resistance to nalidixic acid or tetracycline with one isolate (O?:H8) displaying resistance to aminoglycosides (kanamycin and streptomycin), quinolones (nalidixic acid) and sulphonamides. This study provides further evidence that cattle are a potential source of aEPEC and add to the very limited data currently available on virulence genes and antibiotic resistance in this pathogenic E. coli group in animals.     

Effect of biofilm formation on antimicrobial tolerance of Flavobacterium psychrophilum

Treatment of bacterial fish diseases can be complicated by resistant bacterial biofilms harbouring pathogenic bacteria and causing recurrent exposure of fish to infections. In this study, the effect of biofilm formation on antimicrobial tolerance was examined using three bacterial isolates of the fish pathogen Flavobacterium psychrophilum and two antimicrobial agents, oxytetracycline and flumequine, commonly used in aquaculture. Planktonic and biofilm cells were exposed to a minimum inhibitory concentration (MIC), to a 3 × MIC concentration and to an environmental concentration level of each antimicrobial in 96-well microtitre plates after which growth on agar plates was measured. The type strain NCIMB1947 of F. psychrophilum was further used to study the development of antimicrobial resistance in biofilm cells. The results suggest that at high bacterial densities (>10(7) CFU mL(-1)), biofilm cells of F. psychrophilum are less susceptible to antimicrobial agents. Furthermore, the results imply that biofilm cells of F. psychrophilum may rapidly develop resistance to both oxytetracycline and flumequine if exposed to subinhibitory concentrations of these antimicrobials.     

Dietary levamisole modulates the immune response and disease resistance of Asian catfish Clarias batrachus (Linnaeus)

In order to determine the immunomodulatory effect of dietary levamisole in Asian catfish (Clarias batrachus), fish were fed four different diets for 10 days: a formulated diet as control and the same diet supplemented with 50, 150 or 450 mg levamisole kg^?1 feed. The serum bacterial agglutination titre against Aeromonas hydrophila as a measure of specific immunity, serum haemagglutination titre, natural haemolytic complement activity (ACH₅₀), myeloperoxidase and lysozyme activities, total protein level and oxidative radical production by neutrophils as a measure of non‐specific immunity as well as disease resistance against A. hydrophila challenge to separate vaccinated and non‐vaccinated groups were evaluated at 0, 1, 2 and 3 weeks after last administration of levamisole. Levamisole supplement at the lowest level (50 mg kg^?1) significantly enhanced oxidative radical production and serum myeloperoxidase (MPO) content immediately after 10 days of feeding, which reached peak values after 3 and 2 weeks of feeding respectively. Haemolytic complement and haemagglutination titre were significantly enhanced after 3 and 1 weeks respectively. Haemolytic complement activity and MPO activities were significantly raised to 150 mg kg^?1 after 3 and 2 weeks, respectively. At the highest level of levamisole feeding (450 mg kg^?1) significant decreases in superoxide production and complement activity were measured immediately after levamisole feeding, which returned to the normal level after 1 week post‐ feeding. Fish were challenged with a virulent strain of A. hydrophila at 0, 1, 2 and 3 weeks after levamisole feeding, and the cumulative per cent survival was recorded over 10 days. Feeding levamisole at 50, 150 or 450 mg kg^?1 increased per cent survival in vaccinated fish immediately after levamisole feeding, and survival was significantly higher at 450 mg kg^?1. There was no difference in mortality patterns in non‐vaccinated fish. The results support the use of levamisole at 50 mg kg^?1 feed for 10 days as an immunostimulant in Asian catfish farming.     

Detection of quinolone-resistance genes in Photobacterium damselae subsp. piscicida strains by targeting-induced local lesions in genomes

Quinolone-resistant strains of the fish-pathogenic bacterium, Photobacterium damselae subsp. piscicida are distributed widely in cultured yellowtail, Seriola quinqueradiata (Temminck & Schlegel), in Japan. The quinolone resistance-determining region (QRDR) was amplified with degenerate primers, followed by cassette ligation-mediated PCR. Open reading frames encoding proteins of 875 and 755 amino acid residues were detected in the gyrA and parC genes, respectively. Resistant strains of P. damselae subsp. piscicida carried a point mutation only in the gyrA QRDR leading to a Ser-to-Ile substitution at residue position 83. No amino acid alterations were discovered in the ParC sequence. A mutation in the gyrA gene was also detected in nalidixic acid-resistant mutants of strain SP96002 obtained from agar medium containing increased levels of quinolone. These results suggest that GyrA, as in other Gram-negative bacteria, is a target of quinolone in P. damselae subsp. piscicida. Furthermore, we attempted to detect a point mutation using targeting-induced local lesions in genomes (TILLING), which is a general strategy used for the detection of a variety of induced point mutations and naturally occurring polymorphisms. We developed a new detection method for the rapid and large-scale identification of quinolone-resistant strains of P. damselae subsp. piscicida using TILLING.     

Minimising aerobic respiratory demands could form the basis to sub-lethal copper tolerance by rainbow trout gill epithelial cells in vitro

Mechanisms of Cu tolerance were investigated in respiratory epithelial cell cultures, from rainbow trout gills, by studying O₂ consumption and protein synthesis rates, intracellular Na concentration and TER. The lowest concentration found to reduce O₂ consumption was 25 M Cu. This did not affect either protein synthesis rate or intracellular Na concentration and was interpreted in terms of copper tolerance; i.e., how these two energetically demanding processes are maintained despite a reduction in aerobic ATP supply. The relationship between protein synthesis rate and synthesis cost is exponential and the cost of protein synthesis in gill cells was found to be minimal (i.e., this cell occupies a position on the asymptotic section of the protein synthesis rate/synthesis cost model) and unaffected by 25 M Cu. Thus protein synthesis rates could be maintained since any reduction would represent an insignificant energy saving. Intracellular Na concentrations and O₂ consumption rates were linearly correlated suggesting reducing intracellular maintenance costs would have a greater significance in terms of overall energetic conservation. Intracellular Na maintenance costs, calculated from O₂ consumption rates and intracellular Na concentrations, were found to decline after exposure to 25 M Cu. Since TER was unaffected this implied the reduced costs arose from membrane `channel arrest'. Thus the Na/K ATPase energy demands, associated with maintaining intracellular Na concentration, could be reduced by decoupling metabolic demand and membrane function. Therefore this study may demonstrate how the flexibility of cellular energetics enables gill epithelial cells to tolerate sub-lethal Cu.     

牛蛙脑膜炎黄杆菌病病原菌的研究

从广东省揭西县坪下牛蛙养殖场收集患黄杆菌病的病蛙,其症状是歪头、狂游、双眼白内障、肝脏肿大充血,腹水严重。从病蛙脑髓、眼底等处分离到62菌株。经急性毒性试验证明分离菌株对健康牛蛙有较强的致病性。选用其中有代表性的981229—1等菌株进行系统的生理生化试验结果显示,981229—3等菌株革兰氏染色阴性、短直杆菌、无动力、无芽孢、好氧型、不利用葡萄糖。内毒素试验阳性。经VITEK-AMS-60自动化检测系统鉴定为脑膜炎脓毒性黄杆菌(Flavobacterium meninosepticum)。选取47种抗菌药物,通过K—B纸片扩散法对分离菌进行耐药性试验结果显示,病原菌对40种试验药物耐药(85.11％)、中等敏感药物2种(4.26％)、敏感药物5种(10.64％)。以试管稀释法、测定分离菌株对5种敏感药物的平均 MIC分别是:利福平 0.24、环丙沙星 0.70、氧氟沙星 0.70、氟啶酸 1.20和麦迪霉素7.80μg/ml。     

Field evolution of insecticide resistance in the brown planthopper (Nilaparvata lugens Stål) in China

The brown planthopper (Nilaparvata lugens Stål) is one of the most destructive pests of rice crops in Asian countries including China, Vietnam, Thailand, etc. Evolution of resistance in this pest insect to isoprocarb, buprofezin, pymetrozine, imidacloprid and other neonicotinoid insecticides has been reported. In order to investigate the current status of resistance to commonly used insecticides, nine field populations of N. lugens were collected from Central China, East China and South China, and resistance to insecticides was monitored from 2009 to 2012. All the 9 field populations collected in 2012 had developed extremely high resistance to imidacloprid, with resistance ratios (RR) ranging from 209.3 to 616.6. Resistance to imidacloprid was much higher in 2012 than in 2009. The RR of thiamethoxam varied from 17.4 to 47.1, and the RR of nitenpyram varied from 1.4 to 3.7 in 2012. Of the 9 field populations, six populations showed higher resistance to nitenpyram in 2012 than in 2011. RR for buprofezin varied from 110.1 to 221.6 in 2012 whereas resistance was at a medium level (RR 20.4 ∼ 39.4-fold) in 2009. RR for pymetrozine ranged from 34.9 to 46.8 in 2012. As for isoprocarb, RR ranged from 21.7 to 38.1 in 2012. The obvious increase in resistance to widely applied insecticides indicates that insecticide resistance management strategies are urgently needed to prevent or delay further increase of insecticide resistance in N. lugens.     

Susceptibility of Brazilian populations of Diatraea saccharalis to Cry1Ab and response to selection for resistance

The sugarcane borer, Diatraea saccharalis (F.), is a key pest of sugarcane and other grasses, which is a current focus of research aiming to develop transgenically resistant genotypes. However, the current susceptibility of Brazilian populations of the sugarcane borer to Bt toxins is unknown. Laboratory assays were carried out to characterize the susceptibility of sugarcane borer to the Bt toxin Cry1Ab and to select a resistant strain for additional studies on Cry1Ab resistance. Susceptibility was characterized by exposing neonates of different Brazilian colonies of sugarcane borer to different concentrations in meridic diet using surface application of Cry1Ab toxin. Selection for Cry1Ab resistance was carried out by exposing neonates to Cry1Ab-expressing maize (MON 810). The resistance of the borer populations to Cry1Ab was variable with LC₅₀ and EC₅₀ values reaching about 30-fold. The larvae responded positively to Cry1Ab selection exhibiting a 55-fold increase in resistance after four generations. This suggests the suitability of using leaves containing Bt-expressing genes for selection and the existence of variability of Bt-resistance in populations of the sugarcane borer.     

优质、抗稻瘟病香型水稻三系不育系繁源A的选育

以长丰B为母本,乐丰B为父本杂交配组选育保持系,再与乐丰A测交、回交转育,育成野败籼型香稻三系不育系繁源A,于2013年通过福建省品种审定。结果表明,繁源A不育性稳定、配合力强、异交率高、抗稻瘟病、米质优异有香味。