Ali Sevim

Class 1 and class 2 integrons and plasmid-mediated antibiotic resistance in coliforms isolated from ten rivers in northern Turkey

We aimed to determine the molecular mechanisms of antibiotic resistance in coliforms isolated from ten rivers in northern region of Turkey. A total of 183 isolates were tested for antimicrobial susceptibility by disk diffusion and agar dilution methods. Resistance to ampicillin, streptomycin, trimethoprim, tetracycline, and chloramphenicol was detected in 58%, 51.9%, 24%, 28.4%, and 12.5%, respectively. Twelve (6.5%) phylogenetically distant organisms were detected to harbor self-transmissible plasmids ranging 52 to >147 kb in sizes. Resistances to ampicillin, tetracycline, trimethoprim, streptomycin, and nalidixic acid were commonly transferable traits. Transferable nalidixic acid-resistant strains harbored qnrS gene, which was the first report of plasmid-mediated quinolone resistance in bacteria of environmental origin in Turkey. Fourteen and five coliforms harbored class 1 and class 2 integrons, respectively, and some of them were located on transferable plasmids. Sequence analyses of variable regions of the class 1 and 2 integrons harbored various gene cassettes, dfrA1, dfr2d, dfrA7, dfrA16, dfrA17, aadA1, aadA5, blaoxA-30, and sat1. A gene cassette array, dfrA16 has been demonstrated for the first time in a Citrobacter koseri isolate. Class 1 and class 2-bearing strains were clustered in different groups by BOX-PCR fingerprinting. Rivers in the northern Turkey may act as receptacle for the multi-drug resistant enterobacteria and can serve as reservoirs of the antimicrobial resistance determinants in the environment. The actual risk to public health is the transfer of resistance genes from the environmental bacteria to human pathogens.

Investigation of antibiotic resistance profile and TEM-type β-lactamase gene carriage of ampicillin-resistantEscherichia coli strains isolated from drinking water

Fifty-five ampicillin-resistant (Ampr)Escherichia coli strains were isolated from 51 drinking water points in Rize region containing abundant fresh water sources in Turkey during the years 2000 to 2002 and from January to February 2004. The large number of organisms (nearly 57%) exhibited resistance to three or more antibiotics commonly used in human and veterinary medicine. These strains displayed a multiresistant phenotype. Nearly half of the strains (27%) expressed resistance to ceftazidime, but these strains were not an extended-spectrum β-lactamase-producer according to the results of double-disk synergy test. All isolates were then screened for the carriage of TEM-type β-lactamase gene (blaTEM) by polymerase chain reaction. TEM-type β-lactamase genes were found in six (11%) isolates. Sequence analysis showed TEM-1 type genes. However, isoelectric focusing analysis did not confirm the production of TEM-1 type β-lactamase except for one strain. Conjugation experiments showed that resistance to ampicillin, tetracycline or trimethoprim/sulfamethoxazole was transferable in six (11%) isolates. Emergence of transferable antibiotic resistance andblaTEM-1 gene inE. coli strains from public drinking waters possesses a significant public health risk.

Determination of the bacterial flora as a microbial control agent of Toxoptera aurantii (Homoptera: Aphididae)

Toxoptera aurantii (Homoptera: Aphididae) is one of the most important pests of many agricultural plants such as camellia, cocoa and coffee worldwide. The culturable bacterial flora of the pest was determined to find new microbes that can be used as biocontrol agent against T. aurantii. A total of six bacteria were isolated and identified by a variety of morphological, physiological, biochemical and molecular tests. In addition, an approximately 1,150 bp fragment of the 16S rRNA gene region was sequenced to verify isolate identification. According to the identification studies, the isolates were identified as Bacillus tequilensis Cb1, Chryseobacterium stagni Cb2, Pseudomonas flourescens Cb3, Rahnella aquatilis Cb4, Staphylococcus sp. Cb5 and Cb6. Pathogenicity of the bacterial isolates were carried out against the last instar nymphs of T. aurantii. The highest activity was obtained from Pseudomonas flourescens Cb3 with 50% mortality within 10 days after application (p<0.05). Mortalities of other treatments ranged from 6.6 to 20%. The results presented here show that Pseudomonas flourescens Cb3 appears to be a significant candidate as a possible biocontrol agent against T. aurantii and should be beneficial in the future biocontrol programs of the pest.

Molecular characterization, virulence and horizontal transmission of Beauveria pseudobassiana from Dendroctonus micans (Kug.) (Coleoptera: Curculionidae)

The great spruce bark beetle, Dendroctonus micans (Kugelann) (Coleoptera: Curculionidae), has been a potential threat for Turkey and the entire Eurasian spruce forests for many years. Control strategies which have been applied so far are still insufficient to prevent its damage. A previous study has shown that a Beauveria isolate (ARSEF 9271) proved to be an efficient microbial control agent against the great spruce bark beetle. In this study, this isolate was identified as B. pseudobassiana based on the partial sequence of EF1‐α and ITS sequence. A conidial suspension (1 × 108/ml) of this fungus caused 100% mortality on both larvae and adults of D. micans within 5 and 6 days, respectively. Also, it caused 100% mycosis value on both larvae and adults. Mortality values of horizontal transmission experiments between larvae and adults which were contaminated with 1 × 106/ml spore suspension at 25%, 50%, 75% and 100% rates were determined as 100% after 15 days at 20°C under the laboratory conditions. We also determined the decrease of the damage in spruce wood block (15 × 25 cm) when the contamination rate of the larvae was increased. Our results indicate that B. pseudobassiana ARSEF 9271 seems to be a very promising biocontrol agent against D. micans.

The internal bacterial diversity of stored product pests

Symbiotic bacteria associated with insects play important roles in different physiological processes such as digestion, insect behavior, defense and providing essential nutrition in insect gut. In addition, these bacteria can be used in biocontrol of insect pests using genetic engineering techniques. The first step is to isolate and identify symbiotic bacteria from insects to elucidate their roles, and to use in the development of transgenic strains. For this purpose, we isolated and characterized the bacterial isolates from stored product pests using a combination of conventional tests and 16S rRNA sequence analysis. The bacterial flora of Callosobruchus maculatus included Bacillus pumilus, Staphylococcus sp. and Pantoea sp. Acanthoscelides obtectus flora included Staphylococcus kloosii, Staphylococcus sp., S. saprophyticus and Enterococcus faecalis. The internal flora of Sitotroga cerealella included Staphylococcus succinus, Enterococcus sp. and Staphylococcus sp. Finally, Phthorimaea operculella flora included Bacillus sp., Staphylococcus sciuri, Enterococcus mundtii, E. casseliflavus, Alcaligenes faecalis, Enterobacter sp., Pantoea agglomerans and Pseudomonas fluorescens.

Investigation of the internal bacterial flora of Eurygaster integriceps (Hemiptera: Scutelleridae) and pathogenicity of the flora members

Eurygaster integriceps (Hemiptera: Scutelleridae) is one of the most serious pest of wheat and other cereal crops throughout the Near and Middle East including Turkey. To contribute biological control of this pest, we investigated the culturable bacterial flora of E. integriceps and their virulence against it. We also tested different entomopathogenic fungi (Isaria fumosorosea ARSEF8356, Beauveria bassiana ARSEF8356, Metarhizium brunneum ARSEF8671 and Nomurae rileyi ARSEF1670) against this pest under laboratory conditions. Bacterial isolates were characterized based on morphological, biochemical, physiological and molecular characteristics (16S rRNA sequencing). The isolates were identified as Pantoea sp. (S1, S5, S7, S8, S10 and S11), P. agglomerans (S2, S3 and S4), Pseudomonas sp. S6 and Micrococcus luteus S9. The highest mortality within bacterial isolates was observed from Pantoea sp. S1, P. agglomerans S4 and Pantoea sp. S7 with 100%. Mortality of other bacterial species ranged from 33% to 88%. The highest mortality among the tested entomopathogenic fungi was obtained from Isaria fumosorosea ARSEF 8333 with 100%. Mortality of other fungi ranged from 33% to 50%. Consequently, Pantoea sp. S1, P. agglomerans S4, Pantoea sp. S7, Beauveria bassiana ARSEF 8356 and Isaria fumosorosea ARSEF 8356 seem to be promising candidates in the control of E. integriceps.

Purification and characterization of α-L-arabinofuranosidases from Geobacillus stearothermophilus strain 12

Abstract In order to characterize two α-L-arabinofuranosidases (α-L-AFases), Abf1Geo12 and Abf2Geo12, produced by Geobacillus stearothermophilus strain 12, the genes (abf 1 and abf 2) coding for these enzymes were cloned and sequenced. Based on the protein sequence similarities, approximately 57 kDa two α-L-AFases were assigned to the glycoside hydrolase family 51. To obtain pure enzymes, the abf 1 and abf 2 genes were cloned into pET28a+ expression vector and recombinant α-L-AFases were produced in E.coli BL21(DE3): pLysS. Characterization of recombinant α-L-AFases revealed that Abf1Geo12 and Abf2Geo12 were active in a broad temperature range from 50 to 85°C and from 40 to 80°C, respectively. Also, the Abf1Geo12 was active in a broad pH range from 5.0 to 9.0. The optimum pH and temperature for Abf1Geo12 were determined as pH 6.0 and 65°C, respectively, whereas the optimum pH and temperature for Abf2Geo12 were determined as pH 5.5 and 60°C, respectively. Based on characterization studies, it was determined that the Abf1Geo12 was more stable than Abf2Geo12 and previously identified α-L-AFases from G. stearothermophilus. Using p-nitrophenyl α-L-arabinofuranoside as a substrate, the Km and Vmax values for Abf1Geo12 and Abf2Geo12 were determined as 0.31 mM and 290 U/mg for the former enzyme and 0.19 mM and 213.2 U/mg for the latter enzyme, respectively. The activities of Abf1Geo12 and Abf2Geo12 were strongly inhibited by 1 mM Hg2+. Interestingly, Cu2+ and Co2+ stimulated the activity of Abf1Geo12, but they reduced the activity of Abf2Geo12. The recombinant enzymes released L-arabinose from sugar beet arabinan, arabinobiose, arabinotriose, arabinotetraose and arabinopentaose. Consequently, these characterized two enzymes may be used in industrial fields since they are stable at high temperatures.