PCR-Based Screening of Pathogens in Bombus terrestris Populations of Turkey.

PURPOSE: Bumblebees are an important group of insects in the pollination of various vegetables, fruits, oilseeds, legumes, and the fodder crops. Compared to honeybees, they have a wider choice of hosts and a longer flight period. These bees are used especially for the pollination of plants in greenhouses and are commercially produced for this purpose. Recently, serious decreases have been occurring in bumblebee populations due to various reasons such as pathogens, and some of species are even threatened with extinction. Due to the worldwide decline in pollinator insects, determining the distribution and prevalence of bumblebee pathogens is of great importance. Therefore, this study was conducted to determine the incidence and prevalence of pathogens in Turkish bumblebee populations and how much of each pathogen was in bumblebee samples. METHODS: A total of 172 Bombus terrestris (Linnaeus,1758) samples (21 samples from commercial enterprises, 79 samples from greenhouses and 72 samples from nature) were randomly collected from 3 provinces (Antalya, Mersin and Izmir) where greenhouse cultivation is intensively carried out in Turkey. Eighty-nine of these samples were collected in the spring and eighty-three in the autumn. The presence of four pathogens (Nosema bombi, Crithidia bombi, Apicystis bombi, and Locustacarus buchneri) was investigated by PCR using universal primers. RESULTS: The overall prevalence of Nosema bombi, Crithidia bombi, Apicystis bombi, and Locustacarus buchneri was determined as 7.55%, 9.3%, 11.62%, and 4.65%, respectively. Co-infections (5.81%) were only detected in wild-caught (nature) samples. C. bombi and A. bombi infections were detected at higher rates in the spring samples than in the autumn samples (p < 0.05). There was no significant difference between the spring and autumn samples with respect to the presence of N. bombi and L. buchneri (p > 0.05). CONCLUSION: The results obtained could be important in determining the prevalence and spread rates of the bumblebee diseases in Turkey and to determine appropriate protection measures. The information gathered should increase our knowledge about the presence of these pathogens in Turkey and could contribute to improve apiarist's practice. More studies are needed to determine the transmission pathways of these pathogens between the populations. Also, complex pathogen interactions in bumblebee populations should be considered in the future to improve bumblebee health.

Evaluating molecular epidemiology of carbapenem non-susceptible Klebsiella pneumoniae isolates with MLST, MALDI-TOF MS, PFGE.

BACKGROUND: This study aimed to evaluate antibiotic resistance genes and virulence genes and the clonal relationship of the carbapenem-nonsusceptible Klebsiella pneumoniae strains by molecular methods which are isolated from various clinical specimens from patients treated in tertiary care hospital in Turkey. METHODS: Identification of 32 carbapenem non-susceptible K. pneumoniae were determined by VITEK-2 (BioMérieux, France) automated system. Thirteen colistin-resistant strains were tested with the broth microdilution method. Various antibiotic resistance genes and virulence genes frequently seen in carbapenem-resistant strains were screened by PCR. Immunochromatographic tests used in the rapid diagnosis of carbapenemases were compared with PCR results. In addition, PFGE, MLST and MALDI-TOF MS methods were used to determine the clonal relationship among these strains. RESULTS: PCR demonstrated that 31 of the strains carried at least one of the carbapenemase genes. In one strain, the coexistence of blaOXA-48+NDM was shown. The most common resistance genes were determined as blaSHV (84.3%), blaCTX-M-1 (46.8%), blaOXA-48 (40.6%), blaKPC (40.6%), blaTEM (31.2%), blaNDM (18.8%) respectively. Among the virulence genes; magA (68.7%) was the most common, followed by kpn (59.3%) and K2 (9.3%). Immunochromatographic tests were found to be 100% compatible with PCR results. All colistin-resistant isolates were also found to be resistant by colistin broth microdilution. In PFGE analysis, 25 different genotypes were determined and clustering isolates were collected in 5 different clusters and the clustering rate was 35.4%. In MLST analysis, ST101 type was determined as the most common ST type with a rate of 29%. ST101 is followed by ST16, ST307, ST14, ST147, ST309, ST377, ST395 and ST2096, respectively. The compatibility rate between MALDI-TOF MS and VITEK-2 was found 94.3%, in bacterial identification. In MALDI-TOF MS typing, the maximum similarity between the strains was less than 70% and clustering not shown. CONCLUSION: In addition to OXA-48, which is endemic in our country, it has been determined that KPC, which is more common in the world, is becoming increasingly common in our region. ST101 type was determined as the most common type between the strains. To the best of our knowledge, this is the first study that compares these three methods in our country. There may be differences between bacterial identifications made with VITEK-2 and MALDI-TOF MS. In this study, it was observed that MALDI-TOF MS analyses were not compatible with the typing of strains according to PFGE and MLST analysis results.

PCR-based detection of the honeybee tracheal mite (Acarapis woodi) in Türkiye.

Acarapis woodi (Rennie 1921) (Acari: Tarsonemidae) is one of the mites that settles in the respiratory system of honeybees (Apis mellifera L. (Hymenoptera, Apidae)) and distributed throughout the world. It causes significant economic losses on honey production. In Türkiye, studies on the existence of A. woodi are very limited and so far, no studies on the molecular diagnosis and phylogenetic of it have been reported in Türkiye. This study was conducted to investigate the prevalence of A. woodi in Türkiye, especially in areas where beekeeping is intense. Diagnosis of A. woodi was performed using both microscopic and molecular methods using specific PCR primers. Adult honeybee samples were collected from 1.193 hives in 40 provinces of Türkiye between 2018 and 2019. Based on identification studies, the presence of A. woodi was detected in a total of 3 hives (0.5%) in 2018 and 4 hives (0.7%) in 2019. This is the first report for determination of A. woodi in Türkiye.

Biocidal properties of maltose reduced silver nanoparticles against American foulbrood diseases pathogens.

Bee disease caused by spore-forming Paenibacillus larvae and Paenibacillus alvei is a serious problem for honey production. Thus, there is an ongoing effort to find an effective agent that shows broad biocidal activity with minimal environmental hazard. In this study, the biocidal effect of maltose reduced silver nanoparticles (AgNPs) is evaluated against American foulbrood and European foulbrood pathogens. The results demonstrate that the maltose reduced AgNPs are excellent short and long-term biocides against P. larvae isolates. The long-term effect suggests that the Ag+ ions are released from the AgNPs with increasing time in a controlled manner.

Culturable bacterial microbiota of Plagiodera versicolora (L.) (Coleoptera: Chrysomelidae) and virulence of the isolated strains.

Plagiodera versicolora (Laicharting, 1781) (Coleoptera: Chrysomelidae) is an important forest pest which damages many trees such as willow, poplar, and hazelnut. In order to find new microbes that can be utilized as a possible microbial control agent against this pest, we investigated the culturable bacterial flora of it and tested the isolated bacteria against P. versicolora larvae and adults. We were able to isolate nine bacteria from larvae and adults. The isolates were characterized using a combination of morphological, biochemical, and physiological methods. Additionally, we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results. Based on characterization studies, the isolates were identified as Staphylococcus sp. Pv1, Rahnella sp. Pv2, Rahnella sp. Pv3, Rahnella sp. Pv4, Rahnella sp. Pv5, Pantoea agglomerans Pv6, Staphylococcus sp. Pv7, Micrococcus luteus Pv8, and Rahnella sp. Pv9. The highest insecticidal activity against larvae and adults was obtained from M. luteus Pv8 with 50 and 40 % mortalities within 10 days after treatment, respectively. Extracellular enzyme activity of the bacterial isolates such as amylase, proteinase, lipase, cellulose, and chitinase was also determined. Consequently, our results show that M. luteus Pv8 might be a good candidate as a possible microbial control agent against P. versicolora and were discussed with respect to biocontrol potential of the bacterial isolates.

Reduction in membrane phosphatidylglycerol content leads to daptomycin resistance in Bacillus subtilis.

Daptomycin (DAP) is a cyclic lipopeptide that disrupts the functional integrity of the cell membranes of Gram-positive bacteria in a Ca(2+)-dependent manner. Here we present genetic, genomic, and phenotypic analyses of an evolved DAP-resistant isolate, Dap(R)1, from the model bacterium Bacillus subtilis 168. Dap(R)1 was obtained by serial passages with increasing DAP concentrations, is 30-fold more resistant than the parent strain, and displays cross-resistance to vancomycin, moenomycin, and bacitracin. Dap(R)1 is characterized by aberrant septum placement, notably thickened peptidoglycan at the cell poles, and pleiotropic alterations at both the transcriptome and proteome levels. Genome sequencing of Dap(R)1 revealed 44 point mutations, 31 of which change protein sequences. An intermediate isolate that was 20-fold more resistant to DAP than the wild type had only three of these point mutations: mutations affecting the cell shape modulator gene mreB, the stringent response gene relA, and the phosphatidylglycerol synthase gene pgsA. Genetic reconstruction studies indicated that the pgsA(A64V) allele is primarily responsible for DAP resistance. Allelic replacement with wild-type pgsA restored DAP sensitivity to wild-type levels. The additional point mutations in the evolved strain may contribute further to DAP resistance, serve to compensate for the deleterious effects of altered membrane composition, or represent neutral changes. These results suggest a resistance mechanism by which reduced levels of phosphatidylglycerol decrease the net negative charge of the membrane, thereby weakening interaction with the positively charged Ca(2+)-DAP complex.

DNA-binding properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ(D) proteins.

σ(D) proteins from Aeribacillus pallidus AC6 and Bacillus subtilis bound specifically, albeit weakly, to promoter DNA even in the absence of core RNA polymerase. Binding required a conserved CG motif within the -10 element, and this motif is known to be recognized by σ region 2.4 and critical for promoter activity.

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, bla(oxA-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.

Molecular characterization of antibiotic resistant Escherichia coli strains isolated from tap and spring waters in a coastal region in Turkey.

A hundred and seventeen antibiotic-resistant Escherichia coli strains were isolated from public tap and spring waters which were polluted by fecal coliforms. There were no significant differences between two water sources as to the coliform pollution level (p> 0.05). All E. coli isolates were detected to be resistant to one or more antibiotics tested. Nearly 42% of the isolates showed multiresistant phenotype. Three (2.5%) of these isolates contained class 1 integron. Sequencing analysis of variable regions of the class 1 integrons showed two gene cassette arrays, dfr1-aadA1 and dhfrA17-aadA5. Resistance to ampicillin, tetracycline or trimethoprim-sulfamethoxazole was transferable according to the results of conjugation experiments. The rate of tetracycline resistance was 15%. tet(A)-mediated tetracycline resistance was widespread among tetracycline-resistant E. coli isolates. Genotyping by BOX-polymerase chain reaction (BOX-PCR) showed that some of the strains were epidemiologically related. This is the first report on the prevalence and characterization of class 1 integron-containing E. coli isolates of environmental origin in Turkey.