Search

The aim of this study was to investigate the natural cycle of the new human pathogenic bacteria Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum in Southern Hungary. We collected rodents with live-traps (2010-2013) and questing ticks with flagging in 2012. Small mammals were euthanized, tissue samples were collected and all the ectoparasites were removed and stored in 70% alcohol. We found relatively low overall prevalence of tick infestation (8%). Samples were analysed for A. phagocytophilum and Candidatus N. mikurensis with multiplex quantitative real-time PCR targeting a part of major surface protein 2 (msp2) and the heat shock protein groEL genes, respectively. The overall prevalence in tissue samples was 6.6% (skin) and 5.1% (spleen) for A. phagocytophilum and 1.7% (skin) and 3.4% (spleen) for Candidatus N. mikurensis. Candidatus N. mikurensis was only detected in Apodemus flavicollis and Apodemus agrarius, while A. phagocytophilum was found in A. flavicollis, A. agrarius, Myodes glareolus, Microtus arvalis and Mus musculus samples. Prevalence of A. phagocytophilum in skin samples of A. flavicollis was significantly higher than prevalence of N. mikurensis (p<0.05). Among questing Ixodes ricinus ticks we found three (8.8%) individuals (female, male, nymph) infected with Candidatus N. mikurensis. Five (3.1%) questing ticks had A. phagocytophilum infection (one I. ricinus male, two Dermacentor reticulatus females and two Haemaphysalis concinna females). We found one I. ricinus nymph removed from a male A. flavicollis with A. phagocytophilum infection. Our study provides new data on the occurrence of these pathogens in rodent tissue samples, questing ticks and engorged ticks in Southern Hungary., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

A common physiological response of organisms to environmental conditions is variation in gene expression, especially true for genes encoding for heat shock proteins. In insects, this process has been examined for induced heat or cold stress. The putative long-term imprinted/acquired heat shock protein response due to unfriendly environmental conditions has been far less studied. The Drosophila melanogaster hsp22 gene, which has been extensively reviewed as being sensitive to different changing life conditions, was examined by qRT-PCR, using carboxy-X-rhodamine. In the present study, we focused on the detection of hsp22 level of transcription in three D. melanogaster isolates, collected from sites located near different chemical plants in Romania and subjected to one-year adaptation to laboratory conditions. In all isolates, the hsp22 gene expression was determined using the housekeeping genes Gapdh1 and UbcD10 as internal controls. According to our experimental results, the D. melanogaster hsp22 gene was significantly downregulated compared to the same gene in w(1118)iso, used as a calibrator. We showed that hsp22 could play an important role in relation to stress resistance and adaptation. This study highlights the importance of in vivo studies to demonstrate genome plasticity to overcome different damages induced by any presumed source of stress.

In natural bacterial communities the microbial structure and functions are subjected to dynamic environmental and genetic adaptation. Plasmid-mediated horizontal genes transfer has a major impact on the adaptability of bacteria, exemplified by the interspecific and intergeneric transfer of antibioresistance genes in a variety of aquatic media. The high incidence of resistant bacteria has been documented for fresh waters, marine waters and chronically polluted waters. The aim of this study was to establish the distribution and diversity of plasmids and to study the transfer of plasmids harboring multiple antimicrobial-resistance determinants (R plasmids) belong to 12 multiple antibiotic resistant E. coli strains isolated from river waters. Antimicrobial resistance patterns were performed for aminoglycosides (gentamycin, kanamycin), beta-lactams (ampicillin), cephalosporins (ceftazidime and cefotaxime), tetracycline, nalidixic acid and chloramphenicol by disk diffusion method following NCCLS recommendations. Minimum inhibitory concentrations (MICs) were performed using dilution method in Mueller-Hinton broth with a 0.06-64 micrograms/ml concentration range for all antimicrobials and bacterial inoculum corresponding to 0.5 standard of the McFarland scale. For the data analysis NCCLS breakpoints for resistance and sensitivity were used. Bacterial plasmid isolation was performed by an alkaline lysis method. Genetic characterization was performed by agarose gel electrophoresis and spectrophotometric analysis. R-plasmid transfer frequencies were estimated by conjugation of drug-resistant E. coli strains used as donors with E. coli DH5 alpha F recipient marked with chromosomal resistance to nalidixic acid (Nal). The drug resistance markers possessed by a particular donor strain were sequentially used to screen for R+ transconjugants by incorporation the particular drug in the selective media. All E. coli strains are multiple antibiotic resistant, 65% of them being resistant to all 8 antibiotics tested. Plasmid profile analysis revealed the presence of several plasmids ranging from 3.8 kpb to more than 50 kpb. All aquatic R+ strains transferred two or more of their resistance markers to E. coli DH5 alpha F, transfer of resistance to ampicillin and tetracycline being the most frequent and having a frequency of 10(-4) or greater (expressed as transconjugants/donor). The phenotypic data shows the frequency and dynamic flow of multiple antibioresistant E. coli strains in aquatic media. Electrophoretic patterns analysis reflects the high incidence and diversity of plasmids in aquatic E. coli strains. Plasmid-harboring E. coli strains transferred antibiotic resistance and, hence, possessed conjugative R plasmids. Of these, 80% transferred drug resistance at a frequency of about 10(-4).

Several multiple antibiotic resistant E. coli strains isolated from river and polluted waters were compared for their genetic relatedness. Antibiotic susceptibility testing was performed for gentamycin, kanamycin, ampicillin, tetracycline, chloramphenicol, ceftazidime and cefotaxime, as described by Kirby-Bauer disk diffusion method following NCCLS recommendations. Minimum inhibitory concentrations (MICs) were performed using dilution method in Mueller-Hinton broth with a 0.06-64 micrograms/ml concentration range for all antimicrobials and bacterial inoculum of about 1-2 x 10(8) CFU/ml. For the data analysis NCCLS breakpoints for resistance and sensitivity were used. Genomic DNA was isolated from E. coli strains by CTAB method and digested to completion with HindIII enzyme. Genetic characterization was performed by agarose gel electrophoresis and spectrophotometric analysis. Genetic similarity and clustering were calculated using NISIS program. All E. coli strains isolated from river and polluted waters show a high incidence of multiple antibiotic resistance phenotype, 16% of them being resistant to 7, 6 and 4 antibiotics, 40% to 5 and 8% to 2 antibiotics, respectively. A moderate resistance was observed to kanamycin (higher than 30%) and cefotaxime (68%). The percentage of resistant E. coli strains ranged from 76% (to ampicillin, gentamicin and chloramphenicol) to 85% (to ceftazidime). The best results (resistance about 99%) were obtained with tetracycline. Screening for plasmids relieved the presence into 4 E. coli strains of several plasmids ranging from 3.8 kpb to more than 50 kpb. The number of fragments produced by HindIII digestion of genomic DNA ranged from 11 to 25, with sizes of approximately 22 to more than 750 kb. The phenotypic data shows the dynamic flow of multiple antibioresistant E. coli strains in aquatic media (river and polluted waters). Electrophoretic patterns analysis reflects the incidence and diversity of analyzed plasmids. DNA fingerprinting with genomic DNA RE suggested that, depending of the isolation source, E. coli strains could be grouped in two distinct populations with a different plasmid diversity.

Self-transmissible plasmids conferring multiple antibiotic resistance are wide-spread in coliforms populations. In soil and water, multiple antibiotic resistance is clearly associated with resistance/tolerance to heavy-metals (Hg2+, Cu2+, Pb2+, Zn2+, Ca2+). For different genera the genes for heavy-metals resistance are often plasmid encoded. Since these genes are clustered on the same plasmids, heavy-metals and drugs are environmental factors which exert a selective pressure for the populations of these plasmid-harboring bacteria. The aim of this preliminary study was to find possible correlation between resistance genotype determined by genetic analysis and antibiotic and heavy-metal resistance patterns of 12 E. coli strains isolated from chronically polluted waters. Antimicrobial susceptibility testing was performed for ampicillin, tetracycline, gentamycin, kanamycin, chloramphenicol, ceftazidime and cefotaxime by standard disk diffusion Kirby-Bauer method following NCCLS recommendations. These antibiotics were chosen because of their wide-spread use and importance in the treatment of Gram-negative bacterial infections. MICs values of antibiotics and heavy-metals were determined by dilution method in Mueller-Hinton broth using an inoculum of about 1-2 x 10(8) CFU/ml. The concentration range for antimicrobials and heavy-metals salts (CuSO4, CdCl2, Co(NO3)2, Cr(NO3)3, HgCl2, NiCl2 and ZnSO4) was 0.06-64 [symbol: see text] g/ml, 0.5-256 [symbol: see text] g/ml respectively. Plasmid DNA was isolated from E. coli strains by an alkaline lysis. Genetic characterization was performed by agarose gel electrophoresis and spectrophotometric analysis. All strains are multiple antibiotic resistant, 16% of them being resistant to 3, 4 and 6 antibiotics, 32% to 5 and 8% to all 7 antibiotics, respectively. Multiple tolerance to high levels of Cd2+, Cu2+, Cr3+ and Ni2+ was common among multiple antibioresistant strains. Screening for plasmids relieved the presence of several plasmids ranging from 3.8 kpb to more than 50 kpb. The phenotypic data shows the direct association between multiple antibiotic and heavy-metal resistance for E. coli strains in polluted water. Electrophoretic patterns analysis reflects the high incidence and diversity of analyzed plasmids.

Bacterial resistance by producing of beta-lactamases represents an increasing problem of infections chemotherapy. beta-lactam hydrolyzing activities are detected in virtually all bacteria, from witch Enterobacter cloacae produce chromosomal beta-lactamases included in inducible class AmpC beta-lactamases. The purpose of this preliminary study was to investigate the antibiotic susceptibility of 7 inducible beta-lactamase-producing Enterobacter cloacae strains isolated from aquatic sources (river and polluted waters). The identification to the species level was performed with the API 32E system and susceptibility to antimicrobial agents was tested by the disk diffusion method according to NCCLS recommendations. The following antibiotics were tested: ampicillin, amoxycillin/clavulanic acid, ceftazidime, cefotaxime, cephalotin, cefamandole, cephaclor, imipenem, amikacin, gentamycin, kanamycin, tobramycin, ciprofloxacin, norfloxacin, ofloxacin, nalidixic acid, tetracycline and chloramphenicol. Minimum inhibitory concentrations (MICs) were performed using dilution method in Mueller-Hinton broth with a 0.06-64 micrograms/ml concentration range for all antimicrobials and bacterial inoculum of about 1-2 x 10(8) cfu/ml. For the data analysis NCCLS breakpoints for resistance and sensitivity were used. Interaction of beta-lactamase inhibitor clavulanate with cefotaxime was performed by double-disk synergy test. Detection of inducible beta-lactamase expression was performed by the inductibility disk diffusion test using cefotaxime, ceftazidime and imipenem. Genomic DNA was isolated using CTAB technique and bacterial plasmid isolation was performed by an alkaline lysis method. Genetic characterization was performed by agarose gel electrophoresis and spectrophotometric analysis. The majority of examined E. cloacae strains were sensitive to imipenem, cefamandole, amikacin and quinolones (norfloxacin and ofloxacin), a higher moderate resistance being observed only to nalidixic acid (higher than 50%) and ciprofloxacin (15%). The percentage of resistant strains ranged from 72% (to kanamycin) to 87% (to gentamicin). The best results (resistance about 99%) were obtained with ampicillin, amoxycillin/clavulanic acid, ceftazidime, cefotaxime, cephalotin, cephaclor, tobramycin, tetracycline and chloramphenicol. The disk diffusion tests showed the absence of extended-spectrum beta-lactamases production and the expression of inducible beta-lactamases. Electrophoretic patterns point out the presence of plasmid DNA. Plasmid profile revealed the presence of several different plasmids ranging from 2.5 kpb to more than 30 kpb. The presence of inducible beta-lactamase E. cloacae strains in aquatic media (river and polluted waters) and the closely related pattern of susceptibility among these strains reflect the possible contamination of these sources and the common origin of them.