Ivica Šamanić

Essential oil composition and internal transcribed spacer (ITS) sequence variability of four South-Croatian Satureja species (Lamiaceae).

The purpose of this study was to compare the essential oil profiles of four South-Croatian Satureja species, as determined by GC/FID and GC/MS, with their DNA sequences for an internal transcribed spacer (ITS1-5.8S-ITS2) of the nuclear ribosomal DNA. A phylogenetic analysis showed that S. montana and S. cuneifolia, characterized by a similar essential oil composition, rich in the monoterpene hydrocarbon carvacrol, clustered together with high and moderate bootstrap support. On the contrary, S. subspicata and S. visianii, characterized by quite unique essential oil compositions, clustered together with the moderate bootstrap support. All four Croatian Satureja species clustered in one clade, separately from Macaronesian S. hortensis, although it had essential oil composition similar to that of S. montana and S. cuneifolia. This is the first report on the comparison between the phytochemical and DNA sequence data in Satureja species and useful contribution to the better understanding of interspecies relationships in this genus.

Prevalence and diversity of extended-spectrum-β-lactamase-producing Enterobacteriaceae from marine beach waters

A total of 1,351 Enterobacteriaceae isolates from 144 seawater samples were collected over a four-year period from three public beaches in the eastern Adriatic Sea in Croatia. Approximately 35% of the strains were multidrug-resistant. BlaESBL genes were detected in 4.2% of the isolated Enterobacteriaceae, the main species of which were Escherichia coli, Enterobacter cloacae and Klebsiella pneumoniae. BlaTEM-1+SHV-12 was the most dominant genotype, followed by blaCTX-M-15.Raoultella terrigena and E. intermedius simultaneously harboured blaTEM-1,blaSHV-11/12 and blaCTX-M-15. Isolate fingerprinting revealed that marine E. coli isolates were clonally related to CTX-M-producing strains from a regional university hospital. These results indicate that marine beach waters are reservoirs of ESBL-producing Enterobacteriaceae and thus constitute a public health problem with further potential to act as mediators in gene flow between marine coastal areas and clinical settings.

Aeromonas spp. simultaneously harbouring blaCTX-M-15, blaSHV-12, blaPER-1 and blaFOX-2, in wild-growing Mediterranean mussel (Mytilus galloprovincialis) from Adriatic Sea, Croatia

Aeromonas species are becoming renowned as emerging pathogens by increasingly giving rise to a wide spectrum of food and waterborne infections in humans. Another worrisome feature of aeromonads is the growing frequency of antibiotic resistance as a consequence of their prominent diversity in terms of resistance determinants. This study aimed at determining the antimicrobial resistance pattern, prevalence and characterization of acquired β-lactamases, including extended-spectrum-β-lactamases (ESBLs) and AmpC cephalosporinases, as well as the presence of class 1 and 2 integrons, in Aeromonas isolates from wild-growing Mediterranean mussel (Mytilus galloprovincialis) of the eastern coast of Adriatic Sea, Croatia. Isolates were tested for susceptibility to 16 antibiotics and β-lactam/β-lactamase inhibitor combinations. Cephalosporin-resistant isolates were further screened by PCR for genes encoding AmpC (bla(FOX), bla(CMY), bla(MOX), bla(LAT), bla(BIL), bla(DHA), bla(ACC), bla(MIR), bla(ACT)), ESBLs (bla(TEM), bla(SHV), bla(CTX-M), bla(PER), bla(VEB), bla(GES/IBC), bla(OXA)) and integrases (intI1, intI2, intI3). Location of bla genes was characterized by plasmid DNA fingerprinting and Southern blot hybridization. Plasmids carrying ESBL genes were investigated for transferability by conjugation and PCR-based replicon typed. Out of 147 Aeromonas isolates recovered, 30 (20%) demonstrated multiple resistance profile, with co-resistance most frequently detected against penicillins, piperacillin/sulbactam and tetracycline. ESBL-encoding genes were detected in 21 (13 Aeromonas caviae and 8 Aeromonas hydrophila) isolates, with bla(CTX-M-15) gene identified in 19 and bla(SHV-12) in 12 isolates. Among them, 10 isolates simultaneously harboured bla(CTX-M-15) and bla(SHV-12), while 3 isolates additionally carried an AmpC β-lactamase bla(FOX-2) gene. bla(PER-1) gene was identified in a single isolate also harbouring the bla(CTX-M-15) gene. While bla(SHV-12) was chromosomally encoded, bla(CTX-M-15) was located on conjugative IncFIB-type plasmids of ~40 kb in A. caviae isolates. IntI1 and intI2 genes were detected in 57.1% and 33.3% of ESBL-producing isolates. To the best our knowledge, this is the first report of environmental A. caviae isolates producing CTX-M-15, and isolation of SHV-12-producing A. hydrophila and A. caviae strains worldwide. This is also believed to be the first report of the FOX-2, CTX-M-15 and SHV-12 simultaneous production in aeromonads, highlighting both the potential risk for human health, and a role of these foodborne pathogens as reservoirs of resistance determinants in coastal marine environment.

Triparental origin of triploid onion, Allium × cornutum (Clementi ex Visiani, 1842), as evidenced by molecular, phylogenetic and cytogenetic analyses

BackgroundReconstruction of the parental origins of cultivated plants from wild relatives, especially after long periods of domestication, is not a trivial task. However, recent advances in molecular phylogenetics, among other approaches, have proved to be very informative in analyses of the origin and evolution of polyploid genomes. An established minor garden crop, triploid onion Allium × cornutum (Clementi ex Visiani, 1842) (2n = 3x = 24), is widespread in southeastern Asia and Europe. Our previous cytogenetic analyses confirmed its highly heterozygous karyotype and indicated its possible complex triparental genome origin. Allium cepa L. and Allium roylei Stearn were suggested as two putative parental species of A. × cornutum, whereas the third parental species remained hitherto unknown.ResultsHere we report the phylogenetic analyses of the internal transcribed spacers ITS1-5.8S-ITS2 of 35S rDNA and the non-transcribed spacer (NTS) region of 5S rDNA of A. × cornutum and its relatives of the section Cepa. Both ITS and NTS sequence data revealed intra-individual variation in triploid onion, and these data clustered into the three main clades, each with high sequence homology to one of three other species of section Cepa: A. cepa, A. roylei, and unexpectedly, the wild Asian species Allium pskemense B. Fedtsh. Allium pskemense is therefore inferred to be the third, so far unknown, putative parental species of triploid onion Allium × cornutum. The 35S and 5S rRNA genes were found to be localised on somatic chromosomes of A. × cornutum and its putative parental species by double fluorescent in situ hybridisation (FISH). The localisation of 35S and 5S rDNA in A. × cornutum chromosomes corresponded to their respective positions in the three putative parental species, A. cepa, A. pskemense, and A. roylei. GISH (genomic in situ hybridisation) using DNA of the three putative parental diploids corroborated the results of the phylogenetic study.ConclusionsThe combined molecular, phylogenetic and cytogenetic data obtained in this study provided evidence for a unique triparental origin of triploid onion A. × cornutum with three putative parental species, A. cepa, A. pskemense, and A. roylei.

Evidence for Distinct Functions of MRE11 in Arabidopsis Meiosis

The evolutionary conserved Mre11/Rad50/Nbs1 complex functions as one of the guardians of genome integrity in eukaryotes; it is required for the double-strand break repair, meiosis, DNA checkpoint, and telomere maintenance. To better understand the role of the MRE11 gene in Arabidopsis, we performed comparative analysis of several mre11 alleles with respect to genome stability and meiosis. The mre11-4 and mre11-2 alleles presumably produce truncated MRE11 proteins composed of the first 499 and 529 amino acids, respectively. Although the putative MRE11 truncated proteins differ only by 30 amino acids, the mutants exhibited strikingly different phenotypes in regards to growth morphology, genome stability and meiosis. While the mre11-2 mutants are fully fertile and undergo normal meiosis, the mre11-4 plants are sterile due to aberrant repair of meiotic DNA breaks. Structural homology analysis suggests that the T-DNA insertion in the mre11-4 allele probably disrupted the putative RAD50 interaction and/or homodimerization domain, which is assumed to be preserved in mre11-2 allele. Intriguingly, introgression of the atm-2 mutant plant into the mre11-2 background renders the double mutant infertile, a phenotype not observed in either parent line. This data indicate that MRE11 partially compensates for ATM deficiency in meiosis of Arabidopsis.

Characterization of Environmental CTX-M-15-Producing Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is a widespread environmental microorganism (1) that has emerged as significant opportunistic pathogen (2) due to intrinsic resistance to almost all available antibiotics.…

Antibiotic susceptibility profiles and first report of TEM extended-spectrum β-lactamase in Pseudomonas fluorescens from coastal waters of the Kaštela Bay, Croatia

The aim of this study was to investigate the antibiotic susceptibility profiles and the presence of extended-spectrum-β-lactamases (ESBLs) in Pseudomonas fluorescens isolates from coastal waters of the Kaštela Bay, Croatia. Twenty-two water samples were collected during 2009. Isolates were tested for susceptibilities to 13 antibiotics by Etest. ESBL production was confirmed by double-disk synergy test carried out on Mueller–Hinton agar plates containing efflux pump inhibitor Phe-Arg-β-naphthylamide dihydrochloride. PCR and DNA sequencing analysis were used to identify ESBL-encoding genes. The transferability of cephalosporin resistance was tested by conjugation experiments. Genetic relatedness of ESBL-producing isolates was determined by random amplified polymorphic DNA (RAPD) analysis. Out of 185 P. fluorescens isolates recovered, 70 (37.8%) demonstrated multiresistance phenotype with highest rates of resistance to tetracycline (61.6%), aztreonam (31.9%), meropenem (17.3%), ceftazidime (15.1%) and cefotaxime (12.4%). Ten (5.4%) isolates were identified as ESBL producers. All isolates carried chromosomally located blaTEM-116 gene. RAPD analysis identified four different genotypes. Here, we demonstrated a baseline profiles of antimicrobial resistance of P. fluorescens from coastal waters of the Kaštela Bay, Croatia. To our knowledge, this is the first report of the presence of TEM-type ESBL in P. fluorescens, indicating this bacterium as a reservoir of antibiotic resistance genes with clinical relevance.

Occurrence and antibiotic susceptibility profiles of Burkholderia cepaciacomplex in coastal marine environment

During an environmental study of bacterial resistance to antibiotics in coastal waters of the Kaštela Bay, Adriatic Sea, Croatia, 47 Burkholderia cepacia complex (Bcc) isolates were recovered from seawater and mussel (Mytilus galloprovincialis) samples. All isolates showed multiple antibiotic resistance. Among the isolates, two Burkholderia cenocepacia isolates produced chromosomally encoded TEM-116 extended-spectrum β-lactamase (ESBL). Analysis of outer membrane proteins revealed that decreased expression of a 36-kDa protein could be associated with a high level of β-lactam resistance in both isolates. Phenotypic study of efflux system also indicated an over-expression of Resistance-Nodulation-Cell Division (RND) efflux-mediated mechanism in one of the isolates. This study demonstrated the presence of Bcc inseawater and M. galloprovincialis, which gives evidence that coastal marine environment, including mussels, could be considered as a reservoir for Bcc species. Detection of ESBL-encoding genes indicates the potential role of these bacteria in the maintenance and dispersion of antibiotic resistance genes.

Phylogenetic Relationships among Populations of the Vineyard Snail Cernuella virgata (Da Costa, 1778)

Cernuella virgata (Da Costa, 1778) (Mollusca: Hygromiidae), commonly known as the “vineyard snail,” is endemic species in Mediterranean and Western Europe including the British Isles, but in the Eastern USA and Australia it represents an introduced invasive species. The present work examines the genetic variability and phylogenetic relationships among the four populations of this land snail sampled along the east Adriatic region of Croatia using mitochondrial markers (partial 16S rDNA and COI gene) in addition to traditional methods of shell’s shape analysis. All the three molecular-phylogenetic approaches (median joining haplotype network analysis and Bayesian analysis, as well as maximum likelihood analysis) revealed two-three major subnetworks for both 16S rDNA and COI, with a clear distinction between south Adriatic haplotypes (Pisak) and north Adriatic haplotypes (Krk and Cres). The population from Karlobag was comprised of both north and south haplotypes, thus representing a putative contact zone between these two groups. The morphometric analysis showed that individuals from Cres island population were statistically significantly wider and higher than individuals from Pisak population. Analysis of the SW/SH ratio and the relationship between shell width and shell height showed no differences in shell growth between the two examined populations, indicating equal shell growth and shape, which gives the possibility that differences in size of individuals between those two populations could be influenced by biotic (physiological) or abiotic (environmental) factors. This study represents the first analysis of genetic variability and relatedness among native populations of C. virgata.

Shell Characteristics of Land Snail Eobania vermiculata (Müller, 1774) (Helicidae) from Croatia

Abstract. The land snail genus Eobania P. Hesse, 1913 in Croatia consists of four taxa: Eobania vermiculata vermiculata (O.F. Müller, 1774), E. v. pelagosana (Westerlund, 1894), E. v. figarole (Rensch, 1928) and E. v. kamenensis Berberović, 1963. Only E. v. vermiculata and E. v. pelagosana have been included in this study. The mean shell diameters (D) of the 13 samples ranged from 19.99 mm (sample from the island of Sušac) to 31.78 mm (Šibenik, seaside) and the mean shell height (H) from 17.31 mm (Sušac) to 25.66 mm (Island of Šolta). Shell morphology confirms that all island populations are smaller with examples of nanism on outside islands of Palagruža and Sušac. However, molecular analysis (16S rDNA and COI sequence data) showed no significant differences between the South Adriatic population from the mainland and islands, thus providing no molecular evidence for classification of a single E. vermiculata species into separate subspecies within the investigated area. The observed differences might possibly be attributed to ecological influences. The population with the largest average shell size was approximately 1.5x larger than the population with the smallest average size.