8%, 26.1%, and 22.7% identity, respectively). Iterated PSI-BLAST searches with ORF2 from pHW126 as well as with Rep from pHW104 retrieved sequences CP673451 clinical trial of replication proteins from pSN2-like plasmids and pJW1, indicating that all

these plasmids might form a super-family (Fig. 4B). However, the Rep sequence identity between members of different clades shown in Fig. 4B was around 10% in pair wise alignments and only two amino acids are invariant in all replication proteins of the plasmids analysed (Additional file 2). A final decision whether these plasmids are members of a common super-family is not possible. The very weak similarity of pHW126 to well characterised plasmids raised the question whether pHW126 should be classified as a rolling circle plasmid. However, we observed that increasing the size of pHW126 to more than 5 kb by insertion of foreign DNA fragments rendered this plasmid unstable (data not shown) which is a common phenomenon for rolling circle vectors [47]. To provide further OICR-9429 order experimental evidence a construct containing the rep gene and two copies of the upstream sequences in tandem repeat was generated. These upstream sequences are presumed check details to contain the origin of replication which is usually located 5′ of the rep gene in rolling circle plasmids. This construct was transformed into the recA – strain E. coli INVα F’ and independent clones were grown for 40 generations. Plasmid

DNA prepared from these cultures showed two bands after linearisation with restriction enzymes (Fig. 4C). The larger band of approximately 3.1 kb corresponded to the introduced plasmid. The smaller band, present in variable amounts, had a size of approximately 2.7 kb, consistent with the loss of one copy of the origin of replication. Frequent deletion of one replication origin is evidence for a rolling circle replication mechanism, because replication initiated at the second origin may terminate at the first.

This causes that the part of the plasmid between the two origins to be deleted [47]. As a control a similar construct containing two copies selleck chemicals of the ori from pHW15 (a ColE1 like plasmid replicating by a theta mechanism [6]), was tested in the same way. This construct maintained both origins as indicated by presence of only one band with a size of 3.7 kb (deletion of one ori would have reduced the size to 2.5 kb). These data provide convincing evidence that pHW126 replicates by the rolling circle replication mechanism, and that the origin of replication is located upstream of the rep gene. Both pHW121 and pHW126 showed strikingly low G+C contents of only 37.3% and 31.5%, respectively. Usually the G+C contents of plasmids are correlated with the chromosomal G+C contents of their hosts (Fig. 4B and 4D). pHW121 as well as pHW126 and its close homologues pIGRK, pIGMS31 and pRAO1 clearly deviate from this rule.

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The SCCmec carries the mecA gene, which encodes penicillin binding protein PBP2a, the main causal AICAR nmr factor of methicillin resistance. Different types of SCCmec cassettes and their variants have been identified [10, 11]. The current methods for MRSA detection are based on either the phenotypic expression such as oxacillin resistance, or genotypic characterization. For this study, we used modified broad-range PCR primers that originate from the conserved regions of genes that encode the topoisomerases together with specific oligonucleotide probes located at hyper-variable regions flanked by the primers. Using these primers and probes, single or even multiple infection-causing bacteria could be simultaneously

selleck chemicals llc detected and identified. The bacterial pathogen panel of the assay covered the following species: Acinetobacter baumannii, Enterococcus faecalis, Enterococcus faecium, Haemophilus influenzae, Klebsiella pneumoniae, click here Listeria monocytogenes, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes and selected CNS species. These bacteria are examples of highly virulent, potentially multi-antimicrobial resistant or the most common etiologic agents associated with various life-threatening conditions. Such

conditions include: sepsis, infective endocarditis and central nervous system infection. All these conditions necessitate rapid and accurate diagnostics to improve the chances of a positive outcome for

the patient. We used the ArrayTube™ as a microarray platform for the probes. The ArrayTube™ has been demonstrated to detect and Amisulpride identify bacterial pathogens with a high degree of sensitivity [12–14], differentiate between various pathotypes of the same bacterial species [15] and to be capable of detecting antimicrobial resistance genes [16] from an isolated DNA sample. Furthermore, by including specific primers and probes for the mecA methicillin resistance gene in the same assay, we were able to associate the mecA gene with a particular Staphylococcus species present in the sample. The combination of broad-range PCR and array-based methods provided a sensitive and specific approach for detecting and identifying bacterial pathogens along with finding possible resistance markers. Results Assay design First, we re-designed and modified the bacterial broad-range gyrB/parE primers [4] by using inosines to reduce the level of degeneration. These modifications also facilitated the use of a novel PCR method for the assay (PCR program described in Materials and Methods). The PCR method had two distinct phases: a three-step PCR phase that exponentially produced dsDNA, followed by a two-step PCR phase that took place under two different conditions and which produced ssDNA in a linear manner. The method is based on partly overlapping annealing temperatures of the forward and reverse primers.