Roman Kotłowski

Peptidoglycan hydrolases-potential weapons against Staphylococcus aureus

Bacteria of the genus Staphylococcus are common pathogens responsible for a broad spectrum of human and animal infections and belong to the most important etiological factors causing food poisoning. Because of rapid increase in the prevalence of isolation of staphylococci resistant to many antibiotics, there is an urgent need for the development of new alternative chemotherapeutics. A number of studies have recently demonstrated the strong potential of peptidoglycan hydrolases (PHs) to control and treat infections caused by this group of bacteria. PHs cause rapid lysis and death of bacterial cells. The review concentrates on enzymes hydrolyzing peptidoglycan of staphylococci. Usually, they are characterized by high specificity to only Staphylococcus aureus cell wall components; however, some of them are also able to lyse cells of other staphylococci, e.g., Staphylococcus epidermidis-human pathogen of growing importance and also other groups of bacteria. Some PHs strengthen the bactericidal or bacteriostatic activity of common antibiotics, and as a result, they should be considered as component of combined therapy which could definitely reduced the development of bacterial resistance to both enzymes and antibiotics. The preliminary research revealed that most of these enzymes can be produced using heterologous, especially Escherichia coli expression systems; however, still much effort is required to develop more efficient and large-scale production technologies. This review discusses current state on knowledge with emphasis on the possibilities of application of PHs in the context of therapeutics for infections caused by staphylococci.

PCR Amplification with Primers Based on IS2404 and GC-Rich Repeated Sequence Reveals Polymorphism in Mycobacterium ulcerans

ABSTRACT We describe a simple genotyping method for Mycobacterium ulcerans based on PCR amplification of genomic regions between IS2404 and a frequently repeated GC-rich sequence. Application of this method to a global collection produced 10 M. ulcerans genotypes corresponding to their geographic origin.

PCR-Based Genotyping of Mycobacterium tuberculosis with New GC-Rich Repeated Sequences and IS6110 Inverted Repeats Used as Primers

ABSTRACT In the present study we attempted to develop a PCR-based epidemiological tool for the differentiation of Mycobacterium tuberculosis isolates. Use of the designed primers Mtb1 (5′-CCG-GCG-GGG-CCG-GCG-G) and Mtb2 (5′-CGG-CGG-CAA-CGG-CGG-C) targeting frequently repeated 16-bp sequences in combination with primers sited at the inverted repeats flanking IS6110 allowed differentiation of M. tuberculosis isolates.

Isolation and identification of Mycobacterium avium subspecies silvaticum from a horse

Routine cultivation methods are able to distinguish between isolates of the Mycobacterium avium and the Mycobacterium tuberculosis complex. However, molecular tools are needed to further identify the several subspecies in the M. avium complex, especially for the subspecies avium and silvaticum. A rapid technique using HhaI restriction digestion of a 349 bp amplification product of the 85B antigen (α-antigen) gene was used for the identification of M. avium subsp. silvaticum in a three-year-old gelding presenting with caseous, necrotizing, granulomatous lesions. The result was confirmed by sequencing of the 85B antigen gene.

Expression and intein-mediated purification of novel staphylokinase SakSTAR with reduced immunogenicity and antiplatelet and antithrombin activation.

In an effort to combine the benefits of fibrinolytics, such as staphylokinase (Sak), with those of thrombin inhibitors for the prevention of vessel reocclusion after vascular injury, we produced chimeric protein with plasminogen activator and thrombin-inhibiting properties. This fusion protein was a construct consisting of Sak (SakSTAR) lengthened about 36 amino acids from the C-terminus end of hirudin. We inserted 16 point mutations into the sequence of the gene encoding SakSTAR for reduced antibody binding from 50% to about 17% and inserted two RGD sequences for antiplatelet activity. The inhibition rate of platelet aggregation was 27%. Moreover, we proposed an efficient method of expression and purification in which we used 16 mg/L of anEscherichia coli strain of this novel fusion protein and retained full biologic activities toward plasminogen and thrombin.

Use of Escherichia coli Nissle 1917 producing recombinant colicins for treatment of IBD patients

Patients with Crohns Disease and Ulcerative Colitis infected with Adherent-Invasive Escherichia coli strains constitute the largest group among Inflammatory Bowel Disease subjects, when taking into account all known etiological agents of the disease. A possible link between these pathogenic bacteria and inflammation process has gained the confidence in recently published papers. Observed enteric neuroglial cells apoptosis and epithelial gaps of ileum are probably the key manifestations of inflammation, which has been shown in IBD patients in contrary to the samples taken from healthy individuals. The cascade of consecutive events from bacterial infection via inflammation to excessive apoptosis in IBD patients leads up to the aim of our hypothesis about designing of new therapeutic strategy directed to Adherent-Invasive E. coli strains. The main advantage of biological method, which will rely on application of E. coli Nissle 1917 strain as a carrier for specific recombinant colicins against AIEC strains, could probably cause a long-lasting remission of inflammation in CD and UC patients.

Correlation between the number of Pro-Ala repeats in the EmrA homologue of Acinetobacter baumannii and resistance to netilmicin, tobramycin, imipenem and ceftazidime.

Acinetobacter baumannii coccobacilli are dangerous to patients in intensive care units because of their multidrug resistance to antibiotics, developed mainly in the past decade. This study aimed to examine whether there is a significant correlation between the number of Pro-Ala repeats in the CAP01997 protein, the EmrA homologue of A. baumannii, and resistance to antibiotics. A total of 79 multidrug-resistant A. baumannii strains isolated from patients were analysed. Resistance to antibiotics was determined on Mueller-Hinton agar plates using the Kirby-Bauer disk diffusion method. The number of CCTGCA repeats encoding Pro-Ala repeats in CAP01997 was determined by PCR and capillary electrophoresis. The 3D models of CAP01997 containing Pro-Ala repeats were initially generated using RaptorX Structure Prediction server and were assembled with EasyModeller 4.0. The models were embedded in a model bacterial membrane based on structural information from homologous proteins and were refined using 100-ns molecular dynamics simulations. The results of this research show significant correlation between susceptibility to netilmicin, tobramycin and imipenem and the number of repeated Pro-Ala sequences in the CAP01997 protein, a homologue of the Escherichia coli transporter EmrA. Predicted structures suggest potential mechanisms that confer drug resistance by reshaping the cytoplasmic interface between CAP01997 protein and the critical component of the multidrug efflux pump homologous to EmrB. Based on these results, we can conclude that the CAP01997 protein, an EmrA homologue of A. baumannii, confers resistance to netilmicin, tobramycin and imipenem, depending on the number of Pro-Ala repeats.

A novel method of Mycobacterium tuberculosis complex strain differentiation using polymorphic GC-rich gene sequences.

Tuberculosis is one of the leading infectious diseases. In this work, a new genotyping method of Mycobacterium tuberculosis (Mtb) complex strain is presented. 27 Mtb genomes were analyzed for the presence of length polymorphism within polymorphic GC-rich gene sequences. Four genes, Rv3345c, Rv3507, Rv0747 and Rv3511, showing variation in length depending on the Mtb strain were selected for designing primer sequences flanking variable regions for the PCR method. Identification of 16 genotypes among 27 analyzed genomes demonstrated usefulness of our genotyping method in differentiation of Mtb genomes based on sequence polymorphism in the four PGRS genes.

Intra-operative biopsy in chronic sinusitis detects pathogenic Escherichia coli that carry fimG/H, fyuA and agn43 genes coding biofilm formation

The aim of this study was to investigate whether or not surgical biopsy of sinus tissue in chronic sinusitis, not responsive to treatment, would detect E. coli. We intended to evaluate E. coli virulence genes, therefore dispute the causal role of such an unusual microorganism in chronic sinusitis, as well as consider effective pathogen-targeted therapy. Patients with E. coli isolated by intra-operative puncture biopsy were included in the study. Genetic analysis of E. coli isolates, including phylogenetic grouping and virulence factor characteristics, were done by multiplex PCR. We identified 26 patients with chronic sinusitis, in which 26 E. coli isolates were cultured. The E. coli isolates belonged mainly to pathogenic phylogenetic group B2, and carried multiple virulence genes. Three genes in particular were present in all (100%) of examined isolates, they were (1) marker agn43 gene for forming biofilm, (2) type 1 fimbriae (fimG/H gene) and (3) yersiniabactin receptor (fyuA). Furthermore, a pseudo-phylogenetic tree of virulence genes distribution revealed possible cooperation between agn43, fimG/H, and fyuA in the coding of biofilm formation. Intra-operative-biopsy and culture-based therapy, targeting the isolated E. coli, coincided with long-term resolution of symptoms. This is the first report demonstrating an association between a highly pathogenic E. coli, chronic sinus infection, and resolution of symptoms upon E. coli targeted therapy, a significant finding due to the fact that E. coli has not been considered to be a commensal organism of the oropharynx or sinuses. We postulate that the simultaneous presence of three genes, each coding biofilm formation, may in part account for the chronicity of E. coli sinusitis.