ABSTRACT
AIM: Determination of virulence of enterococci strains isolated from clinical material from humans on pheno- and genotype levels. MATERIALS AND METHODS: 30 strains of enterococci isolated from wound exudate, urine, newborn skin lavage were used in the study. Strain identification was carried out by multiplex PCR. Hemolytic activity was determined by dish method, gelatinase - by dissolution of gelatin column, proteolytic--by biuret method; genes coding virulence factor synthesis (gelE, sprE, cylM, cylB, cylA, cylLs, cylL1, ESP, HYL, ASA)--by using PCR. RESULTS: Clinical isolates of enterococci were assigned to E. faecalis and E. faecium species. Virulence factors on phenotype and genotype levels were detected in both species. CONCLUSION: Genetic determinants of virulence are more widespread among clinical isolates of E.faecalis species. Set of genes coding virulence factors in E. faecalis depends on biotope. Gene coding hyaluronidase synthesis is characteristic for E. faecium. A correlation between phenotypic manifestation of features and enterococci genotype was detected.
Subject(s)
Bacterial Proteins/isolation & purification , Enterococcus faecalis/pathogenicity , Enterococcus faecium/pathogenicity , Gram-Positive Bacterial Infections/microbiology , Virulence Factors/isolation & purification , Adult , Bacterial Proteins/urine , Bacterial Typing Techniques , Enterococcus faecalis/enzymology , Enterococcus faecalis/isolation & purification , Enterococcus faecium/enzymology , Enterococcus faecium/isolation & purification , Female , Humans , Hyaluronoglucosaminidase/isolation & purification , Hyaluronoglucosaminidase/urine , Infant, Newborn , Pregnancy , Skin/microbiology , Urinary Tract Infections/microbiology , Virulence , Virulence Factors/urine , Wounds and Injuries/microbiologyABSTRACT
Murine models of urinary tract infection (UTI) have provided substantial data identifying uropathogenic E. coli (UPEC) virulence factors and assessing their expression in vivo. However, it is unclear how gene expression in these animal models compares to UPEC gene expression during UTI in humans. To address this, we used a UPEC strain CFT073-specific microarray to measure global gene expression in eight E. coli isolates monitored directly from the urine of eight women presenting at a clinic with bacteriuria. The resulting gene expression profiles were compared to those of the same E. coli isolates cultured statically to exponential phase in pooled, sterilized human urine ex vivo. Known fitness factors, including iron acquisition and peptide transport systems, were highly expressed during human UTI and support a model in which UPEC replicates rapidly in vivo. While these findings were often consistent with previous data obtained from the murine UTI model, host-specific differences were observed. Most strikingly, expression of type 1 fimbrial genes, which are among the most highly expressed genes during murine experimental UTI and encode an essential virulence factor for this experimental model, was undetectable in six of the eight E. coli strains from women with UTI. Despite the lack of type 1 fimbrial expression in the urine samples, these E. coli isolates were generally capable of expressing type 1 fimbriae in vitro and highly upregulated fimA upon experimental murine infection. The findings presented here provide insight into the metabolic and pathogenic profile of UPEC in urine from women with UTI and represent the first transcriptome analysis for any pathogenic E. coli during a naturally occurring infection in humans.
Subject(s)
Biomarkers/urine , Escherichia coli Infections/genetics , Escherichia coli Proteins/genetics , Escherichia coli/pathogenicity , Urinary Tract Infections/microbiology , Virulence Factors/genetics , Adult , Aged , Aged, 80 and over , Animals , Blotting, Western , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Infections/microbiology , Escherichia coli Infections/urine , Escherichia coli Proteins/urine , Female , Gene Expression Profiling , Hemagglutination , Humans , Mice , Mice, Inbred CBA , Middle Aged , Oligonucleotide Array Sequence Analysis , RNA, Bacterial/genetics , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Urinary Tract Infections/genetics , Urinary Tract Infections/urine , Virulence Factors/urine , Young AdultABSTRACT
A new approach for the detection of virulence factors of Staphylococcus aureus and Staphylococcus epidermidis using an electrical protein array chip technology is presented. The procedure is based on an enzyme-linked sandwich immunoassay, which includes recognition and binding of virulence factors by specific capture and detection antibodies. Detection of antibody-bound virulence factors is achieved by measuring the electrical current generated by redox recycling of an enzymatically released substance. The current (measured in nanoampere) corresponds to the amount of the target molecule in the analyzed sample. The electrical protein chip allows for a fast detection of Staphylococcus enterotoxin B (SEB) of S. aureus and immunodominant antigen A homologue (IsaA homologue) of S. epidermidis in different liquid matrices. The S. aureus SEB virulence factor could be detected in minimal medium, milk, and urine in a concentration of 1 ng/ml within less than 23 min. Furthermore, a simultaneous detection of SEB of S. aureus and IsaA homologue of S. epidermidis in a single assay could be demonstrated.
