Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms

A rapid procedure was developed for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) directly from sterile sites or mixed flora samples (e.g., nose or inguinal swabs). After a rapid conditioning of samples, the method consists of two main steps: (i) immunomagnetic enrichment in S. aureus and (ii) amplification-detection profile on DNA extracts using multiplex quantitative PCR (5'-exonuclease qPCR, TaqMan). The triplex qPCR assay measures simultaneously the following targets: (i) mecA gene, conferring methicillin resistance, common to both S. aureus and Staphylococcus epidermidis; (ii) femA gene from S. aureus; and (iii) femA gene from S. epidermidis. This quantitative approach allows discrimination of the origin of the measured mecA signal. qPCR data were calibrated using two reference strains (MRSA and methicillin-resistant S. epidermidis) processed in parallel to clinical samples. This 96-well format assay allowed analysis of 30 swab samples per run and detection of the presence of MRSA with exquisite sensitivity compared to optimal culture-based techniques. The complete protocol may provide results in less than 6 h (while standard procedure needs 2 to 3 days), thus allowing prompt and cost-effective implementation of contact precautions.

https://jcm.asm.org/content/jcm/41/1/254.full.pdf

Rapid Detection of Methicillin-Resistant Staphylococcus aureus Directly from Sterile or Nonsterile Clinical Samples by a New Molecular Assay

Fifteen-Year Study of the Changing Epidemiology of Methicillin-Resistant Staphylococcus aureus

In this study we applied serological proteome analysis (Klade, C. S. et al. Proteomics 2001, 1, 890-898) for identification of bacterial vaccine candidate antigens. First, approximately one hundred sera from healthy individuals and patients suffering from Staphylococcus aureus infections were screened for antibodies against staphylococcal lysates and recombinant proteins representing surface antigens. Two pools (healthy donors, patients) each consisting of five sera with the highest antiproteinaceous IgG reactivity were selected. Second, S. aureus COL was grown under different conditions and the number of antigens expressed was monitored by Western blot analysis. Third, surface proteins were enriched by digesting the bacterial cell wall under isotonic conditions and subsequent removal of protoplasts. These protein preparations were resolved by two-dimensional electrophoresis (2-DE) (pI 4-7). 2-DE immunoblotting using the preselected serum pools at 1:10 000-1:100 000 dilutions revealed a number of highly immunogenic staphylococcal proteins. Twenty-one spots were isolated by preparative 2-DE, and analysed by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry sequencing of tryptic peptides. This led to the identification of 15 proteins including known and novel vaccine candidates. Seroreactivity of several antigens including serine-aspartate repeat containing protein D, immuno-dominant staphylococcal antigen and a novel 309 amino acid lipoprotein was independently confirmed by enzyme-linked immunosorbent assay and Western blot analysis of purified recombinant proteins. In conclusion, serological proteome analysis proved to be a powerful tool for the identification of novel staphylococcal antigens, which provide a basis for rational vaccine design.

Identification of vaccine candidate antigens of Staphylococcus aureus by serological proteome analysis.

https://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(15)30039-2/pdf

Methicillin-resistant Staphylococcus aureus: clinical manifestations and antimicrobial therapy

Staphylococcus aureus is recognized as one of the major causes of infections in humans occurring in both the community and the hospital. Up to now one of the most serious aspects as far as treatment of S. aureus infections is concerned is resistance to methicillin, which in clinical terms indicates resistance to all beta-lactam antibiotics. The growing incidence of methicillin-resistant S. aureus (MRSA) infections worldwide, their multidrug resistance, several reservoirs of resistant strains, the facility to spread also outside hospitals and to cause outbreaks requires efficacious infection control measures. For this reason microbiological and epidemiological studies are of crucial importance.

Hryniewicz W

Papers

Epidemiology of MRSA.