The detection of Salmonella using a combined immunomagnetic separation and ELISA end-detection procedure

The aim of this study was to develop a rapid immunoassay to detect Salmonella bacteria. Skimmed milk powder (SMP) in buffered peptone water was inoculated with six Salmonella strains (Salm. typhimurium, Salm. virchow, Salm. enteritidis, Salm. give, Salm. ealing and Salm. arizonae) at three inoculum levels (about 2-200 cfu 25 g(-1) SMP) and incubated (37 degrees C) overnight. Heat-treated salmonella cells were immobilized on paramagnetic particles and detected within 3 h using the Salmonella genus-specific monoclonal antibody M105 in a microtitre plate based assay. The rapid Salmonella detection method combining immunomagnetic separation and ELISA had a total isolation and detection time of less than 24 h, which is significantly shorter than the conventional techniques requiring 72-96 h. The technique had a sensitivity limit of 10(5)-10(6) cfu ml(-1).     

A nonuniform sequential procedure for diagnosing Staphylococcus aureus

The possibility of the sharp differentiation of S. aureus in the nonuniform successive statistical identification procedure with the use of signs ranked by their differential information content. The procedure starting from the signs with greater information content permits the identification of S. aureus in 100% of cases.     

A pentaplex PCR assay for the rapid detection of methicillin-resistant Staphylococcus aureus and Panton-Valentine Leucocidin

Background  

Staphylococcus aureus is a major human pathogen, especially methicillin-resistant S. aureus (MRSA), which causes a wide range of hospital and community-acquired infections worldwide. Conventional testing for detection of MRSA takes 2–5 days to yield complete information of the organism and its antibiotic sensitivity pattern.     

Immunomagnetic separation and conventional culture procedure for detection of naturally occurring Salmonella in raw pork sausages and chicken meat

The aim of the study was to compare immunomagnetic separation (IMS) and conventional selective enrichment procedures using selenite cystine broth (SC) and Rappaport–Vassiliadis broth (RV) in 137 naturally contaminated food samples (69 raw pork sausages and 68 chicken meat). The utilization of SC or IMS appeared to be the most appropriate enrichment procedure: 15 out of 18 Salmonella -positive samples (83·3%) were detected by SC and 12 (66·7%) by IMS; RV yielded only seven positive isolations (38·9%). However, RV yielded the highest count of Salmonella colonies per plate and the lowest interference by competing organisms. IMS could become a reliable alternative to standard enrichment procedures and a combined IMS and selective enrichment broth could increase the chance of Salmonella recovery.     

Lysostaphin lysis procedure for detection of Staphylococcus aureus by the firefly bioluminescent ATP method

The objective of this study was to examine the use of lysostaphin as an ATP-extracting agent for the estimation of Staphylococcus aureus cell number by a rapid bioluminescent ATP method. The results of the study showed that lysostaphin (22 U/ml) was able to lyse most of the S. aureus cells (greater than 99.9%) at room temperature in 1 min; ATP of S. aureus cells extracted by the lysostaphin lysis procedure was stable for 24 h in the presence of EDTA; there was a linear relationship between the ATP content and the number of S. aureus cells (ranging from 10(4) to 10(6) CFU/ml); and the lysis of S. aureus cells by lysostaphin allowed estimation of the number of S. aureus cells in mixed cultures and in meat samples.     

Evaluation of a rapid culture-based screening test for detection of methicillin resistant Staphylococcus aureus

The performance of a culture based assay, BacLite Rapid MRSA for the rapid detection (5 hours) of methicillin resistant Staphylococcus aureus (MRSA) from specimens (n = 377) obtained from nares, throat, wounds and perineum was investigated. Compared to culture based reference methods (chromogenic MRSA ID (bioMerieux)), selective enrichment broth, PBP2' latex agglutination (Oxoid) and VITEK 2 identification (bioMerieux), an overall sensitivity of 71% with a 82% specificity and a negative predictive value (NPV) of 95% was provided. The Baclite test is rapid and easy to use and has the advantage of a culture-based detection method for MRSA.     

Lysostaphin lysis procedure for detection of Staphylococcus aureus by the firefly bioluminescent ATP method.

The objective of this study was to examine the use of lysostaphin as an ATP-extracting agent for the estimation of Staphylococcus aureus cell number by a rapid bioluminescent ATP method. The results of the study showed that lysostaphin (22 U/ml) was able to lyse most of the S. aureus cells (greater than 99.9%) at room temperature in 1 min; ATP of S. aureus cells extracted by the lysostaphin lysis procedure was stable for 24 h in the presence of EDTA; there was a linear relationship between the ATP content and the number of S. aureus cells (ranging from 10(4) to 10(6) CFU/ml); and the lysis of S. aureus cells by lysostaphin allowed estimation of the number of S. aureus cells in mixed cultures and in meat samples.     

Development and application of a triplex-PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus from pigs

A triplex-PCR assay was developed and evaluated for rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) recovered from various biological samples of pig. Three sets of primers were designed to target mecA, 16S rRNA and nuc genes of MRSA. The specific amplification generated three bands on agarose gel, with sizes 280 bp for mecA, 654 bp for 16S rRNA and 481 bp for nuc, respectively. A potential advantage of the PCR assay is its sensitivity with a detection limit of 10² CFU per ml of bacteria. In all, 79 MRSA isolates recovered from various samples of pigs were subjected to the amplification by the triplex-PCR assay and all the isolates yielded three bands corresponding to the three genes under this study. No false-positive amplification was observed, indicating the high specificity of the developed triplex-PCR assay. This assay will be a useful and powerful method for differentiation of MRSA from methicillin-sensitive S. aureus, coagulase-negative methicillin-resistant staphylococci and coagulase-negative methicillin-sensitive staphylococci.     

An improved amperometric immunosensor for the detection and enumeration of protein A-bearing Staphylococcus aureus

An amperometric immunosensor specific to the protein A of Staphylococcus aureus, was developed using the direct electrochemical detection of phenol produced by alkaline phosphatase from phenylphosphate. The immunosensor could detect protein A at 0.01 ng/ml and could reliably detect and quantify pure cultures of protein A-bearing Staph. aureus above 10(3) cfu/ml. A similar sensitivity of detection was obtained with samples of beef and milk.     

A novel method for rapid production and purification of exfoliative toxin A of Staphylococcus aureus

Three strains of Streptococcus dysgalactiae subsp. dysgalactiae (UT516, UT519, ATCC 27957) were used to determine if bovine lactoferrin (Lf) binds to bacterial cells by biotin avidin binding assay (BABA), enzyme-linked immunosorbent assay (ELISA), and binding inhibition assay. Binding assays revealed that all strains of S. dysgalactiae subsp. dysgalactiae (S. dysgalactiae) evaluated in this study bound to Lf. However, differences in Lf binding capability among strains and between methods used were detected. Binding of Lf was not inhibited by transferrin (Tf) and Lf moiety molecules (mannose, galactose, and lactose) but by Lf. This study demonstrates that S. dysgalactiae bound to bovine Lf in a specific manner.     

A rapid procedure for detection of bacterial amino acid decarboxylases

The detection of decarboxylases of arginine, glutamic acid, histidine, lysine, ornithine, phenylalanine, tryptophan, and tyrosine in bacteria by thin-layer chromatography on polyamide sheets is described. The bacteria were grown on agar medium plates supplemented with eight amino acids at pH 5.5 for induction of amino acid decarboxylases, then transferred to amine-production media. The decarboxylation products in the spent media (amines and/or γ-amino-n-butyric acid) were dansylated and the dansyl derivatives were separated by thin-layer chromatography on polyamide sheets. This method requires only two separate incubations of the decarboxylase-induced bacteria in amine-production media for 1 h at 37°C for simultaneous detection of eight bacterial amino acid decarboxylases using 0.4 μl of the spent media.     

A rapid assay procedure for thiamine-binding protein of Escherichia coli

The advantages associated with the described immobilized enzyme reactor include: (1) Use of the common rate equations and kinetic parameters. (2) Detection of significant lag periods. (3) Quantitative measure for non-covalently attached enzyme. (4) The means for washing the immobilized enzyme allows for the repeated use of the same matrix-bound enzyme. (5) Constant temperature control. (6) Both unbound native and matrix-bound enzyme may be reacted under identical conditions. (7) No grinding of the glass-bound enzyme or other matrix fragmentation occurs because no abrasive forces are required for stirring.     

Immunity induced by Staphylococcus aureus surface protein A was protective against lethal challenge of Staphylococcus aureus in BALB/c mice

Staphylococcus aureus is the most common cause of hospital-acquired bacteremia. Due to emergence of antibiotic-resistant strains, these infections present a serious public health threat. In this study, to develop a broadly protective vaccine, we tested whether immune responses induced by several proteins associated with S. aureus toxicity could protect mice from lethal challenge with human clinical S. aureus isolate USA300. We found that the surface protein A (SasA) of S. aureus could protect mice from lethal challenge of the bacteria.     

Immunological detection of penicillin-binding protein 2'' in clinical isolates of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

The additional penicillin-binding protein (PBP 2') that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been detected immunologically in strains from a variety of world-wide locations. This additional protein has also been definitively identified both immunologically and as a PBP in methicillin-resistant strains of S. epidermidis (MRSE). The assay described is rapid, specific and sensitive and has been used to detect PBP 2' in S. haemolyticus but not in beta-lactam resistant Streptococci.