nitrocefin and Staphylococcal-Infections

Staphylococcus epidermidis is the leading coagulase negative staphylococci (CoNS) species associated with healthcare associated infections. In order to de-escalate antimicrobial therapy, isolates of S. epidermidis lacking the blaZ gene should be eligible for targeted antimicrobial therapy. However, testing the susceptibility of coagulase negative staphylococci (CoNS) to penicillin G is no longer recommended by EUCAST, given the low performances for penicillinase detection in CoNS. The objective of this work was to determine a phenotypic method with high performance for detecting penicillinase production in S. epidermidis.. Four techniques for the detection of penicillinase production (disk diffusion, zone edge test, nitrocefin test, Minimal Inhibitory Concentration (MIC) by automated system Vitek2®) were evaluated on 182 S. epidermidis isolates, using identification of blaZ gene by PCR as the reference method. The performance of the methods for penicillinase detection was compared by the sensitivity, the specificity, the negative predictive value and the positive predictive value, and with Cohen's kappa statistical test. Among the 182 S. epidermidis included in this study, 55 carried the blaZ gene. The nitrocefin test, characterized by a poor sensitivity (91%), was therefore excluded from S. epidermidis penicillinase detection. The algorithm proposed here for the penicillinase detection in S. epidermidis involved two common antimicrobial susceptibility techniques: disk diffusion method and MIC by Vitek2® system. Disk diffusion method, interpreted with a 26 mm breakpoint for penicillin G, was associated with a high sensitivity (98%) and specificity (100%). This method was completed with zone edge test for S. epidermidis with penicillin G diameter from 26 to 35 mm (sensitivity of 98%). The Vitek2® system is associated with a low sensitivity (93%) and a high specificity (99%) This low sensitivity is associated with false negative results, in isolates with 0.12 mg/L Penicillin G MIC values and blaZ positive. Thus for penicillin G MIC of 0.06 mg/L or 0.12 mg/L, a second step with disc diffusion method is suggested.. According to our results, the strategy proposed here allows the interpretation of penicillin G susceptibility in S. epidermidis isolates, with an efficient detection of penicillin G resistance.

Topics: Algorithms; Anti-Bacterial Agents; Cephalosporins; Genes, Bacterial; Humans; Microbial Sensitivity Tests; Penicillin G; Penicillin Resistance; Penicillinase; Phenotype; Polymerase Chain Reaction; Sensitivity and Specificity; Staphylococcal Infections; Staphylococcus epidermidis

Methicillin and oxacillin-hydrolyzing enzymes of 6 borderline methicillin-resistant and 1 methicillin-resistant Staphylococcus aureus strains isolated from human clinical samples and 4 borderline methicillin-resistant S. aureus strains isolated from bovine mastitis were investigated. As previous studies suggested the involvement of an additional enzyme besides the penicillinase BlaZ in the determination of borderline resistance, we analyzed the expressed extracellular and membrane-bound β-lactamases with 2-D gel electrophoresis and mass spectrometry. Our analysis showed that the penicillin-hydrolyzing BlaZ alone was responsible for the hydrolysis of both methicillin and oxacillin. All supernatant and membrane fractions contained the same enzyme with slight sequence variations. The size and pI of the proteins were also variable, probably due to spontaneous hydrolysis and/or posttranslational modifications. Interestingly, we found also cytotoxins and other virulence factors in some nitrocefin-hydrolyzing dots, suggesting that those proteins might have a role in the reduction of local antibiotic concentration.

Topics: Animals; Anti-Bacterial Agents; beta-Lactamases; Cattle; Cephalosporins; Drug Resistance, Bacterial; Female; Humans; Membrane Proteins; Methicillin-Resistant Staphylococcus aureus; Penicillinase; Penicillins; Proteomics; Staphylococcal Infections; Staphylococcus aureus

Penicillin resistance identification tests are important in veterinary medicine. Six enzyme assays and a PCR test were compared for the detection of beta-lactamase production or the beta-lactamase gene in 175 staphylococcal isolates. We conclude that the PCR test and two nitrocefin-based assays can be recommended for routine clinical use.

Topics: Animals; beta-Lactamases; Cattle; Cattle Diseases; Cephalosporins; Coagulase; Colorimetry; Indicators and Reagents; Mastitis, Bovine; Microbial Sensitivity Tests; Polymerase Chain Reaction; Sensitivity and Specificity; Staphylococcal Infections; Staphylococcus; Staphylococcus aureus

Resistance of Staphylococcus aureus to penicillin G is common among isolates from bovine mastitis. We determined phenotypic resistance to penicillin G for 151 S. aureus isolates derived from dairy cows with intramammary infection by two methods. The methods were determination of minimum inhibitory concentration (MIC) by a standard agar dilution technique and direct testing of beta-lactamase production using a chromogenic cephalosporin, nitrocefin. The results from these tests were compared with the presence of the beta-lactamase (blaZ) gene in the isolates, which was detected by polymerase chain reaction (PCR). Testing beta-lactamase production with nitrocefin was more predictive for the presence of the blaZ gene than the agar dilution method and the results of the former agreed highly with the presence of the blaZ gene in the isolates. In contrast, the resistance breakpoint generally used in the agar dilution method may be too high for prediction of penicillin resistance in S. aureus isolates with borderline MICs. Using this method, 40% of the isolates possessing the blaZ gene were classified as susceptible; however, majority of these isolates produced beta-lactamase when tested with nitrocefin.

Topics: Animals; Anti-Bacterial Agents; beta-Lactamases; Cattle; Cephalosporins; DNA, Bacterial; Female; Mastitis, Bovine; Microbial Sensitivity Tests; Penicillin G; Penicillin Resistance; Polymerase Chain Reaction; Staphylococcal Infections; Staphylococcus aureus; Statistics, Nonparametric

One hundred ovine milk samples were subjected to bacteriological analysis to detect staphylococci. Twenty-four staphylococcal strains isolated were characterised for methicillin resistance with disk diffusion test (DDT) after incubation at 24 and 48 h, oxacillin agar screen test, Minimal Inhibitory Concentration (MIC), nitrocefin test for beta-lactamase production and PCR for the mecA gene. Nine staphylococcal strains resulted resistant in DDT; some differences in the halo diameter at double incubation period were noted; eight of these strains were resistant in MIC test; just one strain was positive to oxacillin agar screen test. All strains were mecA negative by PCR and positive by nitrocefin test. On the basis of these results methicillin-resistant strains can be classified as beta-lactamase hyperproducers.

Topics: Animals; Bacterial Proteins; beta-Lactamases; Carrier Proteins; Cephalosporins; DNA, Bacterial; Female; Hexosyltransferases; Immunodiffusion; Indicators and Reagents; Mastitis; Methicillin Resistance; Microbial Sensitivity Tests; Milk; Muramoylpentapeptide Carboxypeptidase; Penicillin-Binding Proteins; Peptidyl Transferases; Polymerase Chain Reaction; Sheep; Sheep Diseases; Staphylococcal Infections; Staphylococcus