ABSTRACT
The control of vancomycin-resistant enterococci (VRE) has become an increasing burden on health care resources since their discovery over 20 years ago. Current techniques employed for their detection include time-consuming and laborious phenotypic methods or molecular methods requiring costly equipment and consumables and highly trained staff. An accurate, rapid diagnostic test has the ability to greatly reduce the spread of this organism, which has the ability to colonize patients for long periods, potentially even lifelong. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a technology with the ability to identify organisms in seconds and has shown promise in the identification of other forms of antimicrobial resistance in other organisms. Here we show that MALDI-TOF MS is capable of rapidly and accurately identifying vanB-positive Enterococcus faecium VRE from susceptible isolates. Internal validation of the optimal model generated produced a sensitivity of 92.4% and a specificity of 85.2%. Prospective validation results, following incorporation into the routine laboratory work flow, demonstrated a greater sensitivity and specificity at 96.7% and 98.1%, respectively. In addition, the utilization of MALDI-TOF MS to determine the relatedness of isolates contributing to an outbreak is also demonstrated.
Subject(s)
Disease Outbreaks , Enterococcus faecium/drug effects , Enterococcus faecium/isolation & purification , Gram-Positive Bacterial Infections/epidemiology , Gram-Positive Bacterial Infections/microbiology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Vancomycin Resistance , Enterococcus faecium/chemistry , Humans , Sensitivity and SpecificitySubject(s)
Community-Acquired Infections/diagnosis , Methicillin Resistance , Pneumonia/microbiology , Staphylococcal Infections/diagnosis , Adult , DNA, Bacterial/isolation & purification , Fatal Outcome , Female , Humans , Lung/pathology , Myocardium/pathology , Necrosis , Staphylococcus aureus/genetics , Staphylococcus aureus/isolation & purification , Thrombosis/microbiology , Thrombosis/pathology , Troponin I/bloodABSTRACT
Extended-spectrum beta-lactamases (ESBLs) emerge by point mutation from non-extended-spectrum precursors. The aims of this study were to reveal the basis for variations in resistance levels found in a collection of 21 Klebsiella pneumoniae clinical isolates from Brisbane, Australia. Previous studies have shown that 20 of these isolates possess bla(SHV-11), bla(SHV-2a), and/or bla(SHV-12), and there is an association between the copy numbers of the ESBL-encoding genes and resistance levels. In this study, a real-time PCR method for interrogating the polymorphic sites at codons 238 and 240 was developed, and this confirmed the relationship between mutant gene copy numbers and resistance levels. The bla(SHV) promoter region was cloned from one of the ESBL-expressing isolates, and this showed that bla(SHV) genes exist downstream of two different promoters within this single isolate. These promoters have both been reported previously, and they differ by virtue of the presence or absence of an IS26 insertion. The bla(SHV) copy numbers in cis with the different promoters were measured, and the copy number of the IS26 promoter was correlated with resistance levels. Cloning and analysis of PCR products showed that different bla(SHV) variants existed in cis with individual promoters in individual isolates but that mutant genes were more abundant downstream of the IS26 promoter. There were no ESBL-positive isolates without this promoter. It was concluded that bla(SHV) in cis with the IS26 promoter is located on an amplifiable replicon, and the presence of the IS26 insertion may facilitate the acquisition of an ESBL-positive phenotype.
Subject(s)
Alleles , Gene Dosage , Klebsiella pneumoniae/enzymology , Promoter Regions, Genetic , beta-Lactamases/genetics , Anti-Bacterial Agents/pharmacology , Base Sequence , Drug Resistance, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field , Genetic Variation , Humans , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , Molecular Sequence Data , MutationABSTRACT
AIM: The aim of this study was to assess the discriminatory power and potential turn around time (TAT) of a PCR-based method for the detection of methicillin-resistant Staphylococcus aureus (MRSA) from screening swabs. METHODS: Screening swabs were examined using the current laboratory protocol of direct culture on mannitol salt agar supplemented with oxacillin (MSAO-direct). The PCR method involved pre-incubation in broth for 4 hours followed by a multiplex PCR with primers directed to mecA and nuc genes of MRSA. The reference standard was determined by pre-incubation in broth for 4 hours followed by culture on MSAO (MSAO-broth). RESULTS: A total of 256 swabs was analysed. The rates of detection of MRSA using MSAO-direct, MSAO-broth and PCR were 10.2, 13.3 and 10.2%, respectively. For PCR, the sensitivity, specificity, positive predictive value and negative predictive values were 66.7% (95%CI 51.9-83.3%), 98.6% (95%CI 97.1-100%), 84.6% (95%CI 76.2-100%) and 95.2% (95%CI 92.4-98.0%), respectively, and these results were almost identical to those obtained from MSAO-direct. The agreement between MSAO-direct and PCR was 61.5% (95%CI 42.8-80.2%) for positive results, 95.6% (95%CI 93.0-98.2%) for negative results and overall was 92.2% (95%CI 88.9-95.5%). CONCLUSIONS: (1) The discriminatory power of PCR and MSAO-direct is similar but the level of agreement, especially for true positive results, is low. (2) The potential TAT for the PCR method provides a marked advantage over conventional methods. (3) Further modifications to the PCR method such as increased broth incubation time, use of selective broth and adaptation to real-time PCR may lead to improvement in sensitivity and TAT.
