The polymerase chain reaction (PCR) technique offers a promising alternative to tissue culture for the rapid and sensitive detection of cytomegalovirus (CMV) infection. However, high levels of background amplification detected in samples containing water but no DNA make interpretation of borderline positive samples extremely difficult and reduce the sensitivity of the assay. The signal from amplification of water or positive samples can be eliminated by DNase treatment, but not by filtration through anisotropic membrane, autoclaving, or ultraviolet irradiation. A lag time of 10 to 12 cycles is observed before the reactions with water will show product formation by liquid hybridization detection. The use of nested PCR eliminates the background and, in serial dilutions of a positive sample, shows a 500- to 1000-fold increase in sensitivity by liquid hybridization detection. We suggest that the background signal is arising from small fragments of DNA, which may be produced by autoclaving viral culture material. Such fragments would escape filtration, and overlapping fragments of DNA can prime one another to form complete mosaic sequences that will then amplify. Nested PCR, appropriately controlled for the number of cycles at each step, should successfully overcome such false positives caused by fragmented DNA, no matter if the contamination occurs at the collection site, in processing, or at the facility performing the test.
iIn critically ill patients, the development of pneumonia results in significant morbidity and mortality and additional health care costs. The accurate and rapid identification of the microbial pathogens in patients with pulmonary infections might lead to targeted antimicrobial therapy with potentially fewer adverse effects and lower costs. Major advances in next-generation sequencing (NGS) allow culture-independent identification of pathogens. The present study used NGS of essentially full-length PCRamplified 16S ribosomal DNA from the bronchial aspirates of intubated patients with suspected pneumonia. The results from 61 patients demonstrated that sufficient DNA was obtained from 72% of samples, 44% of which (27 samples) yielded PCR amplimers suitable for NGS. Out of the 27 sequenced samples, only 20 had bacterial culture growth, while the microbiological and NGS identification of bacteria coincided in 17 (85%) of these samples. Despite the lack of bacterial growth in 7 samples that yielded amplimers and were sequenced, the NGS identified a number of bacterial species in these samples. Overall, a significant diversity of bacterial species was identified from the same genus as the predominant cultured pathogens. The numbers of NGSidentifiable bacterial genera were consistently higher than identified by standard microbiological methods. As technical advances reduce the processing and sequencing times, NGS-based methods will ultimately be able to provide clinicians with rapid, precise, culture-independent identification of bacterial, fungal, and viral pathogens and their antimicrobial sensitivity profiles.
ᮀ Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of hospital-and community-associated infections. The formation of adherent clusters of cells known as biofilms is an important virulence factor in MRSA pathogenesis. Previous studies showed that subminimal inhibitory (sub-MIC) concentrations of methicillin induce biofilm formation in the community-associated MRSA strain LAC. In this study we measured the ability sub-MIC concentrations of eight other β-lactam antibiotics and six non-β-lactam antibiotics to induce LAC biofilm. All eight β-lactam antibiotics, but none of the non-β-lactam antibiotics, induced LAC biofilm. The dose-response effects of the eight β-lactam antibiotics on LAC biofilm varied from biphasic and bimodal to near-linear. We also found that sub-MIC methicillin induced biofilm in 33 out of 39 additional MRSA clinical isolates, which also exhibited biphasic, bimodal and linear dose-response curves. The amount of biofilm formation induced by sub-MIC methicillin was inversely proportional to the susceptibility of each strain to methicillin. Our results demonstrate that induction of biofilm by sub-MIC antibiotics is a common phenotype among MRSA clinical strains and is specific for β-lactam antibiotics. These findings may have relevance to the use of β-lactam antibiotics in clinical and agricultural settings.
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