Improved detection of blood stream pathogens by real-time PCR in severe sepsis

Objective: Evaluation of the technical and diagnostic feasibility of commercial multiplex real-time polymerase chain reaction (PCR) for detection of blood stream infections in a cohort of intensive care unit (ICU) patients with severe sepsis, performed in addition to conventional blood cultures. Design: Dual-center cohort study. Setting: Surgical ICU of two university hospitals. Patients and participants: One hundred eight critically ill patients fulfilling the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) severe sepsis criteria were included. Interventions: None. Measurements and results: PCR results obtained in 453 blood samples from 108 patients were compared with corresponding blood culture results. PCR resulted in a twofold higher positivity rate when compared with conventional blood culture (BC) testing (114 versus 58 positive samples). In 40 out of 58 PCR positive assays the results of the corresponding blood cultures were identical to microorganisms detected by PCR. In 18 samples PCR and BC yielded discrepant results. Compared with conventional blood culture the sensitivity and specificity of PCR was 0.69 and 0.81, respectively. Further evaluation of PCR results against a constructed gold standard including conventional microbiological test results from other significant patient specimen (such as bronchio-alveolar lavage fluid, urine, swabs) and additionally generated clinical and laboratory information yielded sensitivity of 0.83 and specificity of 0.93. Conclusions: Our cohort study demonstrates improved pathogen detection using PCR findings in addition to conventional blood culture testing. PCR testing provides increased sensitivity of blood stream infection. Studies addressing utility including therapeutic decision-making, outcome, and cost-benefit following diagnostic application of PCR tests are needed to further assess its value in the clinical settin

Compensatory Adaptation to the Loss of Biological Fitness Associated with Acquisition of Fusidic Acid Resistance in Staphylococcus aureus

Recent studies have shown that individual amino acid exchanges within elongation factor G (EF-G) cause fusidic acid resistance in Staphylococcus aureus. The data from the present study illustrate that the fusidic acid resistance-mediating amino acid substitutions P406L and H457Y are associated with a marked impairment of the biological fitness of S. aureus. In particular, strains producing EF-G derivatives with these mutations showed reduced growth, decreased plasma coagulase activity, and an impaired capability to compete with the isogenic wild-type strain. Second-site mutations within EF-G, such as A67T and S416F, that have been encountered in clinical fusidic acid-resistant isolates containing the amino acid exchanges P406L and H457Y, respectively, were shown not to contribute to resistance. Furthermore, the substitution A67T had no impact on the biological fitness in vitro. The exchange S416F, however, was found to function as a fitness-compensating mutation in S. aureus carrying the substitution H457Y in EF-G. In conclusion, the data presented in this report provide evidence at the molecular level that the deleterious effects of fusidic acid resistance-mediating exchanges within EF-G of S. aureus can be reduced considerably by specific compensating mutations in this target protein. This compensatory adaptation most likely plays a significant role in the stabilization of resistant bacteria within a given population

Biological Cost of Rifampin Resistance from the Perspective of Staphylococcus aureus

Resistance determinants that interfere with normal physiological processes in the bacterial cell usually cause a reduction in biological fitness. Fitness assays revealed that 17 of 18 in vitro-selected chromosomal mutations within the rpoB gene accounting for rifampin resistance in Staphylococcus aureus were associated with a reduction in the level of fitness. There was no obvious correlation between the level of resistance to rifampin and the level of fitness loss caused by rpoB mutations. Among 23 clinical rifampin-resistant S. aureus isolates from six countries, only seven different rpoB genotypes could be identified, whereby the mutation 481His→Asn was present in 21 (91%) of these 23 isolates. The mutation 481His→Asn, in turn, which confers low-level rifampin resistance on its own, was not shown to be associated with a cost of resistance in vitro. The restriction to distinct mutations that confer rifampin resistance in vivo, as demonstrated here, appears to be determined by the Darwinian fitness of the organisms