[Rapid antibiotic susceptibility test in Clinical Microbiology]. / Antibiograma rápido en Microbiología Clínica.

The most widely used antibiotic susceptibility testing methods in Clinical Microbiology are based on the phenotypic detection of antibiotic resistance by measuring bacterial growth in the presence of the antibiotic being tested. These conventional methods take typically 24hours to obtain results. A review is presented here of recently developed techniques for the rapid determination of antibiotic susceptibility. Data obtained with different methods such as molecular techniques, flow cytometry, chemiluminescence, mass spectrometry, commercial methods used in routine work, colorimetric methods, nephelometry, microarrays, microfluids, and methods based on cell disruption and sequencing, are analyzed and discussed in detail.

A two-hour antibiotic susceptibility test by ATP-bioluminescence.

The antibiotic susceptibility test (AST) in Clinical Microbiology laboratories is still time-consuming, and most procedures take 24h to yield results. In this study, a rapid antimicrobial susceptibility test using ATP-bioluminescence has been developed. The design of method was performed using five ATCC collection strains of known susceptibility. This procedure was then validated against standard commercial methods on 10 strains of enterococci, 10 staphylococci, 10 non-fermenting gram negative bacilli, and 13 Enterobacteriaceae from patients. The agreement obtained in the sensitivity between the ATP-bioluminescence method and commercial methods (E-test, MicroScan and VITEK2) was 100%. In summary, the preliminary results obtained in this work show that the ATP-bioluminescence method could provide a fast and reliable AST in two hours.

A two-hour procedure for determining the susceptibility of enterococci and staphylococci to antibiotics by a colourimetric method.

INTRODUCTION: Rapid determination of the antibiotic susceptibility test in bacteria remains a challenge for Clinical Microbiology laboratories. METHODS: An improvement in the colorimetric antimicrobial susceptibility testing performed with resazurin in enterococci and staphylococci has been carried out. The design of method was performed using two collection strains, which have a known susceptibility. This procedure was then validated against standard commercial methods on 15 strains of staphylococci and 15 strains of enterococci from patients. RESULTS: The essential agreement between the colorimetric method and commercial methods (E-test, MicroScan and VITEK2) was 100%. CONCLUSION: Resazurin allows us to obtain a reliable antibiotic susceptibility test in staphylococci and enterococci in less than two hours.

[In vitro activity of ceftaroline against Spanish isolates of Staphylococcus aureus: a multicenter study]. / Estudio multicéntrico sobre la actividad in vitro de ceftarolina frente a Staphylococcus aureus aislados en España.

INTRODUCTION: Ceftaroline fosamil is a new-generation antimicrobial agent of cephalosporins subgroup. It is the first commercially available beta-lactam antibiotic that exhibits activity against methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study is to determine the in vitro Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values of ceftaroline against S.aureus strains (including MRSA). MATERIAL AND METHODS: A multicenter study involving four hospitals representative of the Spanish geography was performed. MIC and MBC values against both the methicillin-resistant and sensitive strains of S.aureus (MRSA and methicillin-sensitive S.aureus [MSSA]) were determined using a broth microdilution method. RESULTS: A total of 266 S.aureus strains were analyzed (95 MRSA and 171 MSSA). Ceftaroline bacterial sensitivity showed a mean MIC of 0.227 µg/ml (SD=0.146; range, 0.06 to 1 µg/ml). All MIC values of the 266 strains tested belonged to the sensitive category (value ≤ 1 µg/ml). Intermediate or resistant strains were not detected. MIC50 and MIC90 values for MRSA were 0.25 and 0.5 µg/ml, respectively (range=0.125-1 µg/ml). MSSA strains showed MIC50 and MIC90 values of 0.125 and 0.25 µg/ml, respectively (range=0.125-0.5 µg/ml). MBC50 and MBC90 values for MRSA were 0.5 and 1 µg/ml, respectively (range=0.125-1 µg/ml). MSSA strains showed MBC50 and MBC90 values of 0.25 and 0.25 µg/ml, respectively (range=0.125-0.5 µg/ml). CONCLUSION: Ceftaroline shows excellent in vitro activity against S.aureus, including MRSA strains. Therefore, this antibiotic may be a promising alternative for the treatment of infections caused by this bacterium.

New procedure for rapid identification of microorganisms causing urinary tract infection from urine samples by mass spectrometry (MALDI-TOF).

INTRODUCTION: Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry is widely established as a technique in clinical microbiology laboratories for the identification of microorganisms. Using this technique, it is also possible to obtain the identification of microorganisms from untreated urine samples. METHODS: In this study, a differential centrifugation protocol and a criterion for validation of the results in order to achieve microbial identification from untreated urine samples are proposed. Additionally, the sensitivity of the analytical procedure in monobacterial urine samples has been evaluated. RESULTS: A 90% sensitivity (confidence interval of 81.96%-94.84%) was obtained in urine samples with bacterial counts of ≥1×10(5)CFU/ml, and it was possible to improve the percentages of direct identifications from urine samples with bacterial counts of <1×10(5)CFU/ml. CONCLUSION: It is concluded that the MALDI-TOF system is both fast and reliable in the identification of individual microorganisms from untreated urine samples with counts of ≥1×10(5)CFU/ml.

Aortic valve-derived calcifyng nanoparticles: no evidence of life.

INTRODUCTION AND OBJECTIVES: Calcifying nanoparticles, also known as "nanobacteria," are very small bacteria-like structures (0.1-0.5 µm) with the ability to facilitate the precipitation and growth of calcium phosphate in pathological conditions and have been associated with aortic valve calcification. The status of nanobacteria is controversial; some have proposed that they are a new class of living organism while others describe calcifying nanoparticles as mineralo-fetuin complexes. The objective of the present study is to elucidate if calcifying nanoparticles are living entities, based on whether or not they have metabolic activity, a characteristic of life, irrespective of their composition. METHODS: Calcifying nanoparticles were grown from 6 different valves randomly chosen among 84 consecutively explanted aortic valves, as described in the literature. The (1)H-NMR spectra were acquired from calcifying nanoparticles culture media to assess metabolic changes and the presence of 16sRNA in the culture media was investigated by real-time polymerase chain reaction. RESULTS: After 6 weeks in culture, calcifying nanoparticles could be seen clearly attached to the surface of culture flasks. All samples were negative for 16sRNA, discarding the presence of known bacteria. (1)H-NMR spectra showed no difference between calcifying nanoparticles and 6-week-old sterile culture media maintained under the same conditions. CONCLUSIONS: Our results show that calcifying nanoparticles cannot be considered as living organisms.

Susceptibility of the Acinetobacter calcoaceticus-A. baumannii complex to imipenem, meropenem, sulbactam and colistin.

The Acinetobacter calcoaceticus-Acinetobacter baumannii complex includes some of the most clinically relevant species of the genus Acinetobacter due to their capacity to cause epidemic nosocomial outbreaks as well as their increasing resistance to antibiotics. Susceptibility of Acinetobacter strains varies greatly depending on origin, thus highlighting the importance of local analyses of susceptibility profiles. Two hundred twenty-one strains of the A. calcoaceticus-A. baumannii complex were identified using biochemical tests and were biotyped. Strain susceptibility to imipenem, meropenem, colistin and sulbactam was studied using agar dilution. Eight different biotypes were found, type 1 accounting for 69.2% of the strains. MIC(50) and MIC(90) to imipenem, meropenem, colistin and sulbactam were 4 and 8 mg/l, 16 and 32 mg/l, 0.5 and 1mg/l, and 8 and 16 mg/l, with susceptibility rates of 64.3, 22.6, 98.2 and 73.8%, respectively. Biotype 1 was the most resistant. A statistically significant difference was observed for the mean MIC of the four predominant biotypes to imipenem, meropenem and sulbactam but not to colistin.