The multidrug-resistant bacterial infections represent a global and complex problem because of the increase in their prevalence and the lack of new antibiotics. In this context, the antimicrobial stewardship programs are based on the collaboration between the infectious disease specialist and the microbiologist, which must merge their competencies to optimize antimicrobial therapy, implement surveillance and control the spread of MDR epidemic in health-care facilities. Moreover, they should create specific diagnostic procedures to obtain a prompt diagnosis of sepsis, with the species-identification of the pathogen and its sensitivity profile to antibiotics.
Our research evaluated a new diagnostic approach for the early determination of the phenotypic antimicrobial susceptibility in Gram-negative bacteria.
The first study aimed to validate an innovative rapid procedure for bacterial antibiogram in sepsis, which is 24 hours quicker than the traditional one. Nowadays indeed, the laboratory can use new technologies molecular biology techniques as a support in sepsis diagnostics; however, culture and microbroth diluition are still the gold standards in sepsis’ microbiological diagnosis. Unfortunately, they require a long time to be finalized, and this is more and more a severe limit since patients’ survival depends on a rapid and correct therapy start. Many authors studied new pathways of identification of resistances, but until now, nobody was able to establish a shared procedure based on evidence. For this reason, in the real-life laboratory, operational protocols are based on the responsibility and the expertise of the single professional.
In our study, we included 145 strains of E. coli, K. pneumoniae and P. aeruginosa, with different phenotypic profiles. They were analyzed with both the gold standard method and the rapid one, comparing the values of Minimal Inhibitory Concentration. The procedure included haemocolture’s bottles inoculum, spot streaking on an agar plate, 5 hours incubation at 37°C, and finally microbroth diluition test preparation (Sensititre® method). MIC data and their interpretation (susceptible, intermediate or resistant) were collected and compared to ones obtained with the gold standard test, and agreement index was calculated together with sensitivity, specificity and prediction parameters.
The aggregate data analysis showed a good performance, regarding precision, the Categorical Agreement (97.9%), the Essential Agreement (99.1%), the sensitivity (96%) and specificity (99%). Furthermore, the errors related to the evaluation of both the single antibiotic and the total of the MIC resulted in being acceptable. However, we observed the presence of some significant Very Major and Major Errors concerning Ampicillin/Sulbactam, Piperacillin/Tazobactam e Fosfomycin: these results suggested us to exclude the three drugs from the new rapid susceptibility test, without precluding the utility in guiding the so called “first line therapy”.
In the second study, we evaluated the application of mass spectrometry to the rapid detection of quinolones’ resistance, with particular attention to the expression of the aminoglycoside acetyltransferase variant AAC(6’)-Ib-cr. We included in the study 72 strains of E. coli, which were spot streaked on an agar plate, incubated for 5 hours at 37°C with norfloxacin disk and finally collected for the MALDI-TOF analysis. For the study, we used the Saramis® software, Vitek® MS (Biomerieux), which allows examining separately every single detected spectrum. In particular, we investigated the presence of spectra related to the native and the acetylated norfloxacin spectra (“indirect” method) and the AAC(6’)-Ib-cr spectra (“direct” method). In both the cases, the analysis did not show satisfactory results, regarding both agreements with the reference test and sensitivity/specificity.
In conclusion, our study demonstrated that the new rapid procedure for the bacterial antimicrobial susceptibility test has an acceptable agreement with the traditional one regarding the major part of the antibiotics that are used in practice as a first line therapy in sepsis. On the contrary, our method for the evaluation of quinolone with mass spectrometry did not show any clinical and microbiological viability. Further studies might be designed in this topic, but they should always consider the need to guarantee a precise and rapid result, together with a targeted diagnosis and care that never neglect expertise, caution, and clinical monitoring.