Rapid species identification and antimicrobial susceptibility testing using Raman spectroscopy.

Infectious diseases remain a serious threat to human life and health as well as having important economical factor. One way of successful combating diseases is designing the most appropriate treatment plan following the correct diagnosis. Therefore, there is a need for a method combining reproducibility, precision and speed. The aim of this work was to evaluate the potential of micro-Raman spectroscopy for identifying bacteria at different taxonomic levels, strains revealing different antibiotic resistance profiles, and for phylogenetic investigation. The project was based on a selection of bacteria: Staphylococcus aureus (6571, Cowanl), Staphylococcus epidermidis (1457, 9142), Escherichia coli including wild- types (strain B, K12, Top 10), transformants expressing ampicillin and kanamycin resistance (Top10Amp, Top10Kan) and clinical isolates expressing extended-spectrum beta- lactamases (ESBL). Following a precise and detailed protocol, Raman spectra were recorded from bacterial colonies grown overnight on a Colombia Blood Agar. In order to remove background fluorescence, rolling-circle filter procedure was applied. The most critical peaks for differentiation between organisms as well as for characterising each microorganism were determined. The spectral data were analyzed using principal component and cluster analysis techniques. As expected, the degree of separation decreased in the order genus→species→strain. It was determined that DNA/RNA, proteins and amino-acids are responsible for the differentiation between strains on a lower level of similarity with more influence of the constituents of the bacterial envelope between more closely related organisms. Raman spectroscopy was capable of differentiating between susceptible and resistant strains as well as monitoring whether the organism has been grown under antibiotic pressure. Based on triplex PCR, clinical isolates of ESBL strains were assigned to one of the phylogenetic group characterising Esherichia genus and it was revealed that within CTX- M TEM-1 there were two distinct clusters of D and B2 groups. Overall we have demonstrated that the combination of micro-Raman spectroscopy, microbiology and bioinformatics has the potential for the successful discrimination of bacteria species and strains, for the determination of antibiotic resistance profiles and investigating phylogenetic grouping in a clinical environment

Localized hypermutation drives the evolution of colistin heteroresistance

These data were created by counting bacterial colonies after serially diluting and plating cultures of Pseudomonas aeruginosa that were incubated in culture medium containing colistin

Efflux pump activity potentiates the evolution of antibiotic resistance across S. aureus isolates

Some bacterial lineages appear to be pre-disposed to evolving antibiotic resistance. Here, the authors show that differential expression of an efflux pump causes widespread variation in evolvability across Staphylococcus aureus isolates, and chemical inhibition of the pump prevents resistance evolution

Gut to lung translocation and antibiotic mediated selection shape the dynamics of Pseudomonas aeruginosa in an ICU patient

Bacteria have the potential to translocate between sites in the human body, but the dynamics and consequences of within-host bacterial migration remain poorly understood. Here we investigate the link between gut and lung Pseudomonas aeruginosa populations in an intensively sampled ICU patient using a combination of genomics, isolate phenotyping, host immunity profiling, and clinical data. Crucially, we show that lung colonization in the ICU was driven by the translocation of P. aeruginosa from the gut. Meropenem treatment for a suspected urinary tract infection selected for elevated resistance in both the gut and lung. However, resistance was driven by parallel evolution in the gut and lung coupled with organ specific selective pressures, and translocation had only a minor impact on AMR. These findings suggest that reducing intestinal colonization of Pseudomonas may be an effective way to prevent lung infections in critically ill patients

Gut to lung translocation and antibiotic mediated selection shape the dynamics of Pseudomonas aeruginosa in an ICU patient

Bacteria have the potential to translocate between sites in the human body, but the dynamics and consequences of within-host bacterial migration remain poorly understood. Here we investigate the link between gut and lung Pseudomonas aeruginosa populations in an intensively sampled ICU patient using a combination of genomics, isolate phenotyping, host immunity profiling, and clinical data. Crucially, we show that lung colonization in the ICU was driven by the translocation of P. aeruginosa from the gut. Meropenem treatment for a suspected urinary tract infection selected for elevated resistance in both the gut and lung. However, resistance was driven by parallel evolution in the gut and lung coupled with organ specific selective pressures, and translocation had only a minor impact on AMR. These findings suggest that reducing intestinal colonization of Pseudomonas may be an effective way to prevent lung infections in critically ill patients

Rapid evolution and host immunity drive the rise and fall of carbapenem resistance during an acute Pseudomonas aeruginosa infection

It is well established that antibiotic treatment selects for resistance, but the dynamics of this process during infections are poorly understood. Here we map the responses of Pseudomonas aeruginosa to treatment in high definition during a lung infection of a single ICU patient. Host immunity and antibiotic therapy with meropenem suppressed P. aeruginosa, but a second wave of infection emerged due to the growth of oprD and wbpM meropenem resistant mutants that evolved in situ. Selection then led to a loss of resistance by decreasing the prevalence of low fitness oprD mutants, increasing the frequency of high fitness mutants lacking the MexAB-OprM efflux pump, and decreasing the copy number of a multidrug resistance plasmid. Ultimately, host immunity suppressed wbpM mutants with high meropenem resistance and fitness. Our study highlights how natural selection and host immunity interact to drive both the rapid rise, and fall, of resistance during infection