Biofilms are of great importance in infection control and healthcare-associated infections (HCAI) due to their inherent tolerance and 'resistance' to antimicrobial therapies. Biofilms have been shown to develop on medical device surfaces and dispersal of single and clustered cells imply a significant risk of microbial dissemination within the host and increased risk of infection. Although routine microbiological testing assists with the diagnosis of a clinical infection, there is no 'gold standard' available to reveal the presence of microbial biofilm from samples collected within clinical settings. Furthermore, such limiting factors as viable but non-culturable (VBNC) microorganisms and small colony variants (SCVs) often prevent a successful detection. In order to increase the chances of detection and provide a more accurate diagnosis, a combination of microbiological culture techniques and molecular methods should be employed. Measures such as antimicrobial coating and surface alterations of medical devices provide promising opportunities in the prevention of biofilm formation on medical devices.

The Gram-negative opportunistic pathogen, Klebsiella pneumoniae, is responsible for causing a spectrum of community-acquired and nosocomial infections and typically infects patients with indwelling medical devices, especially urinary catheters, on which this microorganism is able to grow as a biofilm. The increasingly frequent acquisition of antibiotic resistance by K. pneumoniae strains has given rise to a global spread of this multidrug-resistant pathogen, mostly at the hospital level. This scenario is exacerbated when it is noted that intrinsic resistance to antimicrobial agents dramatically increases when K. pneumoniae strains grow as a biofilm. This review will summarize the findings about the antibiotic resistance related to biofilm formation in K. pneumoniae.

Staphylococcal biofilms are among the main causes of chronic implant-associated infections. We have recently suggested that their transformation into viable but non-culturable (VBNC) forms (i.e. forms capable of resuscitation) could be responsible for the recurrent symptoms. This work aims to establish whether Staphylococcus aureus biofilms can give rise to VBNC forms capable of being resuscitated in suitable environmental conditions, the role of different stressors in inducing the VBNC state and the conditions favouring resuscitation. S. aureus 10850 biofilms were exposed to different concentrations of antibiotic (vancomycin or quinupristin/dalfopristin) and/or to nutrient depletion until loss of culturability. The presence of viable cells and their number were examined by epifluorescence microscopy and flow cytometry. Gene expression was measured by real-time PCR. Resuscitation ability was tested by growth in rich medium containing antioxidant factors. Viable subpopulations were detected in all non-culturable biofilms. However, viable cell numbers and gene expression remained constant for 150 days from loss of culturability in cells from antibiotic-exposed biofilms, but not in those that had only been starved. Resuscitation was obtained in rich medium supplemented with 0.3% sodium pyruvate or with 50% filtrate of a late-log culture. Our findings demonstrate that S. aureus can enter the VBNC state in infectious biofilms. The presence of vancomycin or quinupristin/dalfopristin can inadvertently induce a true VBNC state or its persistence in S. aureus cells embedded in biofilms, supporting previous findings on the role of staphylococcal biofilms in recurrent infections.