Detecting the presence of bacterial DNA by PCR can be useful in diagnosing culture-negative cases of infection, especially in patients with suspected infection and antibiotic therapy.

Failing in bacteria isolation in a significant number of infections might be due to the involvement of microorganisms nonrecoverable in culture media. The presence cannot be ruled out of nondividing cells or even bacterial products still capable of promoting a host immunological response. Antibiotic therapy, for example, might induce a block of bacterial division and the impossibility of recovering cells in culture media. In these cases, a molecular method targeting DNA should be used. In this study, 230 clinical samples with a culture-negative report obtained from 182 patients were examined with a protocol of PCR targeting the bacterial 16S rRNA gene to evaluate the usefulness of molecular methods in differencing culture-negative infections from other pathologies. Amplicons were obtained in 14% of the samples, although this percentage increased (27%) in a subgroup of patients with presumptive diagnosis of infection and ongoing antibiotic therapy. By multiplex PCR, it was shown that detected DNA belonged mostly to Enterobacteriaceae and enterococcal species. Multiple culture-negative, PCR-positive samples and isolation of the same bacterial species in culture in additional samples from the same patient support the clinical significance of the data obtained and highlight the complementary role and usefulness of applying molecular methods in diagnostic microbiology.

Fate of pathogenic bacteria in microcosms mimicking human body sites.

During the infectious process, pathogens may reach anatomical sites where they are exposed to substances interfering with their growth. These substances can include molecules produced by the host, and his resident microbial population, as well as exogenous antibacterial drugs. Suboptimal concentrations of inhibitory molecules and stress conditions found in vivo (high or low temperatures, lack of oxygen, extreme pH) might induce in bacteria the activation of survival mechanisms blocking their division capability but allowing them to stay alive. These "dormant" bacteria can be reactivated in particular circumstances and would be able to express their virulence traits. In this study, it was evaluated the effect of some environmental conditions, such as optimal and suboptimal temperatures, direct light and antibiotic sub-inhibitory concentrations doses of antibiotic, on the human pathogens Escherichia coli and Enterococcus faecalis when incubated in fluids accumulated in the body of patients with different pathologies. It is shown that inoculation in a number of accumulated body fluids and the presence of gentamicin, reliable conditions encountered during pathological states, induce stress-responding strategies enabling bacteria to persist in microcosms mimicking the human body. Significant differences were detected in Gram-negative and Gram-positive species with E. faecalis surviving, as starved or viable but non-culturable forms, in any microcosm and condition tested and E. coli activating a viable but non-culturable state only in some clinical samples. The persistence of bacteria under these conditions, being non-culturable, might explain some recurrent infections without isolation of the causative agent after application of the standard microbiological methods.

Altered intestinal function precedes the appearance of bacterial DNA in serum and ascites in patients with cirrhosis: a pilot study.

OBJECTIVES: Bacterial translocation seems to precede the occurrence of overt bacterial infection in patients with cirrhosis. The presence of bacterial DNA in blood and ascites correlates with bacterial translocation and is frequent in patients with advanced cirrhosis without overt infection. Our aim was to search for bacterial DNA in patients with cirrhosis both with and without ascites, and to study its correlation with abnormal intestinal motility or permeability and the presence of bacterial overgrowth. METHODS: Blood and ascites samples were obtained on day 1, and blood samples were taken twice a day for the following 3 days. Bacterial DNA was assayed by polymerase chain reaction using universal primers for rRNA 16 s. Oro-caecal transit time and bacterial overgrowth were assessed with Lactulose H(2) breath testing. Intestinal permeability was assessed by determining urinary lactulose and mannitol excretion with high performance liquid chromatography. RESULTS: We studied seven patients (six were male, age range was 42-78 years). Aetiology was alcohol in four, HCV in two, HBV in one; ascites was present in four and Child-Pugh grade was A in four and B in three. All patients had increased intestinal permeability, six had decreased transit time and one had bacterial overgrowth. In only one patient (with ascites), polymerase chain reaction was positive for bacterial DNA both in ascites and serum for all 4 days on which samples were taken. CONCLUSION: Increased intestinal permeability and abnormal motility were frequent without evidence of bacterial translocation in cirrhosis even without ascites. They are likely to be facilitators for bacterial translocation and thus precede it.

Inhibition of the resuscitation from the viable but non-culturable state in Enterococcus faecalis.

The viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when exposed to environmental stresses capable of inducing cell growth inhibition and cell death. This state can be summarized as a quiescent form of life waiting for suitable conditions. This strategy has been shown to be activated by medically important bacteria either when present in natural environments or in the human body during the infection process. In this study we have evaluated the effects of antibiotics acting on peptidoglycan or protein synthesis of Enterococcus faecalis in the VBNC state. The activity of the antibiotics was determined by their ability both to inhibit resuscitation (i.e. recovery of cell division) and to bind the molecular target of action. Benzylpenicillin, piperacillin and gentamicin block cell resuscitation at the minimal inhibitory concentrations (MICs) of growing cells, while vancomycin acts only at doses 500 times higher than the MIC. This different behaviour is discussed taking into consideration the mode of action of the antibiotics.

Adhesion to medical device materials and biofilm formation capability of some species of enterococci in different physiological states.

Enterococci may survive in adverse environments including the human body where bacteriocins, antibiotics, iron-limitation and immune response represent stressing conditions for bacteria that cause division block. In those conditions, bacteria present in the human body would hardly be in an exponentially growing phase but would mostly be in physiological states such as starvation or the viable but nonculturable (VBNC) state. The possibility that the starved and VBNC bacteria can maintain their ability to adhere to living and inanimate substrates is the first mandatory step for them potentially to cause an infection process. In this study it is shown that starved and stationary enterococcal cells are able to form biofilms on plastic material albeit with reduced efficiency as compared to growing cells. Moreover, although VBNC enterococcal forms are not capable of forming biofilms, Enterococcus faecalis and other enterococcal species of medical interest maintain their ability to synthesize the polymeric matrix for a limited period of time under adverse environmental conditions. The data presented, together with those regarding the maintenance of the division recovery potential already proved in nonculturable bacteria, further support the possibility for the VBNC and other nondividing bacterial forms to have a role as infectious agents and to constitute a risk to human health.