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.
Viable bacteria were sought in 44 Maki-negative biofilms from central venous catheters (CVCs) using epifluorescence microscopy after live/dead staining. Thirty (77%) samples contained viable but ...non-culturable (VBNC) cells; the majority were positive on real-time PCR specific for Staphylococcus epidermidis (one also for Staphylococcus aureus). Viable cells were significantly (p <0.01) associated with CVCs from febrile patients, three of whom showed S. epidermidis-positive blood cultures, suggesting that CVC-associated biofilms can be reservoirs for staphylococci in the VBNC state. The possible role of VBNC staphylococci in persistent infections related to medical devices requires further investigation.
Despite the recognized potential of long-term survival or even growth of fecal indicators bacteria (FIB) in marine sediments, this compartment is largely ignored by health protection authorities. We ...conducted a large-scale study over approximately 50 km of the Marche coasts (Adriatic Sea) at depths ranging from 2 to 5 m. Total and fecal coliforms (FC) were counted by culture-based methods. Escherichia coli was also quantified using fluorescence in situ hybridization targeting specific 16S rRNA sequences, which yielded significantly higher abundances than culture-based methods, suggesting the potential importance of viable but nonculturable E. coli cells. Fecal coliforms displayed high abundances at most sites and showed a prevalence of E. coli. FC isolates (n = 113) were identified by API 20E, additional biochemical tests, and internal transcribed spacer-PCR. E. coli strains, representing 96% of isolates, were then characterized for genomic relatedness and phylogenetic group (A, B1, B2, and D) of origin by randomly amplified polymorphic DNA and multiplex-PCR. The results indicated that E. coli displayed a wide genotypic diversity, also among isolates from the same station, and that 44 of the 109 E. coli isolates belonged to groups B2 and D. Further characterization of B2 and D isolates for the presence of 11 virulence factor genes (pap, sfa/foc, afa, eaeA, ibeA, traT, hlyA, stx₁, stx₂, aer, and fyuA) showed that 90% of B2 and 65% of D isolates were positive for at least one of these. Most of the variance of both E. coli abundance and assemblage composition (>62%) was explained by a combination of physical-chemical and trophic variables. These findings indicate that coastal sediments could represent a potential reservoir for commensal and pathogenic E. coli and that E. coli distribution in marine coastal sediments largely depends upon the physical and trophic status of the sediment. We conclude that future sampling designs aimed at monitoring the microbiological quality of marine coastal areas should not further neglect the analysis of the sediment and that monitoring of these environments can be improved by including molecular methods as a complement of culture-based techniques.
Fecal indicator bacteria (FIB) are used worldwide to assess water quality in coastal environments, but little is known about their genetic diversity and pathogenicity. This study examines the ...prevalence, antimicrobial resistance, virulence, and genetic diversity of FIB isolated from marine sediments from a central Adriatic seaside resort. FIB, recovered from 6 out of 7 sites, were significantly more abundant at sampling stations 300 m offshore than close to the shore. Escherichia coli accounted for 34.5% of fecal coliforms, and Enterococcus faecalis accounted for 32% of enterococci. Most isolates (27% of E. coli and 22% of enterococci) were recovered from the sediments that had the highest organic content. Multidrug-resistant E. coli (31%) and enterococci (22%) were found at nearly all sites, whereas 34.5% of E. coli and 28% of enterococci harboring multiple virulence factors were recovered from just two sites. Pulsed-field gel electrophoresis typing showed wide genetic diversity among isolates. Human epidemic clones (E. coli ST131 and Enterococcus faecium ST17) were identified for the first time by multilocus sequence typing in an area where bathing had not been prohibited. These clones were from sites far removed from riverine inputs, suggesting a wide diffusion of pathogenic FIB in the coastal environment and a high public health risk.
Epidemic Escherichia coli clones have been recovered in marine sediment along the coast of Marche, an Adriatic region in central Italy. In the present study, E. coli strains from the clam Chamelea ...gallina, sampled from seven natural beds in the same area, were detected. Selected E. coli isolates from all sampling sites were screened for antimicrobial susceptibility, genetic diversity and correlation. The majority (60%) belonged to phylogroups A or B1, 31% to the other groups (B2, C, D, E, F), 8% to cryptic clades, and 1% were untypable. Moreover, 33.3% of isolates were resistant to at least one drug and 11% were multidrug resistant (MDR). The most common resistance was to tetracycline, ampicillin, and streptomycin. No clonality was detected, but the strains' high genetic heterogeneity pointed at multiple sources of microbiological contamination. MLST analysis found potentially pathogenic and even epidemic MDR strains in clams collected in class A (ST746 and ST46) and class B (ST393, ST58 and ST131) areas, indicating that strains of clinical origin are detectable in clams. These data highlight that eating raw or lightly cooked clams may pose a health risk if purification is not performed or is ineffective.
•Multidrug-resistant Escherichia coli isolates of clinical origin from Venus clam.•Potentially pathogenic strains recovered in clams from class A harvesting areas.•Health risks from consumption of raw or lightly cooked contaminated clams.
Streptococcus suis, a major porcine pathogen, is emerging as a zoonotic agent capable of causing severe invasive disease in humans exposed to pigs or pork products. S. suis infection is rare in ...industrialised countries and usually arises as sporadic cases, with meningitis the most common clinical presentation in humans. Recent reports of two cases of meningitis in Sardinia and northeastern Italy prompted this first characterisation of Italian S. suis isolates. Fifty-nine S. suis strains, the two recent human strains and 57 swine clinical isolates collected between 2003 and 2007 from different Italian herds and regions, were tested for antimicrobial susceptibility, PCR-screened for virulence and antibiotic resistance genes, and subjected to molecular typing. Phenotypic and genotypic analysis demonstrated an overall high genetic diversity among isolates, the majority of which were resistant to macrolides (78%) and tetracyclines (90%). The erm(B), tet(O), mosaic tet(O/W/32/O), tet(W), and tet(M) genes were detected. The tet(O/W/32/O) gene, the most frequent tet gene after tet(O), had never been described in the genus Streptococcus before. In addition, a virulent cps2, erm(B) tet(O) clone, belonging to sequence type 1 (ST1) of the ST1 complex, was found to be prevalent and persistent in Italian swine herds. Finally, the two human isolates (both ST1) carrying cps2, erm(B) and tet(W) were seen to be closely related to each other.
Pseudomonas aeruginosa infection is the main cause of morbidity and mortality in cystic fibrosis (CF) patients. Cultural methods, the gold standard for bacterial detection, are hampered by alginate ...overproduction and the possible presence of dormant cells. This work aimed at developing a sensitive and specific diagnostic tool capable of detecting P. aeruginosa in culture-negative sputum from CF patients. DNA was extracted with the QIAamp DNA kit after cell spin-down to remove free DNA. Real-time PCR assays targeting 3 species-specific genes, ecfX, gyrB and oprL, were developed using previously designed and new primers. The LOD was determined by spiking different amounts of P. aeruginosa ATCC9027 in a negative sputum sample. The PCR protocol targeting oprL was found to be unreliable, whereas those targeting ecfX and gyrB proved to be effective (LOD 60 cells/ml and 102 cells/ml, respectively). Examination of 33 randomly selected culture-negative samples yielded 6 positive samples from 3 patients: all 6 were ecfX-positive and 2 were positive for ecfX and gyrB. After 1 and 5 months additional samples from patients 1 and 2 were culture-positive; patient 3 received a 4-month antibiotic cycle and 2 months later he resulted still culture-negative and PCR-positive. These findings highlight the value of the Real-time PCRs developed, especially the one targeting ecfX, in diagnosing P. aeruginosa infection in CF samples. Protocol effectiveness was confirmed by the eventual cultural recovery of P. aeruginosa in the two PCR-positive patients, who were found to have chronic CF. Findings also seem to provide further support for a role of dormant forms in recurrent CF infection.
Coagulase-negative staphylococci (CNS) were the first organisms in which acquired glycopeptide resistance was recognized. Ever since the early reports, it has been apparent that resistance to ...teicoplanin is more common than that to vancomycin and that resistance occurs mostly in species such as Staphylococcus haemolyticus and Staphylococcus epidermidis. The minimum inhibitory concentrations (MICs) of teicoplanin for CNS usually fall over a wide range, and, especially in some methicillin-resistant isolates of the two above-mentioned species, they can reach and even exceed the resistance breakpoint, whereas vancomycin MICs tend to remain more stable over a narrower range within the limits of susceptibility. CNS strains intermediately susceptible and even resistant not only to teicoplanin but also to vancomycin have, however, been isolated, most frequently from patients subjected to prolonged glycopeptide treatment. Laboratory detection of glycopeptide-resistant CNS may be problematic, mainly because susceptibility tests, particularly those for teicoplanin, are influenced by various technical factors, and agar diffusion tests may yield false susceptibility data. In studies with experimental glycopeptides, some molecules have exhibited improved in vitro activity compared with teicoplanin and vancomycin, but these encouraging microbiological findings have not usually been followed by in vivo trials. Stepwise and single-step exposure to teicoplanin and vancomycin has allowed stable clones for which glycopeptide MICs are increased to be obtained from susceptible CNS strains, particularly strains of Staphylococcus haemolyticus and Staphylococcus epidermidis. In these studies, resistance to teicoplanin was generally easier to obtain than resistance to vancomycin, and the levels of teicoplanin resistance were higher. Population studies have demonstrated the usually heterogeneous nature of glycopeptide resistance in CNS. Although glycopeptide-resistant CNS have been shown to differ in several features from their glycopeptide-susceptible counterparts, the exact mechanism of staphylococcal glycopeptide resistance remains unknown.
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