VanA type enterococci from humans, animals and food: species distribution, population structure, Tn1546-typing and location, and virulence determinants

VanA-type human (n = 69), animal (n = 49), and food (n =36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n = 4) and M49 (n = 13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P > 0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P < 0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters

Dormancy within Staphylococcus epidermidis biofilms : a transcriptomic analysis by RNA-seq

The proportion of dormant bacteria within Staphylococcus epidermidis biofilms may determine its inflammatory profile. Previously, we have shown that S. epidermidis biofilms with higher proportions of dormant bacteria have reduced activation of murine macrophages. RNA-sequencing was used to identify the major transcriptomic differences between S. epidermidis biofilms with different proportions of dormant bacteria. To accomplish this goal, we used an in vitro model where magnesium allowed modulation of the proportion of dormant bacteria within S. epidermidis biofilms. Significant differences were found in the expression of 147 genes. A detailed analysis of the results was performed based on direct and functional gene interactions. Biological processes among the differentially expressed genes were mainly related to oxidation-reduction processes and acetyl-CoA metabolic processes. Gene set enrichment revealed that the translation process is related to the proportion of dormant bacteria. Transcription of mRNAs involved in oxidation-reduction processes was associated with higher proportions of dormant bacteria within S. epidermidis biofilm. Moreover, the pH of the culture medium did not change after the addition of magnesium, and genes related to magnesium transport did not seem to impact entrance of bacterial cells into dormancy.The authors thank Stephen Lorry at Harvard Medical School for providing CLC Genomics software. This work was funded by Fundacao para a Ciencia e a Tecnologia (FCT) and COMPETE grants PTDC/BIA-MIC/113450/2009, FCOMP-01-0124-FEDER-014309, FCOMP-01-0124-FEDER-022718 (FCT PEst-C/SAU/LA0002/2011), QOPNA research unit (project PEst-C/QUI/UI0062/2011), and CENTRO-07-ST24-FEDER-002034. The following authors had an individual FCT fellowship: VC (SFRH/BD/78235/2011) and AF (2SFRH/BD/62359/2009)

Detection of viable but non-culturable staphylococci in biofilms from central venous catheters negative on standard microbiological assays

none7To our knowledge this is the first evidence that S. epidermidis and S. aureus can enter the VBNC state. If positively documented, their ability to persist in this state could help to explain the well-known prevalence of both species in recurrent CVC-related infections [4,8,17,18]. Detection of S. epidermidis in 62% of CVCs demonstrated to contain non-culturable bacteria (removed from febrile patients, 27% of whom also had a positive blood culture) supports this hypothesis. Further research into the relationship among VBNC forms found in CVC-associated biofilms, their actual metabolic activity and recurrent infection, as well as careful recording of concurrent clinical data, are required to gain insights into the possible role of VBNC bacterial cells in indwelling medical device-related persistent infections.G. Zandri;S. Pasquaroli;C. Vignaroli;S. Talevi;E. Manso;G. Donelli;F. BiavascoG., Zandri; Pasquaroli, Sonia; Vignaroli, Carla; S., Talevi; E., Manso; G., Donelli; Biavasco, Francesc

Antibiotic pressure can induce the viable but nonculturable state in Staphylococcus aureus growing in biofilms

Objectives: 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. Methods: 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. Results: 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. Conclusions: 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 infection

VanA-Type Enterococci from Humans, Animals, and Food: Species Distribution, Population Structure, Tn1546 Typing and Location, and Virulence Determinants▿

VanA-type human (n = 69), animal (n = 49), and food (n = 36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n = 4) and M49 (n = 13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P > 0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P < 0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters