Investigation on the Synergy between Membrane Permeabilizing Amphiphilic α-Hydrazido Acids and Commonly Used Antibiotics against Drug-Resistant Bacteria.

The growth of (multi)drug resistance in bacteria is among the most urgent global health issues. Monocationic amphiphilic α-hydrazido acid derivatives are structurally simple mimics of antimicrobial peptides (AMPs) with fewer drawbacks. Their mechanism of membrane permeabilization at subtoxic concentrations was found to begin with an initial electrostatic attraction of isolated amphiphile molecules to the phospholipid heads, followed by a rapid insertion of the apolar portions. As the accumulation into the bilayer proceeded, the membrane increased its fluidity and permeability without being subjected to major structural damage. After having ascertained that α-hydrazido acid amphiphiles do not interact with bacterial DNA, they were subjected to synergy evaluation for combinations with conventional antibiotics. Synergy was observed for combinations with tetracycline against sensitive S. aureus and E. coli, as well as with ciprofloxacin and colistin against resistant strains. Additivity with a remarkable recovery in activity of conventional antibiotics (from 2-fold to ≥32-fold) together with largely subtoxic concentrations of α-hydrazido acid derivatives was found for combinations with ciprofloxacin toward susceptible S. aureus and methicillin toward MRSa. However, no potentiation of conventional antibiotics was observed for combinations with linezolid and gentamicin against the corresponding resistant S. aureus and E. coli strains.

Characterization of bacterial communities associated with seabed sediments in offshore and nearshore sites to improve Microbiologically Influenced Corrosion mitigation on marine infrastructures.

Microbiologically Influenced Corrosion (MIC) is one of the main threats for marine infrastructures, leading to severe safety and environmental risks associated with structural failures and/or leakages of dangerous fluids, together with potential huge economic losses and reputational damage for the involved parts. For a safe design and a proper installation of infrastructure systems in contact with the seabed, a deep knowledge of the site-specific microbial community of the sediments should be beneficial. Therefore, in addition to the simple detection or the sole quantification of Sulphate-Reducing Bacteria (SRB), the whole characterization of the microbial members involved in MIC phenomena is desirable. In this study, 16S rRNA-based comparison between bacterial communities thriving in offshore and nearshore marine sediments was performed, with a focus on the main bacterial groups putatively responsible for MIC. The nearshore sediments were significantly enriched in bacterial members associated with human and organic compounds contamination belonging to the Bacteroidota, Desulfobacterota, and Firmicutes phyla, while the offshore sediments hosted Alphaproteobacteria, Nitrospinota, and Nitrospirota members, representative of a low anthropogenic impact. Quantitative PCR targeting the dsrA gene and detailed community analyses revealed that the nearshore sediments were significantly enriched in SRB mainly affiliated to the Desulfobulbus and Desulfosarcina genera potentially involved in biocorrosion, compared to the offshore ones. These results suggest that the bacterial community associated with the high concentration of organic compounds derived by an elevated anthropogenic impact is likely to favour MIC. Such observations highlight the importance of microbiological investigations as prevention strategy against MIC processes, aiming both at characterizing sites for the establishment of new infrastructures and at monitoring those already installed.

Involvement of Acquired Tobramycin Resistance in the Shift to the Viable but Non-Culturable State in Pseudomonas aeruginosa.

Persistent and viable but non-culturable (VBNC) Pseudomonas aeruginosa cells are mainly responsible for the recurrence and non-responsiveness to antibiotics of cystic fibrosis (CF) lung infections. The sub-inhibitory antibiotic concentrations found in the CF lung in between successive therapeutic cycles can trigger the entry into the VBNC state, albeit with a strain-specific pattern. Here, we analyzed the VBNC cell induction in the biofilms of two CF P. aeruginosa isolates, exposed to starvation with/without antibiotics, and investigated the putative genetic determinants involved. Total viable bacterial cells were quantified by the validated ecfX-targeting qPCR protocol and the VBNC cells were estimated as the difference between qPCR and cultural counts. The isolates were both subjected to whole genome sequencing, with attention focused on their carriage of aminoglycoside resistance genes and on identifying mutated toxin-antitoxin and quorum sensing systems. The obtained results suggest the variable contribution of different antibiotic resistance mechanisms to VBNC cell abundance, identifying a major contribution from tobramycin efflux, mediated by MexXY efflux pump overexpression. The genome analysis evidenced putative mutation hotspots, which deserve further investigation. Therefore, drug efflux could represent a crucial mechanism through which the VBNC state is entered and a potential target for anti-persistence strategies.

Class 1 integron and Enterococcus spp. abundances in swine farms from the " Suckling piglets" to the "Fatteners" production category.

Swine farms are considered a hotspot of antimicrobial resistance and may contribute to the spread of antibiotic-resistant and/or pathogenic bacteria into the environment as well as to farm workers. In this study, swine fecal samples have been collected over the primary production, selecting three categories, i.e., "Suckling piglets", "Weaning pigs" and "Fatteners", in six intensive swine farms, for two years. Feces were analysed for the detection and abundance of class 1 integrons (used as proxy of antibiotic resistance and of anthropogenic pollution), and of enterococci [fecal indicator bacteria (FIB) and potentially pathogenic for humans] by quantitative Real Time PCR. Furthermore, Enterococcus faecalis and Enterococcus faecium were isolated, analysed for the presence of the intI1 gene by Real Time PCR and genetically typed by Pulsed-Field Gel Electrophoresis. Both enterococci and class 1 integrons were significantly more abundant in the Suckling piglets (p = 0.0316 and 0.0242, respectively). About 8% of the isolated enterococci were positive for the intI1 gene by Real Time PCR. E. faecalis and E. faecium were found genetically heterogeneous and no specific pattern could be identified as the driver for their presence along the pig primary production. These findings suggest that the "Suckling piglets" category of production represents the key point where to mitigate the risk of transmission of enterococci and class 1 integrons with associated antibiotic resistance genes to humans and spread into the environment.

Zooplankton as a Transitional Host for Escherichia coli in Freshwater.

This study shows that Escherichia coli can be temporarily enriched in zooplankton under natural conditions and that these bacteria can belong to different phylogroups and sequence types (STs), including environmental, clinical, and animal isolates. We isolated 10 E. coli strains and sequenced the genomes of two of them. Phylogenetically, the two isolates were closer to strains isolated from poultry meat than to freshwater E. coli, albeit their genomes were smaller than those of the poultry isolates. After isolation and fluorescent protein tagging of strains ED1 and ED157, we show that Daphnia sp. can take up these strains and release them alive again, thus becoming a temporary host for E. coli. In a chemostat experiment, we show that this association does not prolong bacterial long-term survival, but at low abundances it also does not significantly reduce bacterial numbers. We demonstrate that E. coli does not belong to the core microbiota of Daphnia, suffers from competition by the natural Daphnia microbiota, but can profit from its carapax to survive in water. All in all, this study suggests that the association of E. coli with Daphnia is only temporary, but the cells are viable therein, and this might allow encounters with other bacteria for genetic exchange and potential genomic adaptation to the freshwater environment. IMPORTANCE The contamination of freshwater with feces-derived bacteria is a major concern regarding drinking water acquisition and recreational activities. Ecological interactions promoting their persistence are still very scarcely studied. This study, which analyses the survival of E. coli in the presence of zooplankton, is thus of ecological and water safety relevance.

A proposal for the reference intervals of the Italian microbiota "scaffold" in healthy adults.

Numerous factors, ranging from genetics, age, lifestyle, and dietary habits to local environments, contribute to the heterogeneity of the microbiota in humans. Understanding the variability of a "healthy microbiota" is a major challenge in scientific research. The gut microbiota profiles of 148 healthy Italian volunteers were examined by 16S rRNA gene sequencing to determine the range and diversity of taxonomic compositions in the gut microbiota of healthy populations. Possible driving factors were evaluated through a detailed anamnestic questionnaire. Microbiota reference intervals were also calculated. A "scaffold" of a healthy Italian gut microbiota composition was identified. Differences in relative quantitative ratios of microbiota composition were detected in two clusters: a bigger cluster (C2), which included 124 subjects, was characterized by more people from the northern Italian regions, who habitually practised more physical activity and with fewer dietary restrictions. Species richness and diversity were significantly higher in this cluster (C2) than in the other one (C1) (C1: 146.67 ± 43.67; C2: 198.17 ± 48.47; F = 23.40; P < 0.001 and C1: 16.88 ± 8.66; C2: 35.01 ± 13.40; F = 40.50; P < 0.001, respectively). The main contribution of the present study was the identification of the existence of a primary healthy microbiological framework that is only marginally affected by variations. Taken together, our data help to contextualize studies on population-specific variations, including marginal aspects, in human microbiota composition. Such variations must be related to the primary framework of a healthy microbiota and providing this perspective could help scientists to better design experimental plans and develop strategies for precision tailored microbiota modulation.

A combination of sugar esters and chitosan to promote in vivo wound care.

In recent years, researchers are exploring innovative green materials fabricated from renewable natural substances to meet formulation needs. Among them, biopolymers like chitosans and biosurfactants such as sugar fatty acid esters are of potential interest due to their biocompatibility, biodegradability, functionality, and cost-effectiveness. Both classes of biocompounds possess the ability to be efficiently employed in wound dressing to help physiological wound healing, which is a bioprocess involving uncontrolled oxidative damage and inflammation, with an associated high risk of infection. In this work, we synthesized two different sugar esters (i.e., lactose linoleate and lactose linolenate) that, in combination with chitosan and sucrose laurate, were evaluated in vitro for their cytocompatibility, anti-inflammatory, antioxidant, and antibacterial activities and in vivo as wound care agents. Emphasis on Wnt/ß-catenin associated machineries was also set. The newly designed lactose esters, sucrose ester, and chitosan possessed sole biological attributes, entailing considerable blending for convenient formulation of wound care products. In particular, the mixture composed of sucrose laurate (200 µM), lactose linoleate (100 µM), and chitosan (1%) assured its superiority in terms of efficient wound healing prospects in vivo together with the restoring of the Wnt/ß-catenin signaling pathway, compared with the marketed wound healing product (Healosol®), and single components as well. This innovative combination of biomaterials applied as wound dressing could effectively break new ground in skin wound care.

The Natural Alkaloid Berberine Can Reduce the Number of Pseudomonas aeruginosa Tolerant Cells.

The eradication of recurrent Pseudomonas aeruginosa (PA) lung infection in cystic fibrosis (CF) patients may be hampered by the development of persistent bacterial forms, which can tolerate antibiotics through efflux pump overexpression. After demonstrating the efflux pump inhibitory effect of the alkaloid berberine on the PA MexXY-OprM efflux pump, in this study, we tested its ability (80/320 µg/mL) to enhance tobramycin (20xMIC/1000xMIC) activity against PA planktonic/biofilm cultures. Preliminary investigations of the involvement of MexY in PA tolerance to tobramycin treatment, performed on the isogenic pair PA K767 (wild type)/K1525 (ΔmexY) growing in planktonic and biofilm cultures, demonstrated that the ΔmexY mutant K1525 produced a lower (100 and 10 000 times, respectively) amount of tolerant cells than that of the wild type. Next, we grew broth cultures of PAO1, PA14, and 20 PA clinical isolates (of which 13 were from CF patients) in the presence of 20xMIC tobramycin with and without berberine 80 µg/mL. Accordingly, most strains showed a greater (from 10- to 1000-fold) tolerance reduction in the presence of berberine. These findings highlight the involvement of the MexXY-OprM system in the tobramycin tolerance of PA and suggest that berberine may be used in new valuable therapeutic combinations to counteract persister survival.

Contribution of Drugs Interfering with Protein and Cell Wall Synthesis to the Persistence of Pseudomonas aeruginosa Biofilms: An In Vitro Model.

The occurrence of Pseudomonas aeruginosa (PA) persisters, including viable but non-culturable (VBNC) forms, subpopulations of tolerant cells that can survive high antibiotic doses, is the main reason for PA lung infections failed eradication and recurrence in Cystic Fibrosis (CF) patients, subjected to life-long, cyclic antibiotic treatments. In this paper, we investigated the role of subinhibitory concentrations of different anti-pseudomonas antibiotics in the maintenance of persistent (including VBNC) PA cells in in vitro biofilms. Persisters were firstly selected by exposure to high doses of antibiotics and their abundance over time evaluated, using a combination of cultural, qPCR and flow cytometry assays. Two engineered GFP-producing PA strains were used. The obtained results demonstrated a major involvement of tobramycin and bacterial cell wall-targeting antibiotics in the resilience to starvation of VBNC forms, while the presence of ciprofloxacin and ceftazidime/avibactam lead to their complete loss. Moreover, a positive correlation between tobramycin exposure, biofilm production and c-di-GMP levels was observed. The presented data could allow a deeper understanding of bacterial population dynamics during the treatment of recurrent PA infections and provide a reliable evaluation of the real efficacy of the antibiotic treatments against the bacterial population within the CF lung.

Diffusion and Characterization of Pseudomonas aeruginosa Aminoglycoside Resistance in an Italian Regional Cystic Fibrosis Centre.

AIMS: Extensively-drug-resistant Pseudomonas aeruginosa constitutes a serious threat to patients suffering from Cystic Fibrosis (CF). In these patients, P. aeruginosa lung infection is commonly treated with aminoglycosides, but treatments are largely unsuccessful due a variety of resistance mechanisms. Here we investigate the prevalence of resistance to gentamicin, amikacin and tobramycin and the main aminoglycoside resistance genes found in P. aeruginosa strains isolated at a regional CF centre. RESULTS: A total number of 147 randomly selected P. aeruginosa strains isolated from respiratory samples sent by the Marche regional Cystic Fibrosis Centre to the Microbiology lab, were included in this study. Of these, 78 (53%) were resistant to at least one of the three aminoglycosides tested and 27% were resistant to all three antibiotics, suggesting a major involvement of a chromosome-encoded mechanism, likely MexXY-OprM efflux pump overexpression. A specific pathogenic clone (found in 7/78 of the aminoglycoside resistant strains) carrying ant(2″)-Ia was isolated over time from the same patient, suggesting a role for this additional resistance gene in the antibiotic unresponsiveness of CF patients. CONCLUSIONS: The MexXY-OprM efflux pump is confirmed as the resistance determinant involved most frequently in P. aeruginosa aminoglycoside resistance of CF lung infections, followed by the ant(2″)-Ia-encoded adenylyltransferase. The latter may prove to be a novel target for new antimicrobial combinations against P. aeruginosa.

A Fluorinated Analogue of Marine Bisindole Alkaloid 2,2-Bis(6-bromo-1H-indol-3-yl)ethanamine as Potential Anti-Biofilm Agent and Antibiotic Adjuvant Against Staphylococcus aureus.

Methicillin resistant Staphylococcus aureus (MRSA) infections represent a major global healthcare problem. Therapeutic options are often limited by the ability of MRSA strains to grow as biofilms on medical devices, where antibiotic persistence and resistance is positively selected, leading to recurrent and chronic implant-associated infections. One strategy to circumvent these problems is the co-administration of adjuvants, which may prolong the efficacy of antibiotic treatments, by broadening their spectrum and lowering the required dosage. The marine bisindole alkaloid 2,2-bis(6-bromo-1H-indol-3-yl)ethanamine (1) and its fluorinated analogue (2) were tested for their potential use as antibiotic adjuvants and antibiofilm agents against S. aureus CH 10850 (MRSA) and S. aureus ATCC 29213 (MSSA). Both compounds showed antimicrobial activity and bisindole 2 enabled 256-fold reduction (ΣFICs = 0.5) in the minimum inhibitory concentration (MIC) of oxacillin for the clinical MRSA strain. In addition, these molecules inhibited biofilm formation of S. aureus strains, and compound 2 showed greater eradicating activity on preformed biofilm compared to 1. None of the tested molecules exerted a viable but non-culturable cells (VBNC) inducing effect at their MIC values. Moreover, both compounds exhibited no hemolytic activity and a good stability in plasma, indicating a non-toxic profile, hence, in particular compound 2, a potential for in vivo applications to restore antibiotic treatment against MRSA infections.

Role of Tobramycin in the Induction and Maintenance of Viable but Non-Culturable Pseudomonas aeruginosa in an In Vitro Biofilm Model.

The recurrence of Pseudomonas aeruginosa (PA) biofilm infections is a major issue in cystic fibrosis (CF) patients. A pivotal role is played by the presence of antibiotic-unresponsive persisters and/or viable but non-culturable (VBNC) forms, whose development might be favored by subinhibitory antibiotic concentrations. The involvement of tobramycin and ciprofloxacin, widely used to treat CF PA lung infections, in the abundance of VBNC cells was investigated in PA biofilms models. In vitro biofilms of the laboratory strain PAO1-N and the clinical strain C24 were developed and starved by subculture for 170 days in a non-nutrient (NN) broth, unsupplemented or supplemented with one-quarter minimal inhibitory concentration (MIC) of tobramycin or ciprofloxacin. VBNC cells abundance, estimated as the difference between total live (detected by qPCR and flow cytometry) and colony forming unit (CFU) counts, showed a strain- and drug-specific pattern. A greater and earlier abundance of VBNC PAO1-N cells was detected in all conditions. Exposure of the C24 strain to NN and NN + ciprofloxacin induced only a transient VBNC subpopulation, which was more abundant and stable until the end of the experiment in tobramycin-exposed biofilms. The same response to tobramycin was observed in the PAO1-N strain. These findings suggest that low tobramycin concentrations might contribute to PA infection recurrence by favoring the development of VBNC forms.

Plasmid Replicon Typing of Antibiotic-Resistant Escherichia coli From Clams and Marine Sediments.

Unlike human isolates, environmental Escherichia coli isolates have not been thoroughly investigated for the diversity and transferability of antibiotic-resistant plasmids. In this study, antibiotic-resistant strains from marine sediment (n = 50) and clams (n = 53) were analyzed (i) for their plasmid content using a PCR-based plasmid replicon typing (PBRT) kit and (ii) for the transferability of plasmid-associated antibiotic resistance (AR) traits by mating experiments. Fifteen of the thirty replicons targeted by the PBRT kit were detected in the isolates; 8/15 were identified in both sediment and clam isolates, although at different frequencies. The most frequent replicons in sediment (74%) and in clam strains (66%) alike, were FIA, FIB, or FII, which are associated with the IncF group, followed by the I1α replicon, which was more frequent in clam (24.5%) than in sediment (10%) strains. More than 50% of the strains contained multiple replicons; although 15 were untypable, S1-PFGE analysis demonstrated that 14/15 carried no plasmids. All cryptic strains were successfully typed and were positive for IncF or IncI replicons. Antibiotic-resistant strains accounted for 63% of all isolates and were significantly (p < 0.05) more frequent in phylogroup A. Most (35%) multidrug-resistant (MDR) strains belonged to phylogroup A, too. Although 25/26 MDR strains were positive for IncF plasmids (the exception being a clam strain), the FII-FIB rep combination was predominant (63%) among the sediment isolates, whereas most clam isolates (40%) carried the FII replicon alone. In mating experiments, selected MDR strains carrying FIB, FII, and I1α replicons, used as the donors, transferred multiple ARs together with the IncF or IncI plasmids at high frequency. Since IncI plasmids are common in E. coli and Salmonella enterica isolates from poultry, our findings suggest an animal origin to the E. coli clam strains carrying IncI plasmids. They also suggest a role for IncI plasmids in the spread of ARs among environmental Enterobacteriaceae and, through the food chain, to human isolates. In conclusion, the PBRT kit proved to be a useful tool to identify plasmids carrying antibiotic-resistant genes and to shed light on the factors underpinning their diffusion.

Simple amphiphilic α-hydrazido acids: Rational design, synthesis, and in vitro bioactivity profile of a novel class of potential antimicrobial compounds.

Pursuing the search for a new class of structurally simple mimics of antimicrobial peptides, we optimized a short, cheap and high-yielding synthesis of mono-charged amphiphilic α-hydrazido acid derivatives. The most active derivatives furnished MICs that are among the best values reported in literature for synthetic amphiphilic membranolytic compounds. They exhibited a broad-spectrum in vitro activity against a variety of Gram-positive and Gram-negative bacteria, including two multidrug-resistant strains. In spite of the minimal cationic charge, the best compounds demonstrated to be selective toward bacterial cell membranes over mammalian cell membranes. The relationship between either the antibacterial or the hemolytic activity and the overall lipophilicity furnished an easy way to individuate the best dimensional range for the hydrophobic portions. The importance of a non-disrupted amphiphilicity was also demonstrated. Considering the bioactivity profile and the ease of synthesis, these chemically and proteolitically stable hydrochlorides are suitable for development of a new class of wide-spectrum antibiotics.

Erythromycin-resistant lactic acid bacteria in the healthy gut of vegans, ovo-lacto vegetarians and omnivores.

Diet can affect the diversity and composition of gut microbiota. Usage of antibiotics in food production and in human or veterinary medicine has resulted in the emergence of commensal antibiotic resistant bacteria in the human gut. The incidence of erythromycin-resistant lactic acid bacteria (LAB) in the feces of healthy vegans, ovo-lacto vegetarians and omnivores was analyzed. Overall, 155 LAB were isolated and characterized for their phenotypic and genotypic resistance to erythromycin. The isolates belonged to 11 different species within the Enterococcus and Streptococcus genera. Enterococcus faecium was the dominant species in isolates from all the dietary categories. Only 97 out of 155 isolates were resistant to erythromycin after Minimum Inhibitory Concentration (MIC) determination; among them, 19 isolates (7 from vegans, 4 from ovo-lacto vegetarians and 8 from omnivores) carried the erm(B) gene. The copresence of erm(B) and erm(A) genes was only observed in Enterococcus avium from omnivores. Moreover, the transferability of erythromycin resistance genes using multidrug-resistant (MDR) cultures selected from the three groups was assessed, and four out of six isolates were able to transfer the erm(B) gene. Overall, isolates obtained from the omnivore samples showed resistance to a greater number of antibiotics and carried more tested antibiotic resistance genes compared to the isolates from ovo-lacto vegetarians and vegans. In conclusion, our results show that diet does not significantly affect the occurrence of erythromycin-resistant bacteria and that commensal strains may act as a reservoir of antibiotic resistance (AR) genes and as a source of antibiotic resistance spreading.

Natural Alkaloid Berberine Activity against Pseudomonas aeruginosa MexXY-Mediated Aminoglycoside Resistance: In Silico and in Vitro Studies.

The multidrug efflux system MexXY-OprM, inside the resistance-nodulation-division family, is a major determinant of aminoglycoside resistance in Pseudomonas aeruginosa. In the fight aimed to identify potential efflux pump inhibitors among natural compounds, the alkaloid berberine emerged as a putative inhibitor of MexXY-OprM. In this work, we elucidated its interaction with the extrusor protein MexY and assessed its synergistic activity with aminoglycosides. In particular, we built an in silico model for the MexY protein in its trimeric association using both AcrB (E. coli) and MexB (P. aeruginosa) as 3D templates. This model has been stabilized in the bacterial cytoplasmic membrane using a molecular dynamics approach and used for ensemble docking to obtain the binding site mapping. Then, through dynamic docking, we assessed its binding affinity and its synergism with aminoglycosides focusing on tobramycin, which is widely used in the treatment of pulmonary infections. In vitro assays validated the data obtained: the results showed a 2-fold increase of the inhibitory activity and 2-4 log increase of the killing activity of the association berberine-tobramycin compared to those of tobramycin alone against 13/28 tested P. aeruginosa clinical isolates. From hemolytic assays, we preliminarily assessed berberine's low toxicity.

Characterization of a new transferable MDR plasmid carrying the pbp5 gene from a clade B commensal Enterococcus faecium.

OBJECTIVES: To evaluate the transferability of antibiotic resistance from an MDR clade B Enterococcus faecium and to characterize the genetic elements involved. METHODS: The erm(B)-positive strain E. faecium 37BA (donor) and strains E. faecium 64/3 and Listeria welshimeri 11857RF (recipients) were used in mating experiments. Donors and transconjugants were characterized using MIC assays, PFGE, Southern blotting and hybridization, quantitative RT-PCR (RT-qPCR), next-generation sequencing and PCR mapping. RESULTS: One E. faecium and one L. welshimeri transconjugant were selected for in-depth investigation. Both acquired an ∼40 kb plasmid carrying erm(B). An additional plasmid of ∼200 kb, encoding the full conjugation machinery, was detected in the donor and in the E. faecium transconjugant. Next-generation sequencing revealed a new 40 396 bp plasmid that was designated pEf37BA; it contained 10 antibiotic resistance genes, tet(M), tet(L), erm(B), aadE, sat4, aphA, spw, lsa(E), lnu(B) and pbp5, resulting from the recombination of pM7M2 of E. faecium with an MDR chromosomal region of Erysipelothrix rhusiopathiae. A pbp5-carrying circular form was also detected. The PBP5 amino acid sequence differed from the C46 variant by two mutations (S39T and D644N). Its expression was documented in both transconjugants. pEf37BA persisted in the absence of selective pressure. CONCLUSIONS: The MDR clade B E. faecium plasmid, deriving from the recombination of two different resistance regions, carried a pbp5 element and was transferable to different bacterial species. This finding further documents the dissemination of ampicillin resistance among community-associated E. faecium and the key role of commensal strains in the spread of antibiotic resistance.

Detection of viable but non-culturable Pseudomonas aeruginosa in cystic fibrosis by qPCR: a validation study.

BACKGROUND: Routine culture-based diagnosis of Pseudomonas aeruginosa lung infection in Cystic Fibrosis (CF) patients can be hampered by the phenotypic variability of the microorganism, including its transition to a Viable But Non-Culturable (VBNC) state. The aim of this study was to validate an ecfX-targeting qPCR protocol developed to detect all viable P. aeruginosa bacteria and to identify VBNC forms in CF sputum samples. METHODS: The study involved 115 P. aeruginosa strains of different origins and 10 non-P. aeruginosa strains and 88 CF sputum samples, 41 Culture-Positive (CP) and 47 Culture-Negative (CN). Spiking assays were performed using scalar dilutions of a mixture of live and dead P. aeruginosa ATCC 9027 and a pooled P. aeruginosa-free sputum batch. Total DNA from sputum samples was extracted by a commercial kit, whereas a crude extract was obtained from the broth cultures. Extracellular DNA (eDNA) interference was evaluated by comparing the qPCR counts obtained from DNase-treated and untreated aliquots of the same samples. The statistical significance of the results was assessed by the Wilcoxon test and Student's t test. RESULTS: The newly-developed qPCR protocol identified 96.6% of the P. aeruginosa isolates; no amplification was obtained with strains belonging to different species. Spiking assays supported protocol reliability, since counts always matched the amount of live bacteria, thus excluding the interference of dead cells and eDNA. The protocol sensitivity threshold was 70 cells/ml of the original sample. Moreover, qPCR detected P. aeruginosa in 9/47 CN samples and showed higher bacterial counts compared with the culture method in 10/41 CP samples. CONCLUSIONS: Our findings demonstrate the reliability of the newly-developed qPCR protocol and further highlight the need for harnessing a non-culture approach to achieve an accurate microbiological diagnosis of P. aeruginosa CF lung infection and a greater understanding of its evolution.