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Mycobacterium vaccae is a rapidly growing nonpathogenic species of the Mycobacteriaceae family of bacteria that can cause pulmonary and disseminated disease in particular in immunocompromised individuals. Here we describe a first case of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass-spectrometry (MS) identification of this pathogen in a patient with non-Hodgkin’s lymphoma during chemoimmunotherapy salvage treatment, and its impact on clinical decision making.

A new, bifunctional recombinant protein was expressed as the fusion product of human elastin-like polypeptide (HELP) and the bilirubin-binding protein UnaG. The engineered product displays both the HELP-specific property of forming a functional hydrogel matrix and the UnaG-specific capacity of emitting green fluorescence upon ligand binding. The new fusion protein has been proven to be effective at detecting bilirubin in complex environments with high background noise. A cell culture model of the stress response, consisting of bilirubin released in the cell culture medium, was set up to assess the bilirubin-sensing properties of the functional matrix obtained by cross-linking the HELP moiety. Our engineered protein allowed us to monitor cell induction by the release of bilirubin in the culture medium on a nanomolar scale. This study shows that elastin-like protein fusion represents a versatile platform for the development of novel and commercially viable analytical and biosensing devices.

Aim: This study aims to characterize circulating strains to predict their relationship with sexually transmitted microorganisms, Chlamydia trachomatis, HIV, HCV, Treponema pallidum, HPV, Mycoplasmas, in an Italian multiethnic area, which has revealed a recent increase of Neisseria gonorrhoeae first-line antibiotic resistance. Materials & methods: We performed N. gonorrhoeae multiantigen sequence typing and the N. gonorrhoeae sequence typing for antimicrobial resistance. Results: We identified mutations in genes conferring resistance to cephalosporins, macrolides, fluoroquinolones through por and tbpB loci, and we reported new combinations of already known alleles. N. gonorrhoeae resistance to ciprofloxacin was associated with C. trachomatis coinfection. Conclusion: This study’s data proved the utility of a routine N. gonorrhoeae molecular characterization to monitor the evolution of antibiotic resistance and to detect the most effective clinical treatment.

Bacterial vaginosis involves the presence of a polymicrobial biofilm on the vaginal epithelium, guaranteeing immune escape and spread of antibiotic resistance. To spot known biofilm-forming bacteria, we profiled the vaginal microbiome of sixty-four symptomatic women suffering from a different grade of vaginal disorders and sixty asymptomatic healthy women. Specific microbial profiles distinguished symptomatic from asymptomatic women and characterized the grade of dysmicrobism within the symptomatic group. Lactobacillus crispatus and iners predominated on the healthy vaginal mucosa, while Lactobacillus gasseri predominated in the intermediate dysmicrobism. Furthermore, the intermediate grade of dysmicrobism was characterized by other lactic acid-producers species than Lactobacilli, able to rescue the microbial imbalance, and Ureaplasma parvum-serovar 3. The vaginosis group exhibited the overgrowth of Prevotella bivia, which is known to enhance the biofilm formation by Gardnerella vaginalis, and the presence of Streptococcus anginosus, which is emerging as a new cooperating player of the vaginal biofilm. Identifying specific microorganisms promoting or preventing the biofilm formation could increase the accuracy for a better definition of the vaginal dysmicrobism concept and therapeutic intervention.

Elastin is a fibrous protein that confers elasticity to tissues such as skin, arteries and lung. It is extensively cross-linked, highly hydrophobic and insoluble. Nevertheless, elastin can be hydrolysed by bacterial proteases in infectious diseases, resulting in more or less severe tissue damage. Thus, development of substrates able to reliably and specifically detect pathogen-secreted elastolytic activity is needed to improve the in vitro evaluation of the injury that bacterial proteases may provoke. In this work, two human biomimetic elastin polypeptides, HELP and HELP1, as well as the matrices derived from HELP, have been probed as substrates for elastolytic activity detection. Thirty strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients were analyzed in parallel with standard substrates, to detect proteolytic and elastolytic activity. Results point to the HELP-based 3D matrix as an interesting biomimetic model of elastin to assess bacterial elastolytic activity in vitro. Moreover, this model substrate enables to further elucidate the mechanism underlying elastin degradation at molecular level, as well as to develop biomimetic material-based devices responsive to external stimuli.

In vitro and in vivo studies have demonstrated that UCB (unconjugated bilirubin) is neurotoxic. Although previous studies suggested that both MRP1 (multidrug resistance-associated protein 1) and MDR1 (multidrug resistance protein 1) may protect cells against accumulation of UCB, direct comparison of their role in UCB transport was never performed. To this end, we used an inducible siRNA (small interfering RNA) expression system to silence the expression of MRP1 and MDR1 in human neuroblastoma SH-SY5Y cells. The effects of in vitro exposure to clinically-relevant levels of unbound UCB were compared between unsilenced (control) cells and cells with similar reductions in the expression of MRP1 or MDR1, documented by RT-PCR (reverse transcription-PCR) (mRNA), immunoblotting (protein), and for MDR1, the enhanced net uptake of a specific fluorescent substrate. Cytotoxicity was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] test. MRP1-deficient cells accumulated significantly more UCB and suffered greater cytotoxicity than controls. By contrast, MDR1-deficient cells exhibited UCB uptake and cytotoxicity comparable with controls. At intermediate levels of silencing, the increased susceptibility to UCB toxicity closely correlated with the decrease in the expression of MRP1, but not of MDR1. These data support the concept that limitation of cellular UCB accumulation, due to UCB export mediated by MRP1, but not MDR1, plays an important role in preventing bilirubin encephalopathy in the newborn

Stimuli-responsive hydrogel matrices have attracted great attention in biomedical and biotechnological fields for controlled delivery of bioactive compounds, as well as a vehicle for therapeutic cell spreading. Elastin-derived biomimetic polypeptides are recombinant macromolecules suitable for the realization of smart biomaterials. In this study, we explored the potential of an elastin biomimetic matrix to realize proteolytic stimuli-responsive systems to control the release of substances. Our approach showed that this matrix was susceptible to elastolytic degradation, and it has been successfully employed to obtain an efficient delivery of a model protein. This setup will constitute a therapeutic agent delivery platform to realize devices capable of responding and interacting with biological systems at the molecular level.

Infections caused by Burkholderia cepacia complex in patients with cystic fibrosis (CF) are often related to increased mortality. One of the mechanisms that makes the bacteria more resistant to the action of antibiotics is the ability to produce biofilm. It has been compared the antibiotic resistance of two clinical isolates of a biofilm-producing strain called BTS-2, responsible for chronic pulmonary infection in a CF patient. Bacteria were incubated in both Yeast Extract Mannitol Medium (YEM) and Mueller Hinton Broth (MHB) at 30°C for 24 hours. Biofilm was quantified by spectrophotometer readings (OD570) of the extracted crystal violet. By microdilution broth, we evaluated the minimum concentration of antibiotic inhibiting the growth of the planktonic form (MIC); by the technology “Calgary Biofilm Device” and by Resazurin, we evaluated the susceptibility of sessile forms (BIC). Data demonstrate that the microorganism in the course of time changed its sensitivity to some of the antibiotics tested. The comparison between sessile and planktonic forms of BTS2 pre-incubated in MHB showed that sessile forms increased their resistance to antibiotics in a few cases only. BICs of BTS2 pre-incubated in YEM, where the strain produced an amount of biofilm significantly lower than in MHB, showed a higher susceptibility to 4 of the 10 antibiotics tested. Our data showed that the susceptibility of sessile forms of a strain is not always lower than the susceptibility of its planktonic forms and that culture medium can affect significantly the biofilm production.

The phlogistic-infectious diseases of the lower female genital tract are one of the most widespread obstetricgynecologic issues, due to treatment failures that cause frequent relapses and to the adverse effects of some commonly used drugs.The most common vaginal syndromes are due to uncontrolled growth of bacteria or fungi which replace the normal vaginal flora, causing phlogistic and infectious based diseases. These infections are treated with anti-inflammatory and antibiotic therapy; however, the emergence of resistant strains and the ability of many microorganisms to grow inside biofilms severely reduce the repertoire of useful agents.Thus, in the last years increasing interest has been focused toward compounds of plant origin with anti-microbial properties. In the present work, we studied the antimicrobial activity of fractions obtained from endemic plants of Sardinia towards microorganisms that frequently are involved in vaginal infectious diseases: Streptococcus agalactiae, Gardnerella vaginalis and Candida albicans.

Bacteria belonging to the Burkholderia cepacia complex are mainly isolated from the sputum of cystic fibrosis patients and frequently show a mucoid phenotype.Most of them produce an exopolysaccharide called cepacian, whose repeating unit consists of a branched heptasaccharide carrying from one to three acetyl esters. Two genetic loci, bceI and bceII, consisting of 11 and 9 genes respectively, are involved in cepacian biosynthesis.Three genes located in the bceII locus, named bceOSU, code for different acyltransferases. As the presence of acetyl groups influences the viscosity of cepacian, we compared three strains (two clinical isolates named BTS2 and BTS7, and the reference strain Burkholderia sp. 383) exhibiting differences both in the acetylation pattern and at the genomic level, for the presence of insertion sequences adjacent to bceU.

The Burkholderia cepacia complex (Bcc) consists of several species that cause lung infections in patients with cystic fibrosis but are also capable to colonize immunocompromised patients. Once established, the infection is usually difficult to eradicate, as Bcc is intrinsically resistant to many antibiotics. Besides, the acquisition of additional resistance determinants by horizontal gene transfer makes very difficult the therapeutic approach to these infections. Among horizontally acquired DNAs, integrons have been frequently reported in many Gramnegative bacteria that affect human health, but they have not been found frequently in Burkholderia isolates until now. In the present work we report on a Bcc isolate, recovered from the blood of an immunocompromised patient, that carries a 2.3 kb class I integron already described in a Salmonella enterica isolate eight years ago, coding for aacA4, aadA1 and catB2 in its cassette array.

Burkholderia cepacia is an opportunistic pathogen able to colonize the airways of Cystic Fibrosis (CF) patients, frequently developing chronic infections. In 20% of cases these infections cause severe and poorly controlled pathological situations because of the intrinsic antibiotic resistance expressed by the microorganism. CF patients are often subjected to antibiotic therapy: this facilitates the acquisition of antibiotic resistance determinants by the infecting bacteria. Integrons are mobile genetic elements that are widespread in bacterial populations and favor the acquisition of gene cassettes coding for these determinants.The presence of class 1 integrons was investigated by PCR with primers specific for the 5’ and 3’ ends in Burkholderia isolates recovered from patients in treatment at the CF center of Friuli Venezia Giulia. The same integron, carrying an uncommon allelic form (Ib) of the aacA4 gene in its cassette array and conferring resistance to some aminoglycosides, was found in two independent isolates (different RAPD profiles) infecting two different patients. In both isolates the integron was carried by plasmids and was still present 3 and 6 years later the first finding. Despite the exchange of integrons between bacterial pathogens is fully described, these items were not frequently found in Burkholderia isolates. Although the clinical relevance of the integron we identified is low (a single gene cassette encoding a widespread resistance),we feel concerned that these genetic elements begin to circulate in this bacterial species, as this could make more and more troublesome the treatment of infections notoriously difficult to eradicate.

An epidemiologic survey performed at the Trieste University Hospital (northeastern Italy) between 1999 and 2002 revealed a remarkable spread of an MDR Pseudomonas aeruginosa strain, named TS-832035, which carried the chromosomal integron In70.2 containing four gene cassettes (blaVIM-1, aacA4, aphA15 and aadA1) in its variable region and conferring resistance to ß-lactams, including carbapenems, and to several aminoglycosides. Moreover, some other P. aeruginosa isolates, strictly related to TS-832035 but lacking in the integron In70.2, were detected, but they remained a minor component within the cluster during the three years of surveillance.They showed an MDR phenotype like TS- 832035, differing only for the susceptibility level to carbapenems. The genomic relatedness between TS-832035 and TS-103 was investigated by random amplification of polymorphic DNA (RAPD) typing, pulsed-field gel electrophoresis (PFGE) analysis of SpeI-digested genomic DNA, and multilocus sequence typing (MLST). The cost of the integron In70.2 on the fitness of TS-832035 was determined by performing growth kinetics and direct competition assays against the clonal isolate TS-103 in three media differing for nutrient availability: a rich medium (Luria Bertani (LB) Broth) and a minimal medium (28 g/l K2HPO4, 12 g/l KH2PO4, 0.4 g/l MgSO4, 7H2O, 4 g/l (NH4)2SO4) added with a rich carbon source (0.4% w/v glucose) or with a poorer carbon source (0.4% w/v sodium acetate). Growth kinetic data were obtained by measuring optical density at 600 nm (OD600). For competition assays, the number of CFU/ml of each isolate was estimated by colony-hybridization. We proved the clonality of the two isolates by molecular investigations.The results of the growth kinetics showed the existence of a significant in vitro fitness cost associated with the integron In70.2, more evident in a poorer medium.The sensitivity of the two isolates to the antimicrobial agents tested was the same, except for the different levels of resistance to carbapenems (MIC 16 μg/ml versus 64-128 μg/ml). Although we can not exclude that other factors may have favoured the in vivo spread of TS-832035, our results suggest that the increased level of resistance to carbapenems has conferred on this isolate a selective advantage able to compensate metabolic cost associated to the integron.

The Pseudomonas aeruginosa isolate TS-832035 was responsible for an outbreak that occurred in an Italian hospital between 1999 and 2002. It exhibited a high-level resistance to carbapenems due to the contemporary presence of two independent mechanisms: the production of a carbapenemase, coded by a bla(VIM-1) determinant carried by the chromosomal class 1 integron In70.2 (containing also the aacA4, aphA15, and aadA1 genes in its cassette array), and the lack of the OprD porin. We compared TS-832035 with a strictly related isolate, TS-103, whose resistance to carbapenems was due to the lack of the OprD porin only, as it did not carry In70.2. We evaluated their growth kinetics, in both separate cultures and competition assays, under permissive conditions. These experiments highlighted a significant in vitro fitness cost associated with the integron. On the contrary, none of the resistance determinants other than the bla(VIM-1) seemed to confer a real selective advantage to its host. Comparison of these results with the in vivo behavior, showing that the In70.2-carrying isolates largely prevailed over the In70.2-lacking ones, besides the detection of similar integrons in other Italian clinical isolates, evidenced the need to investigate accurately the causes of their large distribution, as possible soft spots could exist in the ability of their hosts to adapt to the hospital settings.