Germano Orrù

Protocol for Real-Time PCR Identification of Anthrax Spores from Nasal Swabs after Broth Enrichment

ABSTRACT A mass-screening protocol for the diagnosis of anthrax from nasal swabs based on an enrichment step in liquid medium was devised. Incubation for growth was performed in autoclavable vials and racks which allow real-time PCR analysis of sterilized cultures. A dual-color PCR was set up with primers and probes for the chromosomal marker rpoB and the plasmid marker lef. Specific primer and probe sets were designed for the differentiation of Bacillus anthracis from B. cereus and for the differentiation of the Sterne vaccine strain from field isolates and the Ames strain, which was used in the recent anthrax bioterrorist attack. The present protocol thus combines the high specificity and sensitivity of real-time PCR with excellent biosafety and the low hands-on time necessary for the processing of large numbers of samples, which is extremely important during control programs involving the processing of large numbers of samples.

Usefulness of real time PCR for the differentiation and quantification of 652 and JP2 Actinobacillus actinomycetemcomitans genotypes in dental plaque and saliva.

BackgroundThe aim of our study is to describe a fast molecular method, able to distinguish and quantize the two different genotypes (652 and JP2) of an important periodontal pathogen: Actinobacillus actinomycetemcomitans. The two genotypes show differences in the expression of an important pathogenic factor: the leukotoxin (ltx). In order to evidence this, we performed a real time PCR procedure on the ltx operon, able to recognize Aa clinical isolates with different leukotoxic potentials.MethodsThe specificity of the method was confirmed in subgingival plaque and saliva specimens collected from eighty-one Italian (Sardinian) subjects with a mean age of 43.9, fifty five (68 %) of whom had various clinical forms of periodontal disease.ResultsThis procedure showed a good sensitivity and a high linear dynamic range of quantization (107-102 cells/ml) for all genotypes and a good correlation factor (R2 = 0.97–0.98). Compared with traditional cultural methods, this real time PCR procedure is more sensitive; in fact in two subgingival plaque and two positive saliva specimens Aa was only detected with the molecular method.ConclusionA low number of Sardinian patients was found positive for Aa infections in the oral cavity, (just 10 positive periodontal cases out of 81 and two of these were also saliva positive). The highly leukotoxic JP2 strain was the most representative (60 % of the positive specimens); the samples from periodontal pockets and from saliva showed some ltx genotype for the same patient. Our experience suggests that this approach is suitable for a rapid and complete laboratory diagnosis for Aa infection.

Drug Resistance Evolution of a Mycobacterium tuberculosis Strain from a Noncompliant Patient

ABSTRACT The emergence and spread of multidrug-resistant (MDR) Mycobacterium tuberculosis (MT) represents a worldwide health care problem because of the difficulty in treating these infections. Development of drug resistance in MT arises mainly by mutation of chromosomal genes. To investigate the evolution of a MT population during a long-lasting infection, the phenotypic and genotypic changes in the drug resistance of 10 sequential MT isolates from a noncompliant chronically infected patient were investigated. During more than 12 years of active disease, a MDR population developed; molecular typing showed one single parental strain that infected the patient and persisted throughout the disease. Molecular analysis of the drug resistance-related genes revealed that discrete subpopulations evolved over time from the parental strain by acquiring and accumulating resistance-conferring mutations to isoniazid, rifampin, and streptomycin. Overall, these observations indicate that during a chronic infection, several subpopulations may coexist in the same patient with different drug susceptibility profiles.

Microbial biofilm modulation by ultrasound: Current concepts and controversies

Biofilm elimination is often necessary during antimicrobial therapy or industrial medical manufacturing decontamination. In this context, ultrasound treatment has been frequently described in the literature for its antibiofilm effectiveness, but at the same time, various authors have described ultrasound as a formidable enhancer of bacterial viability. This discrepancy has found no solution in the current literature for around 9 years; some works have shown that every time bacteria are exposed to an ultrasonic field, both destruction and stimulation phenomena co-exist. This co-existence proves to have different final effects based on various factors such as: ultrasound frequency and intensity, the bacterial species involved, the material used for ultrasound diffusion, the presence of cavitation effects and the forms of bacterial planktonic or biofilm. The aim of this work is to analyze current concepts regarding ultrasound effect on prokaryotic cells, and in particular ultrasound activity on bacterial biofilm.

Thymosin β-4 in colorectal cancer is localized predominantly at the invasion front in tumor cells undergoing epithelial mesenchymal transition

Objective Thymosin β-4 (Tβ4) is a ubiquitous peptide that plays pivotal roles in the cytoskeletal system and in cell differentiation during embryogenesis. Recently, a role for Tβ4 has been proposed in experimental and human carcinogenesis. This study was aimed at evaluating the correlation between Tβ4 immunoractivity and colorectal cancer, with particular attemption to tumor cells undergoing epithelial-mesenchymal transition. Methods and Results 86 intestinal biopsies were retrospectively analyzed including 76 colorectal adenocarcinomas with evident features of epithelial-mesenchymal transition, and 10 samples of normal colorectal mucosa. Paraffin sections were immunostained for Tβ4 and for E-cadherin. Total RNA was isolated from frozen specimens obtained, at surgery, from the normal colon mucosa, the deeper regions and the superficial tumor regions in four cases of colon cancer. Tβ4 immunoreactivity was detected in the vast majority (59/76) of colon carcinomas, showing a patchy distribution, with well differentiated areas significantly more reactive than the less differentiated tumor zones. We also noted a zonal pattern in the majority of tumors, characterized by a progressive increase in immunostaining for Tβ4 from the superficial toward the deepest tumor regions. The strongest expression for Tβ4 was frequently detected in invading tumor cells with features of epithelial-mesenchymal transition. The increase in reactivity for Tβ4 matched with a progressive decrease in E-cadherin expression in invading cancer cells. At mRNA level, the differences in Tβ4 expression between the surrounding colon mucosa and the tumors samples were not significant. Conclusions Our data show that Tβ4 is expressed in the majority of colon cancers, with preferential immunoreactivity in deep tumor regions. The preferential expression of the peptide and the increase in intensity of the immunostaining at the invasion front suggests a possible link between the peptide and the process of epithelial mesenchymal transition, suggesting a role for Tβ4 in colorectal cancer invasion and metastasis.

Treatment of Tuberculosis in a Region with High Drug Resistance: Outcomes, Drug Resistance Amplification and Re-Infection

Introduction Emerging antituberculosis drug resistance is a serious threat for tuberculosis (TB) control, especially in Eastern European countries. Methods We combined drug susceptibility results and molecular strain typing data with treatment outcome reports to assess the influence of drug resistance on TB treatment outcomes in a prospective cohort of patients from Abkhazia (Georgia). Patients received individualized treatment regimens based on drug susceptibility testing (DST) results. Definitions for antituberculosis drug resistance and treatment outcomes were in line with current WHO recommendations. First and second line DST, and molecular typing were performed in a supranational laboratory for Mycobacterium tuberculosis (MTB) strains from consecutive sputum smear-positive TB patients at baseline and during treatment. Results At baseline, MTB strains were fully drug-susceptible in 189/326 (58.0%) of patients. Resistance to at least H or R (PDR-TB) and multidrug-resistance (MDR-TB) were found in 69/326 (21.2%) and 68/326 (20.9%) of strains, respectively. Three MDR-TB strains were also extensively resistant (XDR-TB). During treatment, 3/189 (1.6%) fully susceptible patients at baseline were re-infected with a MDR-TB strain and 2/58 (3.4%) PDR-TB patients became MDR-TB due to resistance amplification. 5/47 (10.6%) MDR- patients became XDR-TB during treatment. Treatment success was observed in 161/189 (85.2%), 54/69 (78.3%) and 22/68 (32.3%) of patients with fully drug susceptible, PDR- and MDR-TB, respectively. Development of ofloxacin resistance was significantly associated with a negative treatment outcome. Conclusion In Abkhazia, a region with high prevalence of drug resistant TB, the use of individualized MDR-TB treatment regimens resulted in poor treatment outcomes and XDR-TB amplification. Nosocomial transmission of MDR-TB emphasizes the importance of infection control in hospitals.

Molecular Characterization of Anisakis Larvae from Fish Caught Off Sardinia

abstract:  Anisakis spp. larvae are parasitic, and potentially zoonotic, nematodes transmitted by marine fish and cephalopods, which are the main intermediate hosts of the third larval stage. The accidental consumption of infected raw or poorly cooked fish may cause gastroenteric diseases and allergies in humans. The aim of the present study was to use polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) to define the occurrence, species variability, and host preferences of Anisakis spp. larvae in fish caught off the coast of Sardinia. Necropsy was used on 285 samples; 552 Anisakis spp. L3 larvae were isolated from 87 fish that tested positive for this nematode. Anisakis pegreffii was most frequently encountered (90.6%), with a primary preference for Scomber scombrus, Zeus faber, and Trachurus mediterraneus. In contrast, the prevalence of Anisakis physeteris was only 1.3%. A hybrid genotype of Anisakis simplex sensu stricto and Anisakis pegreffii was also observed, which confirms the results of previous studies carried out in the western Mediterranean. Interestingly, no Anisakis simplex s.s. larvae were recovered. These results indicate that the diversity of Anisakis species is low in Sardinia waters, probably because of its geographic position.

Carbohydrate Availability Regulates Virulence Gene Expression in Streptococcus suis

Streptococcus suis is a major bacterial pathogen of young pigs causing worldwide economic problems for the pig industry. S. suis is also an emerging pathogen of humans. Colonization of porcine oropharynx by S. suis is considered to be a high risk factor for invasive disease. In the oropharyngeal cavity, where glucose is rapidly absorbed but dietary α-glucans persist, there is a profound effect of carbohydrate availability on the expression of virulence genes. Nineteen predicted or confirmed S. suis virulence genes that promote adhesion to and invasion of epithelial cells were expressed at higher levels when S. suis was supplied with the α-glucan starch/pullulan compared to glucose as the single carbon source. Additionally the production of suilysin, a toxin that damages epithelial cells, was increased more than ten-fold when glucose levels were low and S. suis was growing on pullulan. Based on biochemical, bioinformatics and in vitro and in vivo gene expression studies, we developed a biological model that postulates the effect of carbon catabolite repression on expression of virulence genes in the mucosa, organs and blood. This research increases our understanding of S. suis virulence mechanisms and has important implications for the design of future control strategies including the development of anti-infective strategies by modulating animal feed composition.

In Vitro evaluation of Enterococcus faecalis adhesion on various endodontic medicaments.

E. faecalis in endodontic infection represents a biofilm type of disease, which explains the bacteria’s resistance to various antimicrobial compounds and the subsequent failure after endodontic treatment. The purpose of this study was to compare antimicrobial activities and bacteria kinetic adhesion in vitro for three endodontic medicaments with a clinical isolate of E. faecalis. We devised a shake culture which contained the following intracanalar preparations: CPD, Endoidrox (EIX), PulpCanalSealer (PCS); these were immersed in a liquid culture medium inoculated with the microorganism. The shake system velocity was able to prevent non-specific bacteria adhesion and simulated the salivary flow. Specimens were collected daily (from both the medium and medicaments) for 10 days; the viable cells were counted by plate count, while the adhesion index AI° [E. faecalis fg DNA] /mm2 was evaluated in the pastes after DNA extraction, by quantitative real time PCR for the 16S rRNA gene. A partial growth inhibition, during the first 24 hours, was observed in the liquid medium and on the medicaments for EIX and subsequently for CPD (six logs). EIX showed the lowest adhesion coefficient (5*102 [fg DNA]/mm2) for nine days and was similar to the control. PCS showed no antimicrobial/antibiofilm properties. This showed that “calcium oxide” base compounds could be active against biofilm progression and at least in the short term (2-4 days) on E. faecalis cells growing in planktonic cultures.

Rapid PCR real-time genotyping of M-Malton alpha1-antitrypsin deficiency alleles by molecular beacons

α1-Antitrypsin deficiency is an autosomal codominant inherited disorder, with increased risk of developing lung and liver disease. The large majority of subjects affected by α1-antitrypsin deficiency carry the PIZZ or PISZ genotypes, which can be easily detected using several molecular methods. Another pathologic allele, the M-Malton variant (also known as Mnichinan and Mcagliari), can mimic the Pi Z clinical phenotype, but this α1-antitrypsin deficiency variant is not easily recognizable and, therefore, seems to be more underrecognized than the Z or S alleles. We report the development of a rapid qualitative fluorescent real-time PCR assay designed for the detection of the M-Malton α1-antitrypsin deficiency alleles using 2 specific molecular beacons. The assay is able to detect in a single tube the homozygous as well the heterozygous genotypes. The procedure combines the great sensitivity of the polymerase chain reaction, the specificity provided by allele-specific molecular beacons, and the throughput of a multicolour fluorescence detection procedure. This technique will be useful for research and molecular diagnostic laboratories involved in the study of α1-antitrypsin deficiency-related diseases.