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The microbiota “organ” is the central bioreactor of the gastrointestinal tract, populated by a total of 1014bacteria and characterized by a genomic content (microbiome), which represents more than 100 times the human genome. The microbiota plays an important role in child health by acting as a barrier against pathogens and their invasion with a highly dynamic modality, exerting metabolic multistep functions and stimulating the development of the host immune system, through well-organized programming, which influences all of the growth and aging processes. The advent of “omics” technologies (genomics, proteomics, metabolomics), characterized by complex technological platforms and advanced analytical and computational procedures, has opened new avenues to the knowledge of the gut microbiota ecosystem, clarifying some aspects on the establishment of microbial communities that constitute it, their modulation and active interaction with external stimuli as well as food, within the host genetic variability. With a huge interdisciplinary effort and an interface work between basic, translational, and clinical research, microbiologists, specialists in “-omics” disciplines, and clinicians are now clarifying the role of the microbiota in the programming process of several gut-related diseases, from the physiological symbiosis to the microbial dysbiosis stage, through an integrated systems biology approach.

We investigated the clonal relatedness of seven multi-drug-resistant (MDR) Klebsiella pneumoniaeisolates, as well as three susceptible K. pneumoniaeisolates collected during hospital outbreaks and outbreak-related microbiological surveillance, respectively. The relatedness among K. pneumoniaeisolates was assessed by pulsed field gel electrophoresis (PFGE) and automated repetitive-sequence-based PCR (rep-PCR) genotyping and the results were compared to a proteomic phenotyping performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). All typing methods agreed on the generation of three different clusters of K. pneumoniaeisogenetic/related MDR strains. After strengthening hospital infection control measures, no other spreading events involving MDR-K. pneumoniaewere reported until the end of the observation period. This preliminary investigation suggests that, in a hierarchical approach to bacterial typing, MALDI-TOF MS proteome profiling might offer a fast and valuable preliminary screening tool able to support microbiologists during nosocomial outbreak surveys.

Pseudomonas aeruginosais an opportunistic pathogen and an ubiquitous environmental bacterium. Fifty-seven days after hospitalization, we isolated three distinct P. aeruginosamorphotypes (smooth, rough and mucoid) from the lower respiratory tract of a patient admitted to a Cardiology Intensive Care Unit (ICU). Moreover, a group of nine colony variants, arising from the three P. aeruginosaisolates growing in laboratory growth media, were also isolated. The resulting 12 isolates were characterised for antibiotic resistance profile and subjected to genotypic analysis by fluorescent-Amplified Fragment Length Polymorphism (f-AFLP) and automated repetitive extragenic palindromic-PCR (rep-PCR) fingerprinting. The three smooth, rough and mucoid morphotypes presented different antibiotic resistance profiles and genotyping analysis showed that they belonged to distinct clones, indicating that at day 57 after the admission the patient was simultaneously colonized by three distinct P. aeruginosaisolates. On the other hand, the nine colony variants presented heterogeneous antibiotic resistance profiles and clustered together with the three parental isolates. The understanding of the link between genotype plasticity and antibiotic resistance may contribute to improving our knowledge of this life-threatening pathogen.

Under conditions of activated type III secretion Shigella flexneriup-regulates the expression of numerous genes, including the virulence plasmid (pINV)-encoded ospBand phoN2genes. ospBand phoN2are virulence-associated genes which are part of a bicistronic transcriptional unit encoding OspB, a protein (effector) of unknown function secreted by the type III secretion (TTS) apparatus, and PhoN2 (apyrase or ATP-diphosphohydrolase), a periplasmic protein involved in polar IcsA localization on the surface of S. flexneri. In this work we used real-time PCR to measure transcription of ospBand phoN2of wild-type S. flexneristrain M90T as well as of derivative mutants impaired in definite virulence traits. The results obtained confirmed and extended previous reports indicating that the expression of ospBand phoN2genes is modulated in a virB-dependent, mxiE-independent manner under conditions of non-activated secretion, while their expression is considerably induced in a mxiE-dependent manner under conditions of activated secretion. That the expression of the ospB-phoN2operon is up-regulated in condition of activated secretion, indicates that probably the expression of these two genes might be important, especially during the later stages of infection of S. flexneri.

Quantitation of neutrophil CD64 expression and procalcitonin (PCT) levels in blood samples have been recently proposed as useful tools for early detection of sepsis. To determine the usefulness of these tests, we analyzed blood samples of 112 patients, admitted to an intensive care unit (ICU), presenting clinical symptoms of sepsis, as well as of 50 healthy controls. At the end of the study, a retrospective analysis showed that only 52 of the 112 ICU-patients presented a real sepsis (positive blood culture). The results obtained indicated that of the 52 patients with sepsis, 50 and 49 presented levels of neutrophil CD64 expression ≥ 2398 molecules per cell (cut-off determined by receiver operator characteristic analysis) and PCT levels > 0.5 ng/ml (cut-off suggested by the manufacturer), respectively. However, the neutrophil CD64 test showed higher specificity in detecting sepsis since 5 out of the 60 ICU-patients without sepsis (negative blood culture), presented CD64 expression levels ≥ 2398 molecules per cell, PCT levels ≥ 0.5 ng/ml were shown in 27 patients. Moreover, while none of the 50 healthy controls presented a neutrophil CD64 level higher than the cut-off value, 5 patients presented PCT levels ≥ 0.5 ng/ml. In conclusion, our data seem to indicate that the quantitation of CD64 expression could be taken into consideration as a sensitive and specific test for early diagnosis of sepsis.

Stenotrophomonas maltophiliais an emerging nosocomial bacterial pathogen which is currently isolated with increasing frequency from the airways of cystic fibrosis (CF) patients. In this study 13 S. maltophiliastrains (11 isolated from the airways of independent CF patients, and two non-CF respiratory reference strains) have been characterized for the expression of several virulence-associated factors. In particular, the ability to form biofilm on abiotic surfaces has been determined and correlated with different features, such as motility, adherence and the ability to invade A549 respiratory epithelial cells. Moreover, the presence of a flagellum-associated gene as well as that of the StmPr1gene, which encodes an extra-cellular protease, have been determined by Southern blot hybridization. Our data indicate that the different degree of biofilm formation exhibited by the 11 CF isolates does not correlate with motility, ability to adhere to and invade A549 cells, or with the presence of flagella. On the other hand, among the CF isolates the StmPr1gene was found only in two strains, both able to establish chronic lung infections in CF patients. Moreover, only four of the strains analyzed show a temperature-independent antibiotic-resistance profile, suggesting either a different origin of these strains or an intervening adaptation to host tissues.

The aim of our study was to evaluate whether the replicate PCR testing may provide more accurate estimates of C. pneumoniaeDNA prevalence in PBMC of patients undergoing carotid endarterectomy. Clinical sensitivity and reproducibility of ompAnested touchdown PCR was also performed. Clinical sensitivity and reproducibility was examined by testing C. pneumoniae-negative PBMC spiked with serial dilutions of semipurified C. pneumoniaeelementary bodies (from 8 to 0.002 IFU/ml). Detection of C. pneumoniaeDNA was performed by ompAnested touchdown PCR. Each clinical and spiked PBMC DNA specimen was analyzed in replicates of 1,3,5 and 10. PCR results of serial dilutions of C. pneumoniaeDNA performed in replicates of 10 were analysed by probit analysis.C. pneumoniaeDNA was detected in 14 of the 30 (46.7%) PBMC clinical specimens examined when 10 replicates were tested. When we analyzed 1, 3 and 5 replicates, 4 (13.3%), 7(23.3%), 12(40%) of the 30 specimens were positive, respectively. The limit of detection of ompAnested PCR touchdown was 0.008 IFU/ml when 10 replicates were tested. The ompAnested PCR had reproducibility scores of 10 for 10 from 8 to 4 IFU/ml concentration, but scores decreased for smaller numbers of IFU/ml. Our results showed that repeat testing of the same specimen increased clinical sensitivity as well as reproducibility of the ompAnested touchdown PCR. In conclusion the replicate PCR testing improves the performance of ompAnested touchdown PCR and provides a more accurate estimates of the prevalence of C. pneumoniaein PBMC of patients with atherosclerotic cardiovascular disease.

Streptococcus mutans, a Gram-positive immobile bacterium, is an oral pathogen considered to be the principal etiologic agent of dental caries. Although some researches suggest that trace metals, including iron, can be associated with dental caries, the function of salivary iron and lactoferrin in the human oral cavity remains unclear. The data reported in this study indicates that iron-deprived saliva (Fe3+<0.1 μM) increases S. mutansaggregation and biofilm formation in the fluid and adherent phases as compared with saliva (Fe3+from 0.1 to 1 μM), while iron-loaded saliva (Fe3+>1 μM) inhibits both phenomena. Our findings are consistent with the hypothesis that S. mutansaggregation and biofilm formation are negatively iron-modulated as confirmed by the different effect of bovine lactoferrin (bLf), added to saliva at physiological concentration (20 μg/ml) in the apo- or iron-saturated form. Even if saliva itself induces bacterial aggregation, iron binding capability of apo-bLf is responsible for the noticeable increase of bacterial aggregation and biofilm development in the fluid and adherent phases. On the contrary, iron-saturated bLf decreases aggregation and biofilm development by supplying iron to S. mutans. Therefore, the iron-withholding capability of apo-Lf or native Lf is an important signal to which S. mutanscounteracts by leaving the planktonic state and entering into a new lifestyle, biofilm, to colonize and persist in the human oral cavity. In addition, another function of bLf, unrelated to its iron binding capability, is responsible for the inhibition of the adhesion of S. mutansfree, aggregated or biofilm on abiotic surfaces. Both these activities of lactoferrin, related and unrelated to the iron binding capability, could have a key role in protecting the human oral cavity from S. mutanspathogenicity.

ABSTRACTEvidence linking Chlamydia pneumoniaeinfection to atherosclerosis and to atherothrombotic events has recently emerged. A primary candidate implicated in these pathogenetic events is the 60-kDa chlamydial heat shock protein (HSP60). Another putative candidate to activate a potential proinflammatory mechanism is the chlamydial outer membrane protein 2 (OMP2). We have generated both HSP60 and OMP2 recombinant antigens in a nondenatured form and shown that (i) the two antigens were highly immunogenic in mice and (ii) murine antisera thus generated recognized the native C. pneumoniaeproteins. We measured by enzyme linked immunosorbent assay (ELISA) and immunoblot assay antibody titers to the recombinant antigens in samples from 219 patients with coronary heart disease (CHD), 179 patients with unstable angina (UA), 40 patients with acute myocardial infarction (AMI), and 100 age-, sex-, and risk factor-matched healthy controls. We also examined whether anti-HSP60 and/or anti-OMP2 antibodies correlated with anti-C. pneumoniaeantibodies assessed by a commercial microimmunofluorescence (MIF) assay. Immunoglobulin G (IgG), but neither IgA nor IgM, antibodies against the two recombinant proteins were detected by ELISA. In particular, anti-HSP60 antibodies were detected in >99% of CHD patients versus 0% of the controls, whereas the proportions of anti-OMP2 positive subjects were >70 and 27%, respectively. Nonetheless, among CHD patients, similar frequencies of positive subjects and titers of anti-HSP60 or anti-OMP2 antibodies were present in UA and AMI subjects. The anti-OMP2, but not the anti-HSP60, antibodies showed high specificity. Consistently, high serological correlation was observed between IgG MIF titers and IgG ELISA reactivity to OMP2 but not to HSP60. Overall, the results of this study demonstrate a strong correlation between CHD and anti-HSP60 IgG levels, as measured by our in-house ELISA. They also suggest that recombinant OMP2 ELISA, because of its high specificity and strong correlation with MIF assay, could be a candidate diagnostic marker for C. pneumoniaeinfection, which would be of potential usefulness for its specificity and nonsubjective nature.

ABSTRACTThe aim of this study was to characterize a Bartonellastrain (BA-1) isolated from a blood culture of an Italian, human immunodeficiency virus-positive patient with bacillary angiomatosis. We analyzed the isolate using molecular biology methods such as whole-cell fatty acid analysis, PCR-restriction fragment length polymorphism analysis, type-specific 16S rRNA PCRs, sequence analysis of the 16S rRNA, pulsed-field gel electrophoresis, and arbitrarily primed PCR. The BA-1 isolate turned out to be a Bartonella quintanastrain, similar but not identical to B. quintanaOklahoma, which was used as a control strain.

ABSTRACTA conjugative IncL/M plasmid (pSEM) conferring resistance to gentamicin, amikacin, kanamycin, sulfonamides, and expanded-spectrum cephalosporins was found in pathogenic strains of Salmonella entericaserotype Typhimurium. Resistance to aminoglycosides was encoded by a sul1-type class 1 integron (In-t3). An extended-spectrum beta-lactamase gene,blaSHV-5, was identified 3.5 kb downstream of the integrase (intI1) gene of In-t3. Nucleotide sequence analysis of the 5.3-kb blaSHV-5–In-t3 region of pSEM highlighted striking similarities with IncL/M plasmids isolated from nosocomial gram-negative pathogens, conferring resistance to expanded-spectrum cephalosporins and aminoglycosides.

ABSTRACTThe presence and genetic content of integrons were investigated for 37 epidemiologically unrelated multiple-drug-resistant strains ofSalmonella entericaserotype Typhimurium from humans. All isolates were resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfonamides, and trimethoprim, as well as to tetracycline and/or nalidixic acid; 20% of them were also resistant to gentamicin and amikacin. Three different class 1 integrons (In-t1, In-t2, and In-t3) were identified by Southern blot hybridization, PCR, and DNA sequencing, and these integrons were found to carry theaadB, catB3, oxa1,aadA1a, aacA4, and aacC1gene cassettes. Integrons In-t1 (aadBand catB3) and In-t2 (oxa1and aadA1a) were both located on a conjugative IncFI plasmid of 140 kb. In-t3 (aacA4,aacC1, and aadAIa) was located on an IncL/M plasmid of 100 kb which was present, in association with the IncFI plasmid, in gentamicin- and amikacin-resistant isolates. Despite the extensive similarity at the level of the antibiotic resistance phenotype, integrons were not found on the prototypic IncFI plasmids carried by epidemic Salmonellastrains isolated during the late 1970s. The recent appearance and the coexistence of multiple integrons on two conjugative plasmids in the sameSalmonellaisolate are examples of how mobile gene cassettes may contribute to the acquisition and dissemination of antibiotic resistance.