Genomic epidemiology of Klebsiella pneumoniae in Italy and novel insights into the origin and global evolution of its resistance to carbapenem antibiotics.

Klebsiella pneumoniae is at the forefront of antimicrobial resistance for Gram-negative pathogenic bacteria, as strains resistant to third-generation cephalosporins and carbapenems are widely reported. The worldwide diffusion of these strains is of great concern due to the high morbidity and mortality often associated with K. pneumoniae infections in nosocomial environments. We sequenced the genomes of 89 K. pneumoniae strains isolated in six Italian hospitals. Strains were selected based on antibiotypes, regardless of multilocus sequence type, to obtain a picture of the epidemiology of K. pneumoniae in Italy. Thirty-one strains were carbapenem-resistant K. pneumoniae carbapenemase producers, 29 were resistant to third-generation cephalosporins, and 29 were susceptible to the aforementioned antibiotics. The genomes were compared to all of the sequences available in the databases, obtaining a data set of 319 genomes spanning the known diversity of K. pneumoniae worldwide. Bioinformatic analyses of this global data set allowed us to construct a whole-species phylogeny, to detect patterns of antibiotic resistance distribution, and to date the differentiation between specific clades of interest. Finally, we detected an ∼ 1.3-Mb recombination that characterizes all of the isolates of clonal complex 258, the most widespread carbapenem-resistant group of K. pneumoniae. The evolution of this complex was modeled, dating the newly detected and the previously reported recombination events. The present study contributes to the understanding of K. pneumoniae evolution, providing novel insights into its global genomic characteristics and drawing a dated epidemiological scenario for this pathogen in Italy.

Antibacterial activity and cytotoxic effect of SIAB-GV3.

The aim of this study was to evaluate the antibacterial activity and cytotoxic effect of the compound SIAB-GV3, a new formulation presenting as an aqueous suspension of silicon dioxide (SiO2) functionalized with silver (Ag+) nanoparticles and benzalkonium. The product is formulated as an adjuvant for the treatment of inflammatory diseases of the oral cavity. This study demonstrates that SIAB-GV3 possesses strong antimicrobial activity against most of the common oral pathogens, in particular against Streptococcus pyogenes, Porphyromonas gingivalis and Aggregatibacter actinimycetemcomitans. The cytotoxic effect against human embryo lung fibroblasts (HELF cells) was evaluated at different times, from 1 h to 168 h, using concentrations of SIAB-GV3 ranging from 50 mg/ml to 0.0008 mg/ml. At the concentration of 10 mg/ml, commonly used in clinical practice, the compound results cyto- toxic after about 2 hours, this time being much longer than the typical time of local application, which is no more than 10 minutes. In conclusion, our findings suggest that SIAB-GV3 has a good antibacterial activity against the most common oral pathogens even at very low concentrations and a low cytotoxic activity, thanks to the synergistic effects of Ag nanoparticles, silicon dioxide and benzalkonium.

Humans parasitized by the hard tick Ixodes ricinus are seropositive to Midichloria mitochondrii: is Midichloria a novel pathogen, or just a marker of tick bite?

Midichloria mitochondrii is an intracellular bacterium found in the hard tick Ixodes ricinus. In this arthropod, M. mitochondrii is observed in the oocytes and in other cells of the ovary, where the symbiont is present in the cell cytoplasm and inside the mitochondria. No studies have so far investigated whether M. mitochondrii is present in the salivary glands of the tick and whether it is transmitted to vertebrates during the tick blood meal. To address the above issues, we developed a recombinant antigen of M. mitochondrii (to screen human sera) and antibodies against this antigen (for the staining of the symbiont). Using these reagents we show that (i) M. mitochondrii is present in the salivary glands of I. ricinus and that (ii) seropositivity against M. mitochondrii is highly prevalent in humans parasitized by I. ricinus (58%), while it is very low in healthy individuals (1·2%). These results provide evidence that M. mitochondrii is released with the tick saliva and raise the possibility that M. mitochondrii is infectious to vertebrates. Besides this, our study indicates that M. mitochondrii should be regarded as a package of antigens inoculated into the human host during the tick bite. This implies that the immunology of the response toward the saliva of I. ricinus is to be reconsidered on the basis of potential effects of M. mitochondrii and poses the basis for the development of novel markers for investigating the exposure of humans and animals to this tick species.

Chlorhexidine-silver sulfadiazine-impregnated central venous catheters: in vitro antibacterial activity and impact on bacterial adhesion.

The aim of this study was to compare the in vitro activity and the impact on bacterial adhesion of two different catheters, one impregnated with chlorhexidine-silver sulfadiazine (C-SS) and the other not impregnated with antibacterial agents. The antimicrobial coating prevented the bacterial colonization by slime positive Staphylococcus epidermidis in the first two days. The antibacterial activity of the effluents from catheters impregnated with C-SS dissipated by day seven. Our results demonstrated that the surface treatment modified the composition of impregnated catheters and determined different contact angle values of the two catheters (impregnated and not impregnated). Examination of coated and uncoated catheter segments by scanning electron microscopy showed a good correlation with the results of adherence experiments. In conclusion, the findings suggest that C-SS coated catheters prevent in vitro bacterial adhesion.

Mycobacterium avium paratuberculosis in Italy: commensal or emerging human pathogen?

BACKGROUND: Specific bacterial infections or alterations of the gut microbiota likely trigger immuno-pathological phenomena associated with Crohn's disease and ulcerative colitis. Mycobacterium avium subspecies paratuberculosis is a candidate etiological agent of Crohn's disease. Definitive causal connection between Mycobacterium avium subspecies paratuberculosis infection and Crohn's disease has not been demonstrated. AIMS: To determine the circulation of Mycobacterium avium subspecies paratuberculosis in Crohn's disease patients and water supplies in an Italian region where this bacterium is endemic in cattle farms. METHODS: Mycobacterium avium subspecies paratuberculosis screening was performed on biopsies from human patients, and from water samples, using two different PCR procedures. RESULTS: In hospitals where multiple specimens were obtained from different sites in the intestine, the prevalence of Mycobacterium avium subspecies paratuberculosis infection was 82.1% and 40% respectively in Crohn's disease and ulcerative colitis patients; in another hospital, where single specimens were obtained from patients, the bacterium was not detected. Control subjects also harboured Mycobacterium avium subspecies paratuberculosis, but at a lower prevalence. Tap water samples collected in the study area contained Mycobacterium avium subspecies paratuberculosis DNA. DISCUSSION: The results of screenings for Mycobacterium avium subspecies paratuberculosis in humans are deeply influenced by both the number and location of the collected biopsies. There is a wide circulation of the organism in the study area, considering the prevalence in humans and its presence in drinking water.

Evaluation of fluoroquinolone resistance mechanisms in Pseudomonas aeruginosa multidrug resistance clinical isolates.

Efflux transporters have a considerable role in the multidrug resistance (MDR) of Pseudomonas aeruginosa, an important nosocomial pathogen. In this study, 45 P. aeruginosa clinical strains, with an MDR phenotype, have been isolated in a hospital of Northern Italy and characterized to identify the mechanisms responsible for their fluoroquinolone (FQ) resistance. These isolates were analyzed for clonal similarity, mutations in genes encoding the FQ targets, overexpression of specific Resistance Nodulation-cell Division efflux pumps, and search for mutations in their regulatory genes. The achieved results suggested that the mutations in genes encoding ciprofloxacin targets represented the main mechanism of FQ resistance of these strains; 97.8% of these isolates showed mutations in gyrA, 28.9% in gyrB, 88.9% in parC, and 6.7% in parE. Another mechanism of resistance was overexpression of the efflux pumps in some representative strains. In particular, overexpression of MexXY-OprM drug transporter was found in five isolates, whereas overexpression of MexCD-OprJ was detected in two isolates; surprisingly, in one of these last two isolates, also overexpression of MexAB-OprM pump was identified.

Control of MRSA infection and colonisation in an intensive care unit by GeneOhm MRSA assay and culture methods.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major nosocomial pathogens. Due to the diffusion of MRSA strains in both hospital and community settings, prevention and control strategies are receiving increased attention. Approximately 25% to 30% of the population is colonised with S. aureus and 0.2% to 7% with MRSA. The BD GeneOhm MRSA real-time PCR assay offers quicker identification of MRSA-colonised patients than do culture methods. METHODS: Ninety-five patients admitted to the Intensive Care Unit of IRCCS Policlinico San Matteo of Pavia (Italy) for a period > 24 h were screened for MRSA colonisation with both the culture method and the GeneOhm assay. RESULTS: Of the 246 nasal swabs collected from 95 patients, 36 samples were found to be positive by both methods (true-positive). 30% of colonised patients had developed the MRSA infection. CONCLUSION: Our results show that the GeneOhm MRSA assay is a valuable diagnostic tool for detecting MRSA quickly in nasal swabs. This study confirms that colonisation represents a high risk factor for MRSA infection, and that good MRSA surveillance in an Intensive Care Unit is therefore an excellent way to prevent MRSA infection.