PCR-based rapid genotyping of Stenotrophomonas maltophilia isolates

<p>Abstract</p> <p>Background</p> <p>All bacterial genomes contain repetitive sequences which are members of specific DNA families. Such repeats may occur as single units, or found clustered in multiple copies in a head-to-tail configuration at specific loci. The number of clustered units per locus is a strain-defining parameter. Assessing the length variability of clusters of repeats is a versatile typing methodology known as multilocus variable number of tandem repeat analysis (MLVA).</p> <p>Results</p> <p><it>Stenotrophomonas maltophilia </it>is an environmental bacterium increasingly involved in nosocomial infections and resistant to most antibiotics. The availability of the whole DNA sequence of the <it>S. maltophilia </it>strain K279a allowed us to set up fast and accurate PCR-based diagnostic protocols based on the measurement of length variations of <it>loci </it>carrying a variable number of short palindromic repeats marking the <it>S. maltophilia </it>genome. On the basis of the amplimers size, it was possible to deduce the number of repeats present at 12 different <it>loci </it>in a collection of <it>S. maltophilia </it>isolates, and therefore label each of them with a digit. PCR-negative regions were labelled 0. Co-amplification of two pairs of <it>loci </it>provided a 4-digit code sufficient for immediate subtyping. By increasing the number of <it>loci </it>analyzed, it should be possible to assign a more specific digit profile to isolates. In general, MLVA data match genotyping data obtained by PFGE (pulsed-field gel electrophoresis). However, some isolates exhibiting the same PCR profiles at all <it>loci </it>display distinct PFGE patterns.</p> <p>Conclusion</p> <p>The utilization of the present protocol allows to type several <it>S. maltophilia </it>isolates in hours. The results are immediately interpretable without the need for sophisticated softwares. The data can be easily reproducible, and compared among different laboratories.</p

Anti-biofilm activity of the fungal phytotoxin sphaeropsidin a against clinical isolates of antibiotic-resistant bacteria

Many pathogens involved in human infection have rapidly increased their antibiotic resistance, reducing the effectiveness of therapies in recent decades. Most of themcan formbiofilms and effective drugs are not available to treat these formations. Natural products could represent an efficient solution in discovering and developing new drugs to overcome antimicrobial resistance and treat biofilm-related infections. In this study, 20 secondary metabolites produced by pathogenic fungi of forest plants and belonging to diverse classes of naturally occurring compounds were evaluated for the first time against clinical isolates of antibiotic-resistant Gram-negative and Gram-positive bacteria. epi-Epoformin, sphaeropsidone, and sphaeropsidin A showed antimicrobial activity on all test strains. In particular, sphaeropsidin A was effective at low concentrations with Minimum Inhibitory Concentration (MIC) values ranging from 6.25 μg/mL to 12.5 μg/mL against all reference and clinical test strains. Furthermore, sphaeropsidin A at sub-inhibitory concentrations decreasedmethicillin-resistant S. aureus (MRSA) and P. aeruginosa biofilmformation, as quantifiedby crystal violet staining. Interestingly,mixtures of sphaeropsidin A and epi-epoformin have shown antimicrobial synergistic effects with a concomitant reduction of cytotoxicity against human immortalized keratinocytes. Our data show that sphaeropsidin A and epi-epoformin possess promising antimicrobial properties

Impact of Clostridium difficile infection on pediatric inflammatory bowel disease.

To determine the prevalence of and explore possible differences in the risk for and symptoms of Clostridium difficile infection between patients with and without inflammatory bowel disease (IBD). STUDY DESIGN: Stool specimens from subjects with and without IBD were evaluated for the presence of C difficile toxins. Demographic information, diagnosis, anatomic location, disease activity, IBD therapy, hospitalizations, and antibiotic and proton pump inhibitor (PPI) exposures were recorded. RESULTS: A total of 193 specimens were collected from 81 patients with IBD and 112 patients without IBD. The prevalence of C difficile infection was significantly greater in the patients with IBD than in those without IBD (P = .004; chi2 = 0.003; odds ratio = 3.3; 95% confidence interval = 1.5 to 7.6). In the patients with IBD, the prevalence of active disease was significantly greater in the C difficile-infected patients than in the uninfected patients (P < .0001). Colonic involvement was found in all patients with IBD. The specific type of IBD, IBD therapy, and antibiotic and PPI exposures that predisposed patients with IBD to C difficile infection were not identified, whereas hospitalization was significantly more frequent in the patients without IBD (P = .025). CONCLUSIONS: Our findings indicate that in children, IBD is associated with an increased prevalence of C difficile infection. The specific risk factors reported in adults were not identified in these children, suggesting the possible involvement of other mechanisms for acquiring the pathogen

Farnesane-type sesquiterpenoids with antibiotic activity from chiliadenus lopadusanus

Chiliadenus lopadusanus Brullo is an Asteraceae plant species endemic to Lampedusa island, the largest island of the Pelage archipelago, Italy. The organic extract of its whole aerial parts, showing antibiotic activity against Staphylococcus aureus and Acinetobacter baumannii, wasfractionated employing bioguided purification procedures affording three main farnesane-type sesquiterpenoids. They were identified by spectroscopic methods (NMR and ESIMS data) as the (E)-3,7,11-trimethyldodeca-1,6,10-triene-3,9-diol, (E)-10-hydroxy-2,6,10-trimethyldodeca-2,6,11-trien-4-one and (E)-10-hydroxy-2,6,10-trimethyl-dodeca-6,11-dien-4-one, commonly named 9-hydroxynerolidol, 9-oxonerolidol, and chiliadenol B, respectively. These three sesquiterpenes, isolated for the first time from C. lopadu-sanus, were tested on methicillin-resistant S. aureus and A. baumannii showing antibacterial and antibiofilm activities. This plant could be used as a source to isolate secondary metabolites as potential new antibiotics