Effects of Megaplasmid Loss on Growth of Neurotoxigenic Clostridium butyricum Strains and Botulinum Neurotoxin Type E Expression

Clostridium butyricum strains that atypically produce the botulinum neurotoxin type E (BoNT/E) possess a megaplasmid of unknown functions in their genome. In this study, we cured two botulinum neurotoxigenic C. butyricum type E strains of their megaplasmids, and compared the obtained megaplasmid-cured strains to their respective wild-type parental strains. Our results showed that the megaplasmids do not confer beta-lactam resistance on the neurotoxigenic C. butyricum type E strains, although they carry several putative beta-lactamase genes. Instead, we found that the megaplasmids are essential for growth of the neurotoxigenic C. butyricum type E strains at the relatively low temperature of 15°C, and are also relevant for growth of strains under limiting pH and salinity conditions, as well as under favorable environmental conditions. Moreover, the presence of the megaplasmids was associated with increased transcript levels of the gene encoding BoNT/E in the C. butyricum type E strains, indicating that the megaplasmids likely contain transcriptional regulators. However, the levels of BoNT/E in the supernatants of the cured and uncured strains were similar after 24 h and 48 h culture, suggesting that expression of BoNT/E in the C. butyricum type E strains is not ultimately controlled by the megaplasmids. Together, our results reveal that the C. butyricum type E megaplasmids exert pleiotropic effects on the growth of their microbial hosts under optimal and limiting environmental conditions, and also highlight the possibility of original regulatory mechanisms controlling the expression of BoNT/E

Use of probiotics in medical devices applied to some common pathologies.

Probiotics, defined as “living microorganisms that, whether ingested in useful amount, may have beneficial effects on human body”, are widely used in various products for human use, such as dietary supplements, medical devices and pharmaceutical products. The European Directive on medical devices (MDs) (DDM 93/42), also includes those MDs containing live microorganisms, particularly probiotics, that may have various destinations of use, including that of assisting the therapy of several human pathologies. In this brief note we analyzed the use of probiotics in MDs and how probiotics administration could represent one of the new frontiers of scientific research on the prevention and treatment of various diseases. We’ll analyze the literature on probiotics based MDs, to review their major targets in the therapy of some of the most common human pathologies: bacterial vaginosis and vaginitis, atopic dermatitis, infant colic, obesity, type 2 diabetes, and pharyngotonsillitis

Drug-resistant tuberculosis in Naples, 2008-2013

Background. Drug-resistant tuberculosis (TB) is an important threat in industrialized countries, but low information is known from Southern Italy. Here, we present the results of a retrospective study on TB cases diagnosed in 2008-2013 in Naples, the biggest city in the South of Italy. Methods. Six hundred ninety Mycobacterium tuberculosis strains were isolated at the Ospedali dei Colli of Naples, and resistance to first-line and second-line drugs was determined. Results. Multidrug-resistant (MDR) TB increased from 2008 to 2013, with most strains being isolated from migrants arriving from 41 countries. Overall MDR-TB rate was 4.5%: Italian-born persons, 2.2%; Romania, 7.5%; Former Soviet Union countries (Ukraine, Russia, Armenia, Georgia), 22.4%; all other foreign countries, 2.0%. Resistance of MDR strains to second-line drugs was high against kanamycin, ofloxacin, capreomycin. Conclusions. MDR-TB increased in 2008-13 and was mostly observed in migrants, indicating the need to intensify diagnosis and treatment of these populations in Naples

Stenotrophomonas maltophilia isolata da pazienti affetti da fibrosi cistica : analisi genotipica e caratterizzazione molecolare di determinanti di virulenza

Stenotrophomonas maltophilia is a an environmental organism, that in recent years has been isolated with increasing frequency as an opportunistic pathogen in nosocomial infections as well as from the airways of cystic fibrosis (CF) patients. Despite its growing clinical importance, the mechanisms adopted by this organism to establish persistent infections have not been clarified yet. The availability of information on genome sequence of two S. maltophilia strains (K279a and R551-3), allow development of new post-genomic approaches direct to understanding of key factors involved in the colonization of respiratory tract of CF patients and of cellular and molecular mechanisms of its pathogenicity. Identification of S. maltophilia genes expressed inside host, is essential for development of new antimicrobial compounds. Moreover, molecular and genetic characterization of S. maltophilia clinical strains is essential to understand pathogenicity mechanism of these organisms and to build detailed epidemiological maps to draw their spread in CF patients. The present study is aimed at determining the genomic and epidemiological relatedness of 52 S. maltophilia strains. Strains included 43 clinical collected, between January 2003 and December 2005, from 43 independent patients (41 were from CF patients, and two from blood cultures of two non-CF patients) attending the Paediatric Hospital “Bambino Gesù” of Rome. Other strains were: K279a and LMG958, two S. maltophilia reference clinical strains, and seven strains of environmental origin (two isolated within the Hospital “Bambino Gesù”). In these strains the presence and expression of the virulence-associated genes were evaluated and their contribution to virulence was assessed using larvae of the wax moth Galleria mellonella as an infection model. To confirm a role of the putative virulence factors identified we constructed K279a (the clinical reference strain whose genome sequence is available) mutants and performed complementation assay with the wild type functional gene. Moreover, to study genomic evolution of S. maltophilia toward a pathogenic lifestyle, we compare the genome of an environmental strain with that of the clinical strain K279a. Genotyping analysis. Data that we obtained indicates PFGE fingerprinting as the most discriminatory technique to characterize genomospecies present in S. maltophilia. All strains produced well-defined PFGE profiles: forty-seven different PFGE pulsotypes were identified, indicating a remarkable genomic diversity among the strains analysed. Three clusters (pulsotype 1, 17 and 33) showed a banding pattern identical to at least 1 other isolate likely indicating either cross-transmission among patients or infection from a common source. However, the results obtained with the RFLP of gyrB gene and with the variable regions of the 16S rRNA gene clearly indicated that certain phylogenetic groups are likely better able to cause infection than others. Identification and characterization of virulence genes involved in S. maltophilia pathogenicity. All 52 S. maltophilia strains were characterized for the presence and expression of type-1 fimbria, esterase, proteases (StmPr1 and StmPr2). Regarding the type-1 fimbria, only the clinical strains showed the presence of the gene coding for this surface structure, indicating that it might be an important virulence factor of this opportunistic pathogen. The expression of esterase activity by most CF-derived S. maltophilia strains, reinforces the hypothesis that esterase, such as fimbria, may play some role in the virulence. In order to verify the role of these potential virulence factors in S. maltophilia pathogenesis, representative S. maltophilia strains unable to express proteases or esterase were assayed in G. mellonella infection. The results indicate that proteases may be relevant virulence factors of S. maltophilia. Moreover, the type-1 fimbria could play an important role since the environmental strains, lacking of smf-1, show the higher lethal dose (LD50). To analyze the relative role of the two proteases (StmPr1 and StmPr2), the OBGTC23 strain, naturally impaired in protease expression, was complemented with the StmPr1 or StmPr2 functional genes and tested in G. mellonella assay. Data obtained showed that StmPr1 protease play a major role in S. maltophilia pathogenesis. Analysis of the role of S. maltophilia virulence factors by mutants construction. In the complex, the results that we obtained using different clinical OBGTC strains, impaired in the expression of one or more hypothetical virulence factors, indicates proteases as important virulence determinants for S. maltophilia. Nevertheless, these natural mutant strains could lack of many others virulence determinants that may be involved in pathogenesis process. Then, to study the effect of the single factors identified and to confirm their role, deletions encompassing different putative virulence genes are introduced by allelic exchange in reference strain K279a, whose genome sequence is available. The proteases mutants showed a strong reduction of the protease activity and were less virulent, compared to wild-type, in G. mellonella larvae, confirming that proteases, particularly StmPr1, are important virulence factors for S. maltophilia. The proteases expression is controlled by the diffusible signal factor (DSF) a quorum sensing signal molecule that controls the expression of several virulence factors. Moreover, the DSF mutant reveals a greater reduction of virulence in G. mellonella infection model, compared to that of proteases mutants, suggesting a DSF involvement in the control of proteases but also of other virulence factors. Finally, despite fimbriae are important to establish chronic infection in airways CF patients, this surface structures may be not important in G. mellonella infection model. Genomic evolution of S. maltophilia toward a pathogenic lifestyle. To identify the specific genetic determinants acquired from S. maltophilia during its evolution towards a more pathogenic lifestyle, we performed a Suppression Subtractive Hybridization: this technique allowed us to identify the genetic sequences present in the clinical strain K279a and absent in the environmental strain LMG959. The majority of the subtracted sequence may have been acquired from other organisms by horizontal gene transfers, since their different G+C content and since the presence of several IS elements. Among the sequence, particularly interesting are genes coding for haemagglutinin, Clp protease, TonB dependent receptor protein, and a putative ankyrin repeat-containing protein, which are known to be important virulence factors in many gram-negative bacteria. Moreover, many of the subtracted sequence represent genes involved in metabolism, DNA restriction/modification system, transmembrane proteins, hypothetical proteins and proteins with unknown function. These differences between S. maltophilia K279a and S. maltophilia LMG959 could be related to niche adaptation or host preference and this accessory genome may represents an advantage for pathogen evolution driven by the need for continuous adaptation to the host in order to evade or suppress coevolving host defense mechanisms, making it an emergent opportunistic pathogen in nosocomial infections

Drug-Resistant Tuberculosis 2020: Where We Stand

The control of tuberculosis (TB) is hampered by the emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) strains, defined as resistant to at least isoniazid and rifampin, the two bactericidal drugs essential for the treatment of the disease. Due to the worldwide estimate of almost half a million incident cases of MDR/rifampin-resistant TB, it is important to continuously update the knowledge on the mechanisms involved in the development of this phenomenon. Clinical, biological and microbiological reasons account for the generation of resistance, including: (i) nonadherence of patients to their therapy, and/or errors of physicians in therapy management, (ii) complexity and poor vascularization of granulomatous lesions, which obstruct drug distribution to some sites, resulting in resistance development, (iii) intrinsic drug resistance of tubercle bacilli, (iv) formation of non-replicating, drug-tolerant bacilli inside the granulomas, (v) development of mutations in Mtb genes, which are the most important molecular mechanisms of resistance. This review provides a comprehensive overview of these issues, and releases up-dated information on the therapeutic strategies recently endorsed and recommended by the World Health Organization to facilitate the clinical and microbiological management of drug-resistant TB at the global level, with attention also to the most recent diagnostic methods

Structure and genetic content of the megaplasmids of neurotoxigenic clostridium butyricum type E strains from Italy.

We determined the genetic maps of the megaplasmids of six neutoroxigenic Clostridium butyricum type E strains from Italy using molecular and bioinformatics techniques. The megaplasmids are circular, not linear as we had previously proposed. The differently-sized megaplasmids share a genetic region that includes structural, metabolic and regulatory genes. In addition, we found that a 168 kb genetic region is present only in the larger megaplasmids of two tested strains, whereas it is absent from the smaller megaplasmids of the four remaining strains. The genetic region unique to the larger megaplasmids contains, among other features, a locus for clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated (cas) genes, i.e. a bacterial adaptive immune system providing sequence-specific protection from invading genetic elements. Some CRISPR spacer sequences of the neurotoxigenic C. butyricum type E strains showed homology to prophage, phage and plasmid sequences from closely related clostridia species or from distant species, all sharing the intestinal habitat, suggesting that the CRISPR locus might be involved in the microorganism adaptation to the human or animal intestinal environment. Besides, we report here that each of four distinct CRISPR spacers partially matched DNA sequences of different prophages and phages, at identical nucleotide locations. This suggests that, at least in neurotoxigenic C. butyricum type E, the CRISPR locus is potentially able to recognize the same conserved DNA sequence of different invading genetic elements, besides targeting sequences unique to previously encountered invading DNA, as currently predicted for a CRISPR locus. Thus, the results of this study introduce the possibility that CRISPR loci can provide resistance to a wider range of invading DNA elements than previously appreciated. Whether it is more advantageous for the peculiar neurotoxigenic C. butyricum type E strains to maintain or to lose the CRISPR-cas system remains an open question

Gene specific probes and primer sets within the <i>C. butyricum</i> type E strain BL5262 contig 1 sequence (http://www.ncbi.nlm.nih.gov/nuccore/NZ_ACOM01000001.1) (757,653 bp).

<p>Gene specific probes and primer sets within the <i>C. butyricum</i> type E strain BL5262 contig 1 sequence (<a href="http://www.ncbi.nlm.nih.gov/nuccore/NZ_ACOM01000001.1" target="_blank">http://www.ncbi.nlm.nih.gov/nuccore/NZ_ACOM01000001.1</a>) (757,653 bp).</p

Analysis of the CRISPR spacers in <i>C. butyricum</i> type E strains BL5262 (http://www.ncbi.nlm.nih.gov/nuccore/NZ_ACOM01000001.1), ISS-86 (GenBank: KF150773) and ISS-190 (GenBank: KF150772).

1<p>Spacers are numbered according to their acquisition order, i.e. the more recently added spacers have the highest numbers.</p>2<p>Spacers 40, 43 and 44 are those of the CRISPR array of strain ISS-86.</p>3<p>Bold underlined nucleotides match the target sequences.</p>4<p>Putative prophage sequences within bacterial genomes were identified through the program Prophinder (<a href="http://aclame.ulb.ac.be/prophinder" target="_blank">http://aclame.ulb.ac.be/prophinder</a>).</p

PFGE patterns of <i>C. butyricum</i> type E strains digested with <i>Nru</i>I restriction enzyme (1a) and Southern hybridization analysis using gene probes <i>dp</i> (1b) and <i>S1</i> (1c).

<p>Strains: ISS-20 (lane 1); ISS-21 (lane 2); ISS-109 (lane 3); ISS-145/1 (lane 4); ISS-86 (lane 5); ISS-190 (lane 6). m.s. (lane 7): molecular standard, <i>Xba</i>I-digested DNA from <i>Salmonella</i> Braenderup strain H9812 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071324#pone.0071324-Hunter1" target="_blank">[13]</a>.</p

Botulinum neurotoxigenic <i>C. butyricum</i> type E strains used in this study.

1<p>References 2, 6, 7, 8, 9.</p>2<p>Reference 6.</p