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

Distribution of epidemic clonal genetic markers among Listeria monocytogenes 4b isolates.

Recent genome sequencing of isolates of Listeria monocytogenes serotype 4b implicated in some major outbreaks of foodborne listeriosis has revealed unique genetic markers in these isolates. The isolates were grouped into two distinct epidemic clones, ECI and ECII. In the present study, selected ECI- and ECII-specific genetic markers were detected in 16 and 15 of 89 L. monocytogenes 4b isolates, respectively. The ECI markers were found in 6 of 34 clinical isolates, 9 of 50 food isolates, and 1 of 5 environmental isolates, and the ECII markers were detected in 7 of 34 clinical isolates, 7 of 50 food isolates, and 1 of 5 environmental isolates. Hence, of the isolates with the epidemic clonal genetic markers, 38% (13 of 34) were of clinical origin, 32% (16 of 50) were of food origin, and 40% (2 of 5) were of environmental origin. The predominance of the epidemic clonal markers among the clinical and environmental isolates supports the hypothesis that these markers are correlated with the pathogenic potential of strains and with their environmental persistence. Several isolates had only one epidemic clonal marker, either the ECI-specific marker 133 or the ECII-specific marker 4bSF18. Pulsed-field gel electrophoresis analysis revealed higher genomic diversity among the strains with ECII-like characteristics than among those strains carrying the ECI-specific genetic markers

Identification of Novel Linear Megaplasmids Carrying a ß-Lactamase Gene in Neurotoxigenic Clostridium butyricum Type E Strains

Since the first isolation of type E botulinum toxin-producing Clostridium butyricum from two infant botulism cases in Italy in 1984, this peculiar microorganism has been implicated in different forms of botulism worldwide. By applying particular pulsed-field gel electrophoresis run conditions, we were able to show for the first time that ten neurotoxigenic C. butyricum type E strains originated from Italy and China have linear megaplasmids in their genomes. At least four different megaplasmid sizes were identified among the ten neurotoxigenic C. butyricum type E strains. Each isolate displayed a single sized megaplasmid that was shown to possess a linear structure by ATP-dependent exonuclease digestion. Some of the neurotoxigenic C. butyricum type E strains possessed additional smaller circular plasmids. In order to investigate the genetic content of the newly identified megaplasmids, selected gene probes were designed and used in Southern hybridization experiments. Our results revealed that the type E botulinum neurotoxin gene was chromosome-located in all neurotoxigenic C. butyricum type E strains. Similar results were obtained with the 16S rRNA, the tetracycline tet(P) and the lincomycin resistance protein lmrB gene probes. A specific mobA gene probe only hybridized to the smaller plasmids of the Italian C. butyricum type E strains. Of note, a ß-lactamase gene probe hybridized to the megaplasmids of eight neurotoxigenic C. butyricum type E strains, of which seven from clinical sources and the remaining one from a food implicated in foodborne botulism, whereas this ß-lactam antibiotic resistance gene was absent form the megaplasmids of the two soil strains examined. The widespread occurrence among C. butyricum type E strains associated to human disease of linear megaplasmids harboring an antibiotic resistance gene strongly suggests that the megaplasmids could have played an important role in the emergence of C. butyricum type E as a human pathogen

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

Evidence that plasmid-borne botulinum neurotoxin type B genes are widespread among Clostridium botulinum serotype B strains.

BACKGROUND: Plasmids that encode certain subtypes of the botulinum neurotoxin type B have recently been detected in some Clostridium botulinum strains. The objective of the present study was to investigate the frequency with which plasmid carriage of the botulinum neurotoxin type B gene (bont/B) occurs in strains of C. botulinum type B, Ab, and A(B), and whether plasmid carriage is bont/B subtype-related. METHODOLOGY/PRINCIPAL FINDINGS: PCR-Restriction fragment length polymorphism was employed to identify subtypes of the bont/B gene. Pulsed-field gel electrophoresis and Southern blot hybridization with specific probes were performed to analyze the genomic location of the bont/B subtype genes. All five known bont/B subtype genes were detected among the strains; the most frequently detected subtype genes were bont/B1 and /B2. Surprisingly, the bont/B subtype gene was shown to be plasmid-borne in >50% of the total strains. The same bont/B subtype gene was associated with the chromosome in some strains, whereas it was associated with a plasmid in others. All five known bont/B subtype genes were in some cases found to reside on plasmids, though with varying frequency (e.g., most of the bont/B1 subtype genes were located on plasmids, whereas all but one of the bont/B2 subtypes were chromosomally-located). Three bivalent isolates carried both bont/A and /B genes on the same plasmid. The plasmids carrying the bont gene were five different sizes, ranging from approximately 55 kb to approximately 245 kb. CONCLUSIONS/SIGNIFICANCE: The unexpected finding of the widespread distribution of plasmids harboring the bont/B gene among C. botulinum serotype B strains provides a chance to examine their contribution to the dissemination of the bont genes among heterogeneous clostridia, with potential implications on issues related to pathogenesis and food safety

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

Attività antimicrobica di oli essenziali contro ceppi di Bacillus spp. e modifica dell’espressione di geni coinvolti nella risposta allo stress

Bacillus spp. comprende specie alteranti e patogene, che possono costituire un pericolo per la qualità e per la sicurezza degli alimenti. Questo lavoro mira a valutare gli effetti del trattamento con oli essenziali (OE) in ceppi di Bacillus spp. isolati da gnocchi ambient, stabili a temperatura ambiente. Tale trattamento rappresenta un evento stressante per la cellula che, a seconda della concentrazione e del tipo di OE applicato, può andare incontro a morte o subire danni, modificando l’espressione di specifici geni. Sei OE (Thymus vulgaris chemiotipo timolo, Thymus vulgaris chemiotipo p-cimene e timolo, Thymbra capitata chemiotipo carvacrolo, Origanum vulgare chemiotipo carvacrolo, Syzygium aromaticum chemiotipo eugenolo e Ocimum basilicum chemiotipo linalolo) sono stati saggiati contro 25 ceppi di Bacillus spp. (B. subtilis, B. cereus, B. thuringiensis e B. paranthracis) isolati da gnocchi ambient. La ricerca delle Minime Concentrazioni Inibenti (MIC) e Battericide (MBC) (1) e della Concentrazione Inibitoria Frazionaria (FIC) attraverso il metodo Checkerboard sono stati utilizzati per valutarne l’attività in singolo o combinata. L’espressione genica è stata valutata attraverso Real-time RT-PCR quantitativa. Gli OE di T. vulgaris (timolo), T. capitata e O. vulgare sono risultati i più attivi, con MIC a 48 ore e MBC tra 0.31-1.25 μL/mL (Fig. 1), e hanno mostrato azione commutativa se combinati a OE di S. aromaticum. I ceppi di B. subtilis sono stati leggermente più sensibili rispetto alle altre specie. L’esposizione di ceppi di B. subtilis e B. cereus a concentrazioni subletali di OE di T. vulgaris (timolo), uno tra i più attivi, ha aumentato l’espressione di geni coinvolti nell’integrità di membrana e nel quorum sensing a partire da 6-12 ore di esposizione. I geni maggiormente sovra-espressi sono stati diversi per le due specie (pbpF per B. subtilis e plcR per B. cereus). In generale, gli OE con chemiotipo timolo e carvacrolo sono risultati i più attivi, anche in combinazione con OE di S. aromaticum ma non con OE di O. basilicum. L’esposizione a OE di timo ha prodotto un evento stressante con aumento della fase lag dei microrganismi, che in questo modo tentano di riparare i danni alla membrana e di restaurare i meccanismi di comunicazione fondamentali per la sopravvivenza. In conclusione, gli OE di T. vulgaris (timolo), T. capitata e O. vulgare potrebbero essere utili per applicazioni alimentari contro Bacillus spp., anche in combinazione con OE meno attivi

<i>Nru</i>I restriction map analysis of 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) as a linear DNA molecule (2a) and as a circular DNA molecule (2b), and relative positions of the <i>S1</i>, <i>latt</i>, <i>bl</i>, <i>mb</i>, and <i>dp</i> gene probes.

<p>The shaded area (∼ 168 kb) corresponds to the genetic region missing from the smaller megaplasmids of strains ISS-20, ISS-21, ISS-109, ISS-145/1.</p

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