The Distinctive Evolution of orfX Clostridium parabotulinum Strains and Their Botulinum Neurotoxin Type A and F Gene Clusters Is Influenced by Environmental Factors and Gene Interactions via Mobile Genetic Elements.

Of the seven currently known botulinum neurotoxin-producing species of Clostridium, C. parabotulinum, or C. botulinum Group I, is the species associated with the majority of human botulism cases worldwide. Phylogenetic analysis of these bacteria reveals a diverse species with multiple genomic clades. The neurotoxins they produce are also diverse, with over 20 subtypes currently represented. The existence of different bont genes within very similar genomes and of the same bont genes/gene clusters within different bacterial variants/species indicates that they have evolved independently. The neurotoxin genes are associated with one of two toxin gene cluster types containing either hemagglutinin (ha) genes or orfX genes. These genes may be located within the chromosome or extrachromosomal elements such as large plasmids. Although BoNT-producing C parabotulinum bacteria are distributed globally, they are more ubiquitous in certain specific geographic regions. Notably, northern hemisphere strains primarily contain ha gene clusters while southern hemisphere strains have a preponderance of orfX gene clusters. OrfX C. parabotulinum strains constitute a subset of this species that contain highly conserved bont gene clusters having a diverse range of bont genes. While much has been written about strains with ha gene clusters, less attention has been devoted to those with orfX gene clusters. The recent sequencing of 28 orfX C. parabotulinum strains and the availability of an additional 91 strains for analysis provides an opportunity to compare genomic relationships and identify unique toxin gene cluster characteristics and locations within this species subset in depth. The mechanisms behind the independent processes of bacteria evolution and generation of toxin diversity are explored through the examination of bacterial relationships relating to source locations and evidence of horizontal transfer of genetic material among different bacterial variants, particularly concerning bont gene clusters. Analysis of the content and locations of the bont gene clusters offers insights into common mechanisms of genetic transfer, chromosomal integration, and development of diversity among these genes.

Use of custom made 3-dimensional printed surgical guide for manubrio-sternal resection of solitary breast cancer metastasis: case report.

A 44-year-old lady with solitary 4.4-cm metastasis to the manubrium from a previously resected invasive ductal carcinoma of the left breast underwent manubrio-sternal resection. We describe our unique approach of using 3-dimensional (3D)-computed tomography (CT) image segmentation planning for reconstructing desirable resection boundaries, design of ideal superficial and deep surgical resection guides, and followed by 3D printing of guides using autoclavable thermoplastic for use during surgery. The surgical guides over the ribs and sternum rapidly and accurately define resection lines intraoperatively, achieve good surgical margins, and could reduce resection and reconstruction related morbidity for performing complex surgical resection of the chest wall. The patient was discharged 2 weeks postoperatively and remained free from local recurrence on CT scan 1-year after resection.

The comparison of electromagnetic with virtual bronchoscopic navigation accuracy at hybrid operating room in patient with sternal wires.

We describe a case of a 63-year-old lady with an incidental small right upper lobe ground glass opacity (GGO) lesion, who had history of open-heart surgery with bypass grafts for coronary artery disease. Video-assisted thoracic surgery (VATS) excisional biopsy with pre-operative localization was planned. Localization by percutaneous approach is challenging due to position of GGO shielded by both the scapular and ribs. Electromagnetic navigation accuracy may be affected by steel sternal wires but its effect has not been reported on literature. Both virtual and electromagnetic navigation bronchoscopy platforms were used with real-time cone-beam CT confirmation in the hybrid operating room to perform dye marking. Both navigation methods successfully marked the GGO for lung resection. Our case demonstrated that the accuracy of electromagnetic navigation in the presence of steel sternal wire is satisfactory for dye marking and the use of intra-operative cone-beam CT in hybrid operating room is invaluable for the success of navigational bronchoscopy.

Genomic Characterization of Newly Completed Genomes of Botulinum Neurotoxin-Producing Species from Argentina, Australia, and Africa.

Botulinum neurotoxin-producing clostridia are diverse in the types of toxins they produce as well as in their overall genomic composition. They are globally distributed, with prevalent species and toxin types found within distinct geographic regions, but related strains containing the same toxin types may also be located on distinct continents. The mechanisms behind the spread of these bacteria and the independent movements of their bont genes may be understood through examination of their genetic backgrounds. The generation of 15 complete genomic sequences from bacteria isolated in Argentina, Australia, and Africa allows for a thorough examination of genome features, including overall relationships, bont gene cluster locations and arrangements, and plasmid comparisons, in bacteria isolated from various areas in the southern hemisphere. Insights gained from these examinations provide an understanding of the mechanisms behind the independent movements of these elements among distinct species.

Evidence for surgical resections in oligometastatic lung cancer.

With the advent of advanced technology in performing diagnostics for lung cancer, an incremental increase in the number of patients with oligometastatic disease is currently being managed with intent to cure. As treatment of selected types of patients with oligometastasis show favourable outcomes, the past notion of managing these patients palliatively is fast becoming extinct. Selection of patients based on established criterion together with surgical metastasectomy combined with multiple ablative techniques with or without systemic chemotherapy offers a reasonable rate of treatment success which provides basis for treating such patient population. As more evidence becomes available to suggest that the oligometastatic state of lung cancer does exist, and are potentially curable, a better understanding of the condition is necessary for clinicians, and surgeons to provide optimal care. In this review we present some of the clinical basis which may cause a paradigm shift in management of patients with oligometastatic lung disease.

Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay.

Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha positive [ha+] or orfX+). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters.IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and to provide information about the type (ha+ or orfX+) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest.

Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia.

BACKGROUND: Clostridium botulinum is a diverse group of bacteria characterized by the production of botulinum neurotoxin. Botulinum neurotoxins are classified into serotypes (BoNT/A-G), which are produced by six species/Groups of Clostridia, but the genetic background of the bacteria remains poorly understood. The purpose of this study was to use comparative genomics to provide insights into the genetic diversity and evolutionary history of bacteria that produce the potent botulinum neurotoxin. RESULTS: Comparative genomic analyses of over 170 Clostridia genomes, including our draft genome assemblies for 59 newly sequenced Clostridia strains from six continents and publicly available genomic data, provided in-depth insights into the diversity and distribution of BoNT-producing bacteria. These newly sequenced strains included Group I and II strains that express BoNT/A,/B,/E, or/F as well as bivalent strains. BoNT-producing Clostridia and closely related Clostridia species were delineated with a variety of methods including 16S rRNA gene, concatenated marker genes, core genome and concatenated multi-locus sequencing typing (MLST) gene phylogenies that related whole genome sequenced strains to publicly available strains and sequence types. These analyses illustrated the phylogenetic diversity in each Group and the diversity of genomic backgrounds that express the same toxin type or subtype. Comparisons of the botulinum neurotoxin genes did not identify novel toxin types or variants. CONCLUSIONS: This study represents one of the most comprehensive analyses of whole genome sequence data for Group I and II BoNT-producing strains. Read data and draft genome assemblies generated for 59 isolates will be a resource to the research community. Core genome phylogenies proved to be a powerful tool for differentiating BoNT-producing strains and can provide a framework for the study of these bacteria. Comparative genomic analyses of Clostridia species illustrate the diversity of botulinum-neurotoxin-producing strains and the plasticity of the genomic backgrounds in which bont genes are found.

First report of an infant botulism case due to Clostridium botulinum type Af.

Most infant botulism cases worldwide are due to botulinum toxin types A and B. Rarely, Clostridium botulinum strains that produce two serotypes (Ab, Ba, and Bf) have also been isolated from infant botulism cases. This is the first reported case of infant botulism due to C. botulinum type Af worldwide.

On the origin of calcium oxalate monohydrate papillary renal stones.

Calcium oxalate monohydrate (COM) papillary calculi can be initiated by subepithelial calcification of the renal papillae. Hydroxyapatite disruption of the papillary epithelial layer can become the nidus of a COM papillary calculus. This study evaluated the causes of papillary tissue calcifications in 60 patients with calcium oxalate lithiasis, 30 with COM papillary and 30 with calcium oxalate dihydrate (COD) calculi. Urinary redox potential was higher in the COM than the COD group, suggesting that the former is more deficient in antioxidants due to increased oxidative stress. Urinary calcium was significantly higher in the COD group, whereas urinary oxalate was significantly higher in the COM group, suggesting a greater degree of oxidative injury of renal cells. Evaluations of their diets showed that both groups consumed low amounts of phytate-rich products. Of chronic diseases possibly associated with urolithiasis, only the prevalence of gastroduodenal ulcer differed significantly, being higher in the COM group and suggesting that epithelial lesions are common to gastroduodenal ulcers and COM papillary renal stones. Occupational exposure to cytotoxic products occurred in 47 % of the COM and 27 % of the COD group, but this difference was not statistically significant. These findings indicate that oxidative stress is associated with injury to papillary tissue and that this is the origin of intrapapillary calcifications. The continuation of this process is due to modulators and/or deficiencies in inhibitors of crystallization. Identifying and eliminating the causes of injury may prevent recurrent episodes in patients with papillary COM calculi.

Analysis of a unique Clostridium botulinum strain from the Southern hemisphere producing a novel type E botulinum neurotoxin subtype.

BACKGROUND: Clostridium botulinum strains that produce botulinum neurotoxin type E (BoNT/E) are most commonly isolated from botulism cases, marine environments, and animals in regions of high latitude in the Northern hemisphere. A strain of C. botulinum type E (CDC66177) was isolated from soil in Chubut, Argentina. Previous studies showed that the amino acid sequences of BoNT/E produced by various strains differ by < 6% and that the type E neurotoxin gene cluster inserts into the rarA operon. RESULTS: Genetic and mass spectral analysis demonstrated that the BoNT/E produced by CDC66177 is a novel toxin subtype (E9). Toxin gene sequencing indicated that BoNT/E9 differed by nearly 11% at the amino acid level compared to BoNT/E1. Mass spectrometric analysis of BoNT/E9 revealed that its endopeptidase substrate cleavage site was identical to other BoNT/E subtypes. Further analysis of this strain demonstrated that its 16S rRNA sequence clustered with other Group II C. botulinum (producing BoNT types B, E, and F) strains. Genomic DNA isolated from strain CDC66177 hybridized with fewer probes using a Group II C. botulinum subtyping microarray compared to other type E strains examined. Whole genome shotgun sequencing of strain CDC66177 revealed that while the toxin gene cluster inserted into the rarA operon similar to other type E strains, its overall genome content shared greater similarity with a Group II C. botulinum type B strain (17B). CONCLUSIONS: These results expand our understanding of the global distribution of C. botulinum type E strains and suggest that the type E toxin gene cluster may be able to insert into C. botulinum strains with a more diverse genetic background than previously recognized.

Genetic diversity among Clostridium botulinum strains harboring bont/A2 and bont/A3 genes.

Clostridium botulinum type A strains are known to be genetically diverse and widespread throughout the world. Genetic diversity studies have focused mainly on strains harboring one type A botulinum toxin gene, bont/A1, although all reported bont/A gene variants have been associated with botulism cases. Our study provides insight into the genetic diversity of C. botulinum type A strains, which contain bont/A2 (n = 42) and bont/A3 (n = 4) genes, isolated from diverse samples and geographic origins. Genetic diversity was assessed by using bont nucleotide sequencing, content analysis of the bont gene clusters, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sequences of bont genes obtained in this study showed 99.9 to 100% identity with other bont/A2 or bont/A3 gene sequences available in public databases. The neurotoxin gene clusters of the subtype A2 and A3 strains analyzed in this study were similar in gene content. C. botulinum strains harboring bont/A2 and bont/A3 genes were divided into six and two MLST profiles, respectively. Four groups of strains shared a similarity of at least 95% by PFGE; the largest group included 21 out of 46 strains. The strains analyzed in this study showed relatively limited genetic diversity using either MLST or PFGE.

Equine botulinum antitoxin for the treatment of infant botulism.

Infant botulism is the most common form of human botulism in Argentina and the United States. BabyBIG (botulism immune globulin intravenous [human]) is the antitoxin of choice for specific treatment of infant botulism in the United States. However, its high cost limits its use in many countries. We report here the effectiveness and safety of equine botulinum antitoxin (EqBA) as an alternative treatment. We conducted an analytical, observational, retrospective, and longitudinal study on cases of infant botulism registered in Mendoza, Argentina, from 1993 to 2007. We analyzed 92 medical records of laboratory-confirmed cases and evaluated the safety and efficacy of treatment with EqBA. Forty-nine laboratory-confirmed cases of infant botulism demanding admission in intensive care units and mechanical ventilation included 31 treated with EqBA within the 5 days after the onset of signs and 18 untreated with EqBA. EqBA-treated patients had a reduction in the mean length of hospital stay of 23.9 days (P = 0.0007). For infants treated with EqBA, the intensive care unit stay was shortened by 11.2 days (P = 0.0036), mechanical ventilation was reduced by 11.1 days (P = 0.0155), and tube feeding was reduced by 24.4 days (P = 0.0001). The incidence of sepsis in EqBA-treated patients was 47.3% lower (P = 0.0017) than in the untreated ones. Neither sequelae nor adverse effects attributable to EqBA were noticed, except for one infant who developed a transient erythematous rash. These results suggest that prompt treatment of infant botulism with EqBA is safe and effective and that EqBA could be considered an alternative specific treatment for infant botulism when BabyBIG is not available.

Presence of Clostridium botulinum spores in Matricaria chamomilla (chamomile) and its relationship with infant botulism.

Nowadays, infant botulism is the most important form of human botulism in some countries. This illness affects infants younger than 52 weeks of age. The infection occurs in the intestinal tract; therefore, ingestion of Clostridium botulinum spores with food is proposed. In some countries, people use chamomile tea as a household remedy for intestinal colics and given this tea to infants. Chamomile can be contaminated with C. botulinum and could be a vehicle of its spores. Our aim was to study the prevalence and spore-load of C. botulinum in chamomile. We analysed 200 samples; the 7.5% of them were contaminated with botulinum spores. However, prevalence of these spores was significantly higher in chamomile sold by weight in herbal stores (unwrapped chamomile) than prevalence in chamomile sold in tea bags (p=0.0055). The spore-load detected in all positive samples was 0.3-0.4 spores per gram of chamomile. We identified C. botulinum types A, B, and F in the 53.3%, 6.7%, and 13.3%, respectively. Chamomile (principally, unwrapped chamomile) is a potencial vehicle of C. botulinum spores, and ingestion of chamomile tea could represent a risk for infant botulism.

Distribution of botulinum toxin-producing clostridia in soils of Argentina.

We studied the presence of botulinum toxin-producing clostridia in 2,009 soil samples from five geographical regions of Argentina. The prevalence was 23.5%, and the distribution was not homogeneous among the regions. We observed a great multiplicity of serological types and a higher prevalence in nonvirgin soils than in virgin soils.

Botulismo del lactante / Infant botulism

En el presente trabajo se expone la experiencia clínica, terapéutica y evolutiva de 6 casos de botulismo del lactante acaecidos desde marzo de 1982 hasta enero de 1986 en la ciudad de Mendonza, Argentina. La edad fluctuó entre 2 y 4 meses. Se identificó toxina botulínica tipo A en sangre de 4 niños y en las heces de 5. En los 6 casos se aisló Clostridium botulinum tipo A de las heces. El cuadro clínico que nos permite sospechar el diagnóstico tempranamente se manifiesta con una tríada: hipotonía, constipación y reflejo fotomotor disminuido o ausente. La alta contaminación de nuestros suelos y la atención creciente que se presta a esta patología incidirán seguramente incrementando su diagnóstico

Botulismo del lactante: presentación en un niño de 40 días / Infant botulism: report of a child aged 40 days

Se describemn los signos clínicos, evolución y tratamiento de un niño de 40 días con butolismo. Es el octavo diagnosticado en Mendoza, el de menor edad de esta casuística y el tercero que sobrevive. Se señalan los métodos de diagnóstico y l seguimiento de laboratorio. También se refiere la búsqueda de Clostridium botulinum en el medio ecológico del niño. Se considera la posibilidad de que la lactancia materna brinde una protección relativa

Botulismo del lactante / Infant botulism

En el presente trabajo se expone la experiencia clínica, terapéutica y evolutiva de 6 casos de botulismo del lactante acaecidos desde marzo de 1982 hasta enero de 1986 en la ciudad de Mendonza, Argentina. La edad fluctuó entre 2 y 4 meses. Se identificó toxina botulínica tipo A en sangre de 4 niños y en las heces de 5. En los 6 casos se aisló Clostridium botulinum tipo A de las heces. El cuadro clínico que nos permite sospechar el diagnóstico tempranamente se manifiesta con una tríada: hipotonía, constipación y reflejo fotomotor disminuido o ausente. La alta contaminación de nuestros suelos y la atención creciente que se presta a esta patología incidirán seguramente incrementando su diagnóstico (AU)

Botulismo del lactante: presentación en un niño de 40 días / Infant botulism: report of a child aged 40 days

Se describemn los signos clínicos, evolución y tratamiento de un niño de 40 días con butolismo. Es el octavo diagnosticado en Mendoza, el de menor edad de esta casuística y el tercero que sobrevive. Se señalan los métodos de diagnóstico y l seguimiento de laboratorio. También se refiere la búsqueda de Clostridium botulinum en el medio ecológico del niño. Se considera la posibilidad de que la lactancia materna brinde una protección relativa (AU)