Application of MALDI-TOF mass spectrometry, and DNA sequencing-based SLST and MLST analysis for the identification of Cronobacter spp. isolated from environmental surveillance samples.

Cronobacter spp. are emerging infectious foodborne bacteria that can cause acute meningitis and necrotizing enterocolitis in neonates and immunocompromised individuals. Although, little is known about its reservoirs or transmission routes, it has been linked to powdered infant formula worldwide. Three Cronobacter spp. (C. sakazakii, C. malonaticus, and C. turicensis) have been described as more virulent, and isolated frequently from infant meningitis cases. The estimated mortality rates are as high as 80% in infants. Thus, surveillance and typing of Cronobacter spp. isolated from food and environmental samples is essential to prevent contamination and spread of this pathogen. In this study, we have characterized 83 Cronobacter isolates recovered from various environmental samples by conventional microbiologic protocols. Species identification was accomplished by VITEK 2 system and real-time PCR analysis. Subsequently, these isolates were analyzed using VITEK MS system. Single locus sequence typing (SLST) was achieved by characterizing the regions of 16S rRNA and rpoB genes. Multilocus sequence typing (MLST) was performed by sequence characterization of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, and pps) using ABI 3500XL Genetic Analyzer. VITEK MS system identified, the majority of isolates as Cronobacter sakazakii with a high confidence value (99.9%). MLST analysis ascertained 12 distinct clonal complexes (CC1, CC4, CC8, CC13, CC17, CC21, CC31, CC40, CC52, CC64, CC73, and CC83) for the recovered C. sakazakii isolates. The results suggest that the MALDI-TOF MS is a reliable diagnostic tool for rapid species identification whereas 7-loci MLST is a powerful technique to discriminate and differentiate Cronobacter spp. isolates.

Interlaboratory validation of an improved method for detection of Cyclospora cayetanensis in produce using a real-time PCR assay.

A collaborative validation study was performed to evaluate the performance of a new U.S. Food and Drug Administration method developed for detection of the protozoan parasite, Cyclospora cayetanensis, on cilantro and raspberries. The method includes a sample preparation step in which oocysts are recovered from produce using an enhanced produce washing solution containing 0.1% Alconox and a commercially available method to disrupt the C. cayetanensis oocysts and extract DNA. A real-time PCR assay targeting the C. cayetanensis 18S rDNA gene with an internal amplification control to monitor PCR inhibition provides species-specific identification. Five laboratories blindly analyzed a total of 319 samples consisting of 25 g of cilantro or 50 g of raspberries which were either uninoculated or artificially contaminated with C. cayetanensis oocysts. Detection rates for cilantro inoculated with 200, 10, and 5 oocysts, were 100%, 80%, and 31%, respectively. For raspberries, the detection rates for samples inoculated with 200, 10, and 5 oocysts were 100%, 90% and 50%, respectively. All uninoculated samples, DNA blank extracts, and no-template PCR controls were negative. Reproducibility between laboratories and analysts was high and the method was shown to be an effective analytical tool for detection of C. cayetanensis in produce.

Rhinoplasty using autologous costal cartilage.

Most Latin American patients looking to have a primary septorhinoplasty share common characteristics in relation to an incorrect projection of the nasal tip complex and a low dorsal line. Thus, the frequent use of structural techniques and of surgical enhancement techniques becomes necessary to improve the nasal contour. In cases of secondary septorhinoplasty, it is also usual in our practice not to have sufficient septal cartilage available or with the required quality to give structure and support to the nasal tip complex, handle the nasal dorsum, and simultaneously correct postseptorhinoplasty deformities. For these reasons, in our practice costal cartilage represents an excellent option as autologous graft material. We present our experience using autologous costal cartilage for structural and nonstructural purposes in 286 selected patients who underwent open rhinoplasty between 2004 and 2011. We emphasize preoperative analyses, we discuss the criteria for selecting costal graft as graft material, we show key aspects of the dynamic of the surgery, and we consider the possibility of using autologous costal graft in combination with heterologous grafts. In this work we also establish the disadvantages of costal cartilage as graft material in specific areas of the surgical anatomy of the nose.

A Single-Laboratory Performance Evaluation of MALDI-TOF MS in Rapid Identification of Staphylococcus aureus, Cronobacter sakazakii, Vibrio parahaemolyticus, and Some Closely Related Bacterial Species of Public Health Importance.

BACKGROUND: Staphylococcus is a genus of Gram-positive bacteria, known to cause food poisoning and gastrointestinal illness in humans. Additionally, the emergence of methicillin-resistant S. aureus (MRSA) strains has caused a major health care burden worldwide. Cronobacter is a group of Gram-negative bacteria that can survive in extreme dry conditions. Cronobacter sakazakii is known to contaminate powdered infant formula and cause life-threatening infections in neonates. Vibrio is a genus of human-pathogenic Gram-negative bacteria that can cause foodborne illness by consuming undercooked or raw seafood. Vibrio parahaemolyticus can cause serious gastrointestinal disease in humans. Thus, rapid identification of Staphylococcus spp., Cronobacter spp., and Vibrio spp. is crucial for the source tracking of contaminated food, as well as to measure the transmission dynamics of these bacterial pathogens causing foodborne diseases and outbreaks. OBJECTIVE: This single-laboratory performance evaluation study used the VITEK MS system to evaluate the potential of MALDI-TOF MS technology for rapid identification of S. aureus-like, C. sakazakii-like, and V. parahaemolyticus-like isolates of public health importance. METHOD: A total of 226 isolates recovered from various food, environmental surveillance samples, and other sources were identified by bioMérieux VITEK 2 and VITEK MS systems as Staphylococcus spp., Cronobacter spp., and Vibrio spp. Five American Type Culture Collection (ATCC) reference Gram-positive and Gram-negative bacterial isolates were also tested to complete the study. In addition, for some Staphylococcus spp. isolates, whole genome sequencing (WGS) and DNA sequencing of 16S rRNA partial region were also performed for species identification. RESULTS: The VITEK MS system was able to provide species identification to all 96 isolates of Staphylococcus spp. and to all 29 isolates of Vibrio spp. examined with a high confidence value (99.9%). Similarly, species identification was observed for the majority of spots (245 of 303) for the 101 Cronobacter spp. isolates (∼82.0%) with a high confidence value (99.9%), and genus level identification was noticed for the rest of the Cronobacter spp. isolates (18.0%; 58 of the 303 spots) analyzed. Species identification data generated by VITEK 2 system were comparable to data obtained by the VITEK MS system. CONCLUSIONS: The VITEK MS system is a reliable high-throughput platform that can rapidly identify Staphylococcus, Vibrio, and Cronobacter to the genus level, as well as S. aureus, C. sakazakii, V. parahaemolyticus, and other closely related foodborne isolates and bacterial isolates from additional sources, in most cases. HIGHLIGHTS: The VITEK MS system can be used in the rapid genus and species identification of human-pathogenic Staphylococcus spp., Cronobacter spp., and Vibrio spp. isolates.

MALDI-TOF Mass Spectrometry and 16S rRNA Gene Sequence Analysis for the Identification of Foodborne Clostridium Spp.

BACKGROUND: Clostridium is a genus of Gram-positive, spore-forming, anaerobic bacteria comprising approximately 100 species. Some Clostridium spp. (C. botulinum, C. perfringens, C. tetani, and C. difficile) have been recognized to cause acute food poisoning, botulism, tetanus, and diarrheal illness in humans. Thus, rapid identification of Clostridium spp. is critical for source-tracking of contaminated food and to understand the transmission dynamics of these foodborne pathogens. OBJECTIVE: This study was carried out to rapidly identify Clostridium-like isolates by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS and rRNA sequencing methods. METHOD: Thirty-three Clostridium-like isolates were recovered from various baby food and surveillance samples. Species identification of these isolates was accomplished using the VITEK MS system. Sequence characterization of the 16S rRNA region was done on an ABI 3500xL Genetic Analyzer. RESULTS: The VITEK MS system identified 28 of the 33 Clostridium-like isolates with a high confidence value (99.9%); no identification was observed for the remaining five isolates. Nucleotide sequencing of the 16S rRNA region identified all 33 Clostridium-like isolates. Furthermore, while characterizing the 16S rRNA gene, 11 distinct Clostridium spp. (Clostridium aciditolerans, Clostridium aerotolerans, Clostridium argentinense, Clostridium beijerinckii, Clostridium bifermentans, Clostridium butyricum, Clostridium cochlearium, Clostridium difficile, Clostridium perfringens, Clostridium sporogenes, and Clostridium subterminale) were recognized among the 33 Clostridium-like isolates. One of the Clostridium-like isolates was identified as Citrobacter amalonaticus by both diagnostic methods. The generated 16S rRNA sequences matched completely (100%) with sequences available in GenBank for Clostridium and Citrobacter species. Species identification attained using the VITEK MS for the Clostridium-like isolates was comparable to that from the 16S rRNA sequencing-based data. CONCLUSIONS: The VITEK MS and 16S rRNA sequence analysis can be implemented in the species identification of Clostridium spp. isolates of public health importance. HIGHLIGHTS: MALDI-TOF MS and 16S rRNA sequencing can be used in the species identification of Clostridium species.

Rapid Detection of Staphylococcus aureus and Related Species Isolated from Food, Environment, Cosmetics, a Medical Device, and Clinical Samples Using the VITEK MS Microbial Identification System.

Staphylococcus spp. is considered as one of the most common human-pathogenic bacteria, causing illnesses ranging from nonthreatening skin infections to lethal diseases, including sepsis, pneumonia, bloodstream infections, and food poisoning. The emergence of methicillin-resistant Staphylococcus aureus strains has increased morbidity and mortality and resulted in a major healthcare burden worldwide. Single and multilocus sequence typing have been extensively used in the identification of Staphylococcus species. Nevertheless, these assays are relatively time-consuming and require high-quality DNA. Matrix-assisted laser desorption ionization-time-of-flight has been used recently for the rapid identification of several bacterial species. In this study, we have examined 47 Staphylococcus isolates recovered from food, environment, clinical samples, cosmetic products, and a medical device and 3 American Type Culture Collection Staphylococcus reference isolates using bioMérieux VITEK MS and VITEK 2 systems to determine isolate identity. Sequencing of the 16S ribosomal RNA gene was performed to confirm and compare the species identification data generated by VITEK 2 and VITEK MS systems. Although the VITEK 2 system could not identify one of the isolates, VITEK MS identified all 50 Staphylococcus spp. isolates tested. Results of this study clearly suggest that VITEK MS can be used in the rapid identification of Staphylococcus isolates of public health importance.

Identification of Lysinibacillus fusiformis Isolated from Cosmetic Samples Using MALDI-TOF MS and 16S rRNA Sequencing Methods.

Background: Lysinibacillus fusiformis is a Gram-positive, rod-shaped bacterium that can cause tropical ulcers, severe sepsis, and respiratory illnesses in humans. Objective: In this study, we analyzed cosmetic samples for the presence of human pathogenic microorganisms. Methods: Five unopened jars of exfoliating cream were examined initially by microbiological methods. Afterward, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS and 16S ribosomal RNA (rRNA) sequencing techniques were applied to characterize the recovered isolates. Results: Of the eight recovered Gram-positive bacterial subs, the VITEK® MS could provide genus-level identification to five subs and species-level identification to two subs (L. fusiformis with a 99.9% confidence value); one sub was unidentified. Subsequently, the deoxyriboneucleic acid sequencing of the 16S rRNA gene was done on an ABI 3500XL Genetic Analyzer for the confirmation of species identification. An analysis of sequencing data revealed a complete absence of genetic variation among the eight subs sequenced at this locus and confirmed the eight bacterial subs to be L. fusiformis, as their respective 16S rRNA sequences were identical to the available sequence in public domain (GenBank accession No. KU179364). Conclusions: Our results suggest that the VITEK MS and the 16S rRNA sequencing can be used for the identification of human pathogenic bacteria of public health importance. Highlights: We characterized eight isolates of Lysinibacillus spp. from cosmetics by MALDI-TOF MS and 16S rRNA sequence analyses.

Application of MALDI-TOF MS Systems in the Rapid Identification of Campylobacter spp. of Public Health Importance.

Campylobacteriosis is an infectious gastrointestinal disease caused by Campylobacter spp. In most cases, it is either underdiagnosed or underreported due to poor diagnostics and limited databases. Several DNA-based molecular diagnostic techniques, including 16S ribosomal RNA (rRNA) sequence typing, have been widely used in the species identification of Campylobacter. Nevertheless, these assays are time-consuming and require a high quality of bacterial DNA. Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS is an emerging diagnostic technology that can provide the rapid identification of microorganisms by using their intact cells without extraction or purification. In this study, we analyzed 24 American Type Culture Collection reference isolates of 16 Campylobacter spp. and five unknown clinical bacterial isolates for rapid identification utilizing two commercially available MADI-TOF MS platforms, namely the bioMérieux VITEK® MS and Bruker Biotyper systems. In addition, 16S rRNA sequencing was performed to confirm the species-level identification of the unknown clinical isolates. Both MALDI-TOF MS systems identified the isolates of C. jejuni, C. coli, C. lari, and C. fetus. The results of this study suggest that the MALDI-TOF MS technique can be used in the identification of Campylobacter spp. of public health importance.

Molecular Surveillance of Cronobacter spp. Isolated from a Wide Variety of Foods from 44 Different Countries by Sequence Typing of 16S rRNA, rpoB and O-Antigen Genes.

Cronobacter spp. are emerging infectious bacteria that can cause acute meningitis and necrotizing enterocolitis in neonatal and immunocompromised individuals. Although this opportunistic human-pathogenic microorganism has been isolated from a wide variety of food and environmental samples, it has been primarily linked to foodborne outbreaks associated with powdered infant formula. The U.S. Food and Drug Administration use the presence of these microbes as one of the criteria to assess food adulteration and to implement regulatory actions. In this study, we have examined 195 aliquots of enrichments from the nine major categories of foods (including baby and medical food, dairy products, dried food, frozen food, pet food, produce, ready-to-eat snacks, seafood, and spices) from 44 countries using conventional microbiological and molecular techniques. The typical colonies of Cronobacter were then identified by VITEK2 and real-time PCR. Subsequently, sequence typing was performed on the 51 recovered Cronobacter isolates at the 16S rRNA, rpoB and seven O-antigen loci for species identification in order to accomplish an effective surveillance program for the control and prevention of foodborne illnesses.

Injerto cartilagenoso autólogo extensor caudal en el manejo de la proyección y rotación de la punta nasal en rinoseptoplastia / Flow extension autologoces graft cartilagenoso in the prevention and management of the rotation of the nasal tip in rhinoplasty

La rotación y la proyección de la punta nasal representan dos de los parámetros más importantes a ser tomados en cuenta durante la rinoseptoplastia. Nos permite establecer un complejo de punta nasal cercano a lo considerado estéticamente ideal y además establece directrices acerca del manejo del dorso nasal. En el siguiente trabajo expones nuestra experiencia con el uso de injerto cartilaginoso antólogo, utilizando como extensor del borde caudal del septum nasal, como método para mejorar la rotación y proyección de la punta nasal en casos seleccionados. Se realizó un estudio prospectivo de un grupo seleccionado de 37 pacientes entre el año 2003 y el año 2007, con criterios para la realización de dicha técnica quirúrgica, esta es descrita de manera sistemática. En el 95 por ciento de los casos se obtuvieron resultados satisfactorios. Se discuten las ventajas y desventajas de la técnica quirúrgica

Rinoplastia abierta vs rinoplastia cerrada: proposición de flujograma clínico que racionalice la selección del abordaje quirúrgico / Rhinoplasty open vs closed rhinoplasty: proposition of clinical flujograma that he/she rationalizes the selection of the surgical boarding

La rinoseptoplastia, o cirugía cosmética nasal representa uno de los procedimientos quirúrgicos más complejos en la práctica de nuestra especialidad. Entre los factores que constituyen a hacer de este procedimiento un reto constante encontramos: los diferentes parámetros estéticos individuales a los que nos enfrentamos en cada caso, los ideales estéticos de cada paciente y lo que cada uno de ellos espera de la cirugía, la necesidad de lidiar con aspectos estéticos puros conjuntamente con aspectos funcionales del complejo de la nariz y de los senos paranasales e incluso en muchas ocasiones con aspectos funcionales de la rinofaringe y orofaringe, la necesidad de manejar en un mismo tiempo quirúrgico estético-funcional enfermedades propias del conplejo de la nariz y los senos paranasales de diversas etiologías, la necesidad de manejar quirúrgicamente múltiples tipos de tejidos con características histológicas diferentes, con actividades fisiológicas diversas y con procesos de cicatrización individuales y asincrónicos; todo esto sin mencionar los factores íntimamente relacionados con los diferentes abordajes y múltiples filosofías acerca de las técnicas quirúrgicas a ser empleadas y la puesta en práctica de dichas técnicas dentro de la sala de operaciones. Este conjunto de razones antes expuesta ha hecho que a lo largo de la historia de este procedimiento quirúrgico, desde que fue descrito en el Papiro de Ebers Egipcio (que data del año 3500 antes de cristo) hasta el presente evidencie en la literatura médica la divergencia franca entre autores en cuanto al abordaje ideal, la gran cantidad de técnicas quirúrgicas disponibles, e incluso consideraciones técnicas relacionadas a los factores étnicos y al sexo del paciente a ser sometido al procedimiento quirúrgico.

Manejo de la punta nasal en rinoplastia abierta a través de la técnica tongue-in-groove / Management of the nasal tip in open rhinoplasty through the technical tongue in groove

El manejo de la punta nasal representa el aspecto más complejo del procedimiento quirúrgico para la mayoría de los cirujanos rinoplásticos. Es por ello que no solo constituye una de las aristas principales de una cirugía estética nasal exitosa, si no que además se presenta como un reto quirúrgico permanente para el cirujano. Más allá de la técnica escogida para acercarse a los estándares estéticos ideales, el manejo de la punta nasal amerita un análisis pre-operatorio riguroso, seguido de un diagnóstico detallado, lo cual permitirá escoger el tipo de abordaje a utilizar dependiendo del plan quirúrgico establecido a fin de corregir las deformidades estructurales individuales presentes en esta sub-unidad nasal. En el presente estudio los autores presentan la técnica tongue-in-groove como una herramienta útil en el manejo quirúrgico de la punta nasal, basado en la experiencia de 42 pacientes sometidos a rinoseptoplastia abierta, 28 pertenecientes al sexo masculino y 14 pertenecientes al sexo femenino, con alteraciones estructurales y estéticas específicas de la punta nasal, se discuten además los factores relacionados con el análisis y diagnóstico preoperatorio, aspectos concernientes a la técnica quirúrgica así como sus ventajas y desventajas.