Enhancing Escherichia coli abiotic stress resistance through ornithine lipid formation.

Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications. KEY POINTS: • Ornithine lipid production in E. coli increases biomass yield under phosphate limitation. • Engineered strains show an enhanced production phenotype under low pH stress. • Transcriptome analysis and CCCP experiments revealed reduced proton leakage.

Plasmids of the incompatibility group FIBK occur in Klebsiella variicola from diverse ecological niches.

Plasmids play a fundamental role in the evolution of bacteria by allowing them to adapt to different environments and acquire, through horizontal transfer, genes that confer resistance to different classes of antibiotics. Using the available in vitro and in silico plasmid typing systems, we analyzed a set of isolates and public genomes of K. variicola to study its plasmid diversity. The resistome, the plasmid multilocus sequence typing (pMLST), and molecular epidemiology using the MLST system were also studied. A high frequency of IncF plasmids from human isolates but lower frequency from plant isolates were found in our strain collection. In silico detection revealed 297 incompatibility (Inc) groups, but the IncFIBK (216/297) predominated in plasmids from human and environmental samples, followed by IncFIIK (89/297) and IncFIA/FIA(HI1) (75/297). These Inc groups were associated with clinically important ESBL (CTX-M-15), carbapenemases (KPC-2 and NDM-1), and colistin-resistant genes which were associated with major sequence types (ST): ST60, ST20, and ST10. In silico MOB typing showed 76% (311/404) of the genomes contained one or more of the six relaxase families with MOBF being most abundant. We identified untypeable plasmids carrying blaKPC-2, blaIMP-1, and blaSHV-187 but for which a relaxase was found; this may suggest that novel plasmid structures could be emerging in this bacterial species. The plasmid content in K. variicola has limited diversity, predominantly composed of IncFIBK plasmids dispersed in different STs. Plasmid detection using the replicon and MOB typing scheme provide a broader context of the plasmids in K. variicola. This study showed that whole-sequence-based typing provides current insights of the prevalence of plasmid types and their association with antimicrobial resistant genes in K. variicola obtained from humans and environmental niches.

PCR system for the correct differentiation of the main bacterial species of the Klebsiella pneumoniae complex.

Accurate recognition of the closely related species Klebsiella pneumoniae, Klebsiella quasipneumoniae and Klebsiella variicola by phenotypic, biochemical and automated tests is notoriously unreliable in hospitals' diagnostic laboratories. A comparative genomics approach was conducted for the correct differentiation of the main bacterial species in the K. pneumoniae complex. Analysis of the deduced proteomes of 87 unique genomes of the Klebsiella in public databases, was used for the identification of unique protein family members. This allowed the design of a multiplex-PCR assay for the correct differentiation of these three species from different origins. This system allowed us to determine the prevalence of K. pneumoniae, K. quasipneumoniae and K. variicola among a collection of 552 clinical isolates. Of these, 87.3% (482/552) isolates corresponded to K. pneumoniae, 6.7% (33/552) to K. quasipneumoniae and 5.9% (33/552) to K. variicola. The multiplex-PCR results showed a 100% accuracy for the correct identification of the three species evaluated, which was validated with rpoB phylogenetic sequence analysis.

Xanthogranuloma in the External Auditory Canal in Childhood, an Unexpected Lesion.

Pedunculated lesions in the external auditory canal present a broad differential diagnosis. It is crucial to rule out malignant neoplasms and consider the possibility of xanthogranulomas, which are very rare in this location. Management is determined by associated complications, and otolaryngologists must consider this for appropriate treatment.

Host population structure and species resolution reveal prophage transmission dynamics.

Much knowledge about bacteriophages has been obtained via genomics and metagenomics over the last decades. However, most studies dealing with prophage diversity have rarely conducted phage species delimitation (aspect 1) and have hardly integrated the population structure of the host (aspect 2). Yet, these two aspects are essential in assessing phage diversity. Here, we implemented an operational definition of phage species (clustering at 95% identity, 90% coverage) and integrated the host's population structure to understand prophage diversity better. Gathering the most extensive data set of Acinetobacter baumannii phages (4,152 prophages + 122 virulent phages, distributed in 46 countries in the world), we show that 91% (875 out of 963) of the prophage species have four or fewer prophages per species, and just five prophage species have more than 100 prophages. Most prophage species have a narrow host range and are geographically restricted; yet, very few have a broad host range being well spread in distant lineages of A. baumannii. These few broad host range prophage species are not only cosmopolitan but also the most abundant species. We also noted that polylysogens had very divergent prophages, belonging to different prophage species, and prophages can easily be gained and lost within the bacterial lineages. Finally, even with this extensive data set, the prophage diversity has not been fully grasped. Our study highlights how integrating the host population structure and a solid operational definition of phage species allows us to better appreciate phage diversity and its transmission dynamics. IMPORTANCE: Much knowledge about bacteriophages has been obtained via genomics and metagenomics over the last decades. However, most studies dealing with prophage diversity have rarely conducted phage species delimitation (aspect 1) and have hardly integrated the population structure of the host (aspect 2). Yet, these two aspects are essential in assessing phage diversity. Here, we implemented an operational definition of phage species (clustering at 95% identity, 90% coverage) and integrated the host's population structure to understand prophage diversity better. Gathering the most extensive data set of Acinetobacter baumannii phages, we show that most prophage species have four or fewer prophages per species, and just five prophage species have more than 100 prophages. Most prophage species have a narrow host range and are geographically restricted; yet, very few have a broad host range being well spread in distant lineages of A. baumannii. These few broad host range prophage species are cosmopolitan and the most abundant species. Prophages in the same bacterial genome are very divergent, and prophages can easily be gained and lost within the bacterial lineages. Finally, even with this extensive data set, the prophage diversity has not been fully grasped. This study shows how integrating the host population structure and clustering at the species level allows us to better appreciate phage diversity and its transmission dynamics.

Acinetobacter junii: an emerging One Health pathogen.

Acinetobacter junii is an opportunistic human and animal pathogen severely understudied. Here, we conducted the largest genomic epidemiological study on this pathogen to date. Our data show that this bacterium has spread globally. Also, we found that some human and non-human isolates are not well differentiated from one another, implying transmission between clinical and non-clinical, non-human settings. Remarkably, human but also some non-human isolates have clinically important antibiotic resistance genes, and some of these genes are located in plasmids. Given these results, we put forward that A. junii should be considered an emerging One Health problem. In this regard, future molecular epidemiological studies about this species will go beyond human isolates and will consider animal-, plant-, and water-associated environments. IMPORTANCE: Acinetobacter baumannii is the most well-known species from the genus Acinetobacter. However, other much less studied Acinetobacter species could be important opportunistic pathogens of animals, plants and humans. Here, we conducted the largest genomic epidemiological study of A. junii, which has been described as a source not only of human but also of animal infections. Our analyses show that this bacterium has spread globally and that, in some instances, human and non-human isolates are not well differentiated. Remarkably, some non-human isolates have important antibiotic resistance genes against important antibiotics used in human medicine. Based on our results, we propose that this pathogen must be considered an issue not only for humans but also for veterinary medicine.

Genomic and Phylogenomic Characterization of Carbapenem-resistant Pseudomonas aeruginosa 'High-risk' Clone O4/ExoS+/ST654 Circulating in Chilean Hospitals.

INTRODUCTION: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a serious threat to public health. Globally, carbapenemases-producing CRPA isolates mainly belong to 'high-risk' clones; however, the molecular epidemiology of CRPA isolates circulating in Chile are scarce, where this pathogen is the main aetiological agent of ventilator-associated pneumonia. OBJECTIVES: To characterize the phylogenomics and molecular features of ST654 CRPA isolates collected in Chile between 2016 and 2022. METHODS: Eighty-nine CRPA isolates collected in different Chilean hospitals from clinical specimens between 2005 and 2022 were analysed. Antibiotic susceptibility tests and carbapenemases production were carried out on the CRPA ST654 isolates. Also, they were subjected to whole-genome sequencing, from which in silico analyses were performed. RESULTS: Thirty-four strains (38.2%) belonged to the ST654 high-risk clone, being the most predominant lineage of the collection. Most of these isolates belonged to a subclade including KPC producers that also clustered with strains from Argentina and the United States, whereas few VIM and NDM co-producers clustered in two different smaller subclades. The isolates exhibited a broad resistome encompassing genes mediating resistance to several other clinically relevant drugs. Additionally, all the 34 ST654 isolates were ExoS+ as a virulence factor and associated to the O4-serotype. CONCLUSIONS: Our report represents the most comprehensive phylogenomic study of a CRPA high-risk clone ST654 to date. Our analyses suggest that this lineage is undergoing a divergent evolutionary path in Chile, because most of the isolates were KPC producers and were O4 serotype, differing from previous descriptions, which underline the relevance of performing molecular surveillance on this pathogen.

Acinetobacter baumannii from grass: novel but non-resistant clones.

Acinetobacter baumannii is one the most worrisome nosocomial pathogens, which has long been considered almost mainly as a hospital-associated bacterium. There have been some studies about animal and environmental isolates over the last decade. However, little effort has been made to determine if this pathogen dwells in the grass. Here, we aim to determine the evolutionary relationships and antibiotic resistance of clones of A. baumannii sampled from grass to the major human international clones and animal clones. Two hundred and forty genomes were considered in total from four different sources for this study. Our core and accessory genomic epidemiology analyses showed that grass isolates cluster in seven groups well differentiated from one another and from the major human and animal isolates. Furthermore, we found new sequence types under both multilocus sequence typing schemes: two under the Pasteur scheme and seven for the Oxford scheme. The grass isolates contained fewer antibiotic-resistance genes and were not resistant to the antibiotics tested. Our results demonstrate that these novel clones appear to have limited antibiotic resistance potential. Given our findings, we propose that genomic epidemiology and surveillance of A. baumannii should go beyond the hospital settings and consider the environment in an explicit One Health approach.

Molecular and Genomic Insights of mcr-1-Producing Escherichia coli Isolates from Piglets.

The use of colistin in food-producing animals favors the emergence and spread of colistin-resistant strains. Here, we investigated the occurrence and molecular mechanisms of colistin resistance among E. coli isolates from a Mexican piglet farm. A collection of 175 cephalosporin-resistant colonies from swine fecal samples were recovered. The colistin resistance phenotype was identified by rapid polymyxin test and the mcr-type genes were screened by PCR. We assessed the colistin-resistant strains by antimicrobial susceptibility test, pulse-field gel electrophoresis, plasmid profile, and mating experiments. Whole-Genome Sequencing data was used to explore the resistome, virulome, and mobilome of colistin-resistant strains. A total of four colistin-resistant E. coli were identified from the cefotaxime-resistant colonies. All harbored the plasmid-borne mcr-1 gene, which was located on conjugative 170-kb IncHI-2 plasmid co-carrying ESBLs genes. Thus, high antimicrobial resistance rates were observed for several antibiotic families. In the RC2-007 strain, the mcr-1 gene was located as part of a prophage carried on non-conjugative 100-kb-plasmid, which upon being transformed into K. variicola strain increased the polymyxin resistance 2-fold. The genomic analysis showed a broad resistome and virulome. Our findings suggest that colistin resistance followed independent acquisition pathways as clonal and non-genetically related mcr-1-harboring strains were identified. These E. coli isolates represent a reservoir of antibiotic resistance and virulence genes in animals for human consumption which could be potentially propagated into other interfaces.

Exploring the environmental traits and applications of Klebsiella variicola.

Klebsiella variicola has been found in various natural niches, alone or in association with other bacteria, and causes diseases in animals and plants with important economic and environmental impacts. K. variicola has the capacity to fix nitrogen in the rhizosphere and soil; produces indole acetic acid, acetoin, and ammonia; and dissolves phosphorus and potassium, which play an important role in plant growth promotion and nutrition. Some members of K. variicola have properties such as halotolerance and alkalotolerance, conferring an evolutionary advantage. In the environmental protection, K. variicola can be used in the wastewater treatment, biodegradation, and bioremediation of polluted soil, either alone or in association with other organisms. In addition, it has the potential to carry out industrial processes in the food and pharmaceutical industries, like the production of maltose and glucose by the catalysis of debranching unmodified oligosaccharides by the pullulanase enzyme. Finally, this bacterium has the ability to transform chemical energy into electrical energy, such as a biocatalyst, which could be useful in the near future. These properties show that K. variicola should be considered an eco-friendly bacterium with hopeful technological promise. In this review, we explore the most significant aspects of K. variicola and highlight its potential applications in environmental and biotechnological processes.

Short-term audiological outcomes of stapedotomy: microdrill at low revolutions versus manual perforator to perform a small footplate fenestra. A prospective observational study.

<b>Introduction:</b> Stapedotomy is currently the surgical technique of choice for treating otosclerosis. Despite this, there is no agreement about the best technique to perform a small fenestra footplate, therefore multiple procedures have been proposed. The aim of this study was to investigate the hearing outcomes of microdrill and manual perforator. <br><b>Material and Methods:</b> An observational prospective study was carried out on patients who underwent stapedotomy. We analyzed the hearing threshold in two groups of patients according to the way the fenestra footplate was realized by microdrill or manual perforator. <br><b>Results:</b> A total of 113 patients were evaluated. Postoperative hearing gain of the microdrill group was 23.29 (18.58) dB HL 95% CI (18.40-28.18), while in the manual perforator group, it was 22.67 (12.91) dB HL 95% CI (19.07-26.26). Both groups were statistically significant. Postoperative bone conductive hearing threshold at the frequencies of 0.5, 1 and 2 KHz and postoperative air conductive hearing threshold at the frequencies of 2 and 4 KHz showed statistically significant differences in the manual perforator group. The closure of air-bone gap was higher in the microdrill group with statistically significant differences. <br><b>Conclusion:</b> Both manual perforator and microdrill have good hearing outcomes at six months after surgery. The manual perforator has better audiological outcomes than microdrill. Hence, the manual perforator is a safe technique and can be used in centers that do not have other methods to make the small fenestra.

RhizoBindingSites, a Database of DNA-Binding Motifs in Nitrogen-Fixing Bacteria Inferred Using a Footprint Discovery Approach.

Basic knowledge of transcriptional regulation is needed to understand the mechanisms governing biological processes, i.e., nitrogen fixation by Rhizobiales bacteria in symbiosis with leguminous plants. The RhizoBindingSites database is a computer-assisted framework providing motif-gene-associated conserved sequences potentially implicated in transcriptional regulation in nine symbiotic species. A dyad analysis algorithm was used to deduce motifs in the upstream regulatory region of orthologous genes, and only motifs also located in the gene seed promoter with a p-value of 1e-4 were accepted. A genomic scan analysis of the upstoream sequences with these motifs was performed. These predicted binding sites were categorized according to low, medium and high homology between the matrix and the upstream regulatory sequence. On average, 62.7% of the genes had a motif, accounting for 80.44% of the genes per genome, with 19613 matrices (a matrix is a representation of a motif). The RhizoBindingSites database provides motif and gene information, motif conservation in the order Rhizobiales, matrices, motif logos, regulatory networks constructed from theoretical or experimental data, a criterion for selecting motifs and a guide for users. The RhizoBindingSites database is freely available online at rhizobindingsites.ccg.unam.mx.

Molecular epidemiology of Klebsiella variicola obtained from different sources.

Klebsiella variicola is considered an emerging pathogen in humans and has been described in different environments. K. variicola belongs to Klebsiella pneumoniae complex, which has expanded the taxonomic classification and hindered epidemiological and evolutionary studies. The present work describes the molecular epidemiology of K. variicola based on MultiLocus Sequence Typing (MLST) developed for this purpose. In total, 226 genomes obtained from public data bases and 28 isolates were evaluated, which were mainly obtained from humans, followed by plants, various animals, the environment and insects. A total 166 distinct sequence types (STs) were identified, with 39 STs comprising at least two isolates. The molecular epidemiology of K. variicola showed a global distribution for some STs was observed, and in some cases, isolates obtained from different sources belong to the same ST. Several examples of isolates corresponding to kingdom-crossing bacteria from plants to humans were identified, establishing this as a possible route of transmission. goeBURST analysis identified Clonal Complex 1 (CC1) as the clone with the greatest distribution. Whole-genome sequencing of K. variicola isolates revealed extended-spectrum ß-lactamase- and carbapenemase-producing strains with an increase in pathogenicity. MLST of K. variicola is a strong molecular epidemiological tool that allows following the evolution of this bacterial species obtained from different environments.

Measuring plasma levels of three microRNAs can improve the accuracy for identification of malignant breast lesions in women with BI-RADS 4 mammography.

A BI-RADS category of 4 from a mammogram indicates suspicious breast lesions, which require core biopsies for diagnosis and have an approximately one third chance of being malignant. Human plasma contains many circulating microRNAs, and variations in their circulating levels have been associated with pathologies, including cancer. Here, we present a novel methodology to identify malignant breast lesions in women with BI-RADS 4 mammography. First, we used the miRNome array and qRT-PCR to define circulating microRNAs that were differentially represented in blood samples from women with breast tumor (BI-RADS 5 or 6) in comparison to controls (BI-RADS 1 or 2). Next, we used qRT-PCR to quantify the level of this circulating microRNAs in patients with mammograms presenting with BI-RADS category 4. Finally, we developed a machine learning method (Artificial Neural Network - ANN) that receives circulating microRNA levels and automatically classifies BI-RADS 4 breast lesions as malignant or benign. We identified a minimum set of three circulating miRNAs (miR-15a, miR-101 and miR-144) with altered levels in patients with breast cancer. These three miRNAs were quantified in plasma from 60 patients presenting biopsy-proven BI-RADS 4 lesions. Finally, we constructed a very efficient ANN that could correctly classify BI-RADS 4 lesions as malignant or benign with approximately 92.5% accuracy, 95% specificity and 88% sensibility. We believe that our strategy of using circulating microRNA and a machine learning method to classify BI-RADS 4 breast lesions is a non-invasive, non-stressful and valuable complementary approach to core biopsy in women with BI-RADS 4 lesions.

Morfología radicular y mediciones apicales en primeros molares en una población maya / Root morphology and apical measurements of first molars in a Mayan population

El conocimiento preciso de la morfología radicular dental son claves para el éxito en las terapias endodónticas. El objetivo de este estudio fue evaluar las distintas variaciones de la morfología radicular y apical en primeros molares superiores e inferiores de una población maya moderna. Se observaron microfotografías provenientes de 80 primeros molares superiores e inferiores, presentadas en cortes transversales a 1 y 2,5 mm del vértice apical, así como de las porciones 1/3, 1⁄2, 2/3 del largo radicular y en la unión amelocementaria. Se determinó el número de raíces, conductos y configuración de Vertucci, así como forma y medidas de diámetro mayor y menor a 1 mm del vértice apical. El 90 % de primeros molares superiores reportó la presencia de 3 raíces y el 10 % mostró 2. En tanto que el total de primeros molares inferiores (100 %) reportó 2 raíces. En general, en las raíces mesiales prevaleció el Tipo II de Vertucci mientras que en las distales y palatinas la Tipo I. En el corte a 1 mm del vértice apical la forma redonda fue la de mayor prevalencia en los conductos de primeros molares superiores (44,5 %), seguida de la irregular (34,1 %) y la oval (21,4 %); mientras que en los primeros molares inferiores la más prevalente fue la irregular (54, 5 %), seguida de la redonda (23,9 %) y oval (21,6 %). Los diámetros mayor y menor de los conductos mesiales a 1 mm apical midieron 0,46 y 0,23 mm y 0,64 y 0,25 mm en molares superiores e inferiores respectivamente. La frecuencia de MB2 en primeros molares superiores fue del 77,8 %. Los conductos con mayor variabilidad fueron los mesiales. Se observó baja frecuencia de conductos ovales. Los diámetros mayores de los conductos mesiales en general, fueron superiores a 0,45 mm.

Accurate knowledge of dental root morphology is a key to success in endodontic therapies. The aim of this study was to evaluate the different variations of the root and apical morphology in upper and lower first molars of a modern Mayan population. Photomicrographs were taken from 80 upper and lower first molars, presented in cross sections at 1 and 2.5 mm from the apical vertex, as well as from the 1/3, 1⁄2, 2/3 portions of the root length and at the cementoenamel junction. The number of roots, canal and Vertucci`s configuration was determined, as well as the shape and measurements of the major and minor diameters at 1 mm from the apex. In this study 90 % of upper first molars reported the presence of 3 roots and 10 % showed 2, while the total lower first molars (100 %) reported 2 roots. In general, Vertucci`s Type II prevailed in the mesial roots while Type I was prevalent in the distal and palatal ones. In the 1 mm cut of the apical vertex, the round shape was the most prevalent in the canal of the first upper molars (44.5 %), followed by irregular (34.1 %) and oval (21.4 %); while in the first lower molars the most prevalent one was irregular (54.5 %), followed by round (23.9 %) and oval (21.6 %). The major and minor diameters of the mesial canals at 1 mm apical recorded 0.46 and 0.23 mm and 0.64 and 0.25 mm in upper and lower molars respectively. The frequency of MB2 in upper first molars was 77.8 %. The canals with greater variability were the mesial ones. Low frequency of oval canals was observed. The largest diameters of the mesial canal in general were greater than 0.45 mm.

Relato de caso de linfoma anaplásico de células grandes associado a implante mamário em paciente brasileira / Report of a case of anaplastic large cell lymphoma associated with a breast implant in a Brazilian patient

O linfoma anaplásico de células grandes (ALCL) associado a implantes mamários é um distúrbio linfoproliferativo das células T que foi recentemente reconhecido como uma entidade independente na classificação de linfomas da Organização Mundial de Saúde (OMS). Apesar do pequeno número de descrições, o número de casos está crescendo rapidamente. Das poucas centenas de casos que foram publicados até agora, muito poucos vieram do Brasil e nenhum foi relatado às autoridades locais. Encontramos um caso recentemente, e acreditamos que seu relato à comunidade local de cirurgia plástica poderá chamar a sua atenção para essa patologia emergente. O prognóstico é muito bom na maior parte dos casos diagnosticados. Contudo, ainda se sabe pouco sobre como e por que os implantes de silicone poderiam desencadear uma resposta linfoide, culminando num ALCL.

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a T-cell lymphoproliferative disorder that has recently been recognized as an independent entity in the World Health Organization (WHO) classification of lymphomas. Despite the small number of reports to date, the number of cases is rapidly increasing. Of the few hundred cases that have been reported so far, very few came from Brazil and none have been reported to the local authorities. We encountered a case of BIA-ALCL and believe that its report to the local plastic surgery community could raise awareness to this emerging pathology. The prognosis is very good in most of the diagnosed cases. However, little is known about how and why silicone implants could trigger a lymphoid response that results in ALCL.

Duplo contorno uterino: sinal radiológico de gravidez na histerossalpingografia / Double-outlined uterine cavity: radilogical sign of pregnancy in the hysterosalpingography

Resumo: Na literatura, os achados histerossalpingráficos clássicos para gestaçäo säo cavidade uterina ampla e falha de enchimento intracavitária, correspondente ao saco gestacional. Entratanto, existe outro sinal radiológico muito sugestivo de gravidez, embora incomum, conhecido como duplo contorno uterino. Relatamos, neste trabalho, três casos de duplo contorno uterino caracterizados durante a realizaçäo da historossalpingografia com contraste iodado e discutimos alguns aspectos da radiaçäo fetal intra-útero. Foram observados dois casos de gestaçäo tópico e um caso de gestaçäo ectópica (tubária).