Tenebrio molitor as a model system to study Staphylococcus spp virulence and horizontal gene transfer.

Invertebrates can provide a valuable alternative to traditional vertebrate animal models for studying bacterial and fungal infections. This study aimed to establish the larvae of the coleoptera Tenebrio molitor (mealworm) as an in vivo model for evaluating virulence and horizontal gene transfer between Staphylococcus spp. After identifying the best conditions for rearing T. molitor, larvae were infected with different Staphylococcus species, resulting in dose-dependent killing curves. All species tested killed the insects at higher doses, with S. nepalensis and S. aureus being the most and least virulent, respectively. However, only S. nepalensis was able to kill more than 50% of larvae 72 h post-infection at a low amount of 105 CFU. Staphylococcus infection also stimulated an increase in the concentration of hemocytes present in the hemolymph, which was proportional to the virulence. To investigate T. molitor's suitability as an in vivo model for plasmid transfer studies, we used S. aureus strains as donor and recipient of a plasmid containing the gentamicin resistance gene aac(6')-aph(2″). By inoculating larvae with non-lethal doses of each, we observed conjugation, and obtained transconjugant colonies with a frequency of 1.6 × 10-5 per donor cell. This study demonstrates the potential of T. molitor larvae as a reliable and cost-effective model for analyzing the virulence of Staphylococcus and, for the first time, an optimal environment for the plasmid transfer between S. aureus carrying antimicrobial resistance genes.

Interspecies transfer of plasmid-borne gentamicin resistance between Staphylococcus isolated from domestic dogs to Staphylococcus aureus.

As preconized by the One Health concept, the intimate relationship between pets and owners is a common source for the trade of microorganisms with zoonotic potential, and with them, antimicrobial resistance genes. In this work, we evaluated the presence of antimicrobial resistance genes, that are usually within mobile genetic elements, in a laboratory collection of 79 canine Staphylococcus strains, mostly Staphylococcus pseudintermedius and Staphylococcus coagulans. Resistance to tetracycline was observed in 34% of the strains, followed by resistance to erythromycin (21%) and gentamicin (19%). These phenotypes were partially correlated with the presence of the tetracycline resistance genes tet(M) and tet(K) in 64% and 44% of all strains, respectively; erythromycin resistance genes erm(A) and erm(C) in 53% and 23%; and gentamicin resistance gene aac(6')-aph(2″) in 26% of the strains. At least 45% of the strains harbored high- and/or low-molecular weight plasmids, whose transfer may be facilitated by their widespread biofilm-forming capacity, and absence of restrictive CRISPR systems. We selected eight plasmid-bearing and multidrug resistant strains, which were submitted to plasmid curing by stress with SDS. No strain lost resistance during stressing cultivation but, by conjugation experiments, the S. pseudintermedius strain 27 transferred its plasmid-borne resistance to gentamicin, conferred by the aac(6')-aph(2″) gene, to Staphylococcus aureus. The frequent empirical use of gentamicin to treat skin and ear infections in domestic dogs is likely to select resistant strains. Also, as demonstrated by our study, these strains can serve as gene reservoirs for human pathogens, such as S. aureus.

Sampling Methods and Risk Stratification Regarding Environmental Contamination by SARS-CoV-2.

INTRODUCTION: Transmission of COVID-19 through close contact and droplets is well established, but the influence of aerosol and surface contamination remains to be determined. Literature is scarce and inconsistent about the viable virus particles free-distance from infected patients, as well as about different swabbing methods for surface contamination evaluation. The aim of this study was to evaluate the most sensitive method for the assessment of surface contamination, classify the likelihood of environmental contamination in risk zones and compare the environmental contamination between oxygenation and ventilatory support. MATERIAL AND METHODS: Swabs from potentially contaminated surfaces in a COVID-19 ward, with patients treated with different types of oxygen and ventilatory support, were collected. Three types of swabs were compared in order to evaluate the most sensitive collection method. For risk zone categorization, areas were divided according to the distance from the patient. RESULTS: Of the 63 swabs collected, 17 (27%) tested positive for the presence of SARS-CoV-2. The highest positivity rate was observed with the sterile premoistened swab with saline (n = 8; 38%), but without statistically significant differences. The highest number of positive samples were collected from the high-risk zones, specifically those located one meter from the patient (n = 13; 48%), with statistically significant differences. Only the rooms of patients supported with non-invasive ventilation or high-flow nasal cannula had evidence of bedroom contamination, with 45% and 27% of swab positivity, with statistically significant differences. DISCUSSION: Our findings favour the premoistened swab without transport medium for surface contamination assessment, even though without statistical differences. A statistically significant trend supporting the division in risk zones, according to the distance from the patient, was also identified. The higher positivity rate from the non-invasive ventilation and high-flow nasal cannula bedrooms suggests a significant association between ventilatory strategies and surface contamination, probably due to higher particle dispersion. CONCLUSION: Our findings support the use of the sterile premoistened swab without preservation medium, the classification of risk areas considering the distance from the patient, and the variability of RNA dispersion between oxygenation and ventilatory support.

Introdução: A transmissão da COVID-19 através do contacto e gotículas está bem estabelecida, mas a importância da sua transmissão através do aerossol e da contaminação das superfícies permanece por determinar. A literatura é escassa e inconsistente em relação à distância mínima livre de partículas víricas, desde um paciente, e também acerca dos mais adequados métodos de colheita de zaragatoas para avaliação da contaminação das superfícies. Os objectivos deste estudo foram avaliar qual o método mais sensível para avaliação da contaminação de superfícies, classificar a contaminação ambiental de acordo com zonas de risco e comparar a contaminação ambiental sob diferentes dispositivos para oxigenoterapia e suporte ventilatório. Material e Métodos: Realizamos colheitas de zaragatoas em superfícies potencialmente contaminadas numa ala COVID-19, onde se encontravam doentes sob diferentes dispositivos para oxigenoterapia e suporte ventilatório. Para avaliar o método de recolha mais sensível para verificação da contaminação das superfícies, comparámos três tipos de zaragatoas. Para a classificação das zonas de risco, dividimos as áreas de acordo com a distância ao doente. Resultados: Das 63 zaragatoas, 17 (27%) testaram positivo para SARS-CoV-2 (27%). A maior positividade foi observada na zaragatoa estéril pré-humedecida com soro fisiológico (n = 8; 38%), mas sem significância estatística. O maior número de amostras positivas obteve-se nas zonas de alto risco, especialmente aquelas a um metro do paciente (n = 13; 48%), com diferenças significativas. Apenas os quartos dos doentes sob ventilação não invasiva e cânula nasal de alto fluxo tiveram evidência de contaminação com 45% e 27% de positividade das zaragatoas, e significância estatística. Discussão: Os nossos resultados favorecem a zaragatoa estéril pré-humedecida sem meio de preservação para avaliação da contaminação das superfícies, embora sem significância estatística. Os resultados suportam também com significância estatística a divisão em zonas de risco de acordo com a distância ao doente. A maior positividade obtida nos quartos dos pacientes que se encontravam a utilizar ventilação não invasiva e cânula nasal de alto fluxo sugere uma associação, com significância, entre as estratégias ventilatória e a contaminação ambiental, provavelmente relacionada com uma maior dispersão das partículas. Conclusão: Os nossos resultados apoiam o uso da zaragatoa estéril pré-humedecida sem meio de preservação, a classificação das áreas de risco considerando a distância ao doente, e a variabilidade da dispersão do RNA entre diferentes dispositivos para oxigenoterapia e ventilação.

Staphylococcus nepalensis, a commensal of the oral microbiota of domestic cats, is a reservoir of transferrable antimicrobial resistance.

Staphylococcus nepalensis is a commensal bacterium from the oral microbiota of domestic cats, with a still obscure clinical importance. In this work, we analysed the ability of feline strains of S. nepalensis to transfer antimicrobial resistance genes to Staphylococcus aureus isolated from humans through plasmids. To this end, we first analysed all publicly available genomes from cat staphylococci using computational methods to build a pan-resistome. Genes that encode resistance to erythromycin, gentamicin, mupirocin and tetracycline, common to human and cat staphylococci and previously described to be located in mobile genetic elements, were chosen for the next analyses. We studied 15 strains of S. nepalensis, which were shown to be genetically different by GTG5-PCR. As observed by disc diffusion, resistance to tetracycline was widespread (80 %), followed by resistance to erythromycin (40 %), gentamicin (27 %) and mupirocin (7 %). The strains were positive for several antimicrobial resistance genes and more than half of them harboured plasmids. The loss of plasmids and resistance genes in some strains were induced by stress with SDS. Through conjugation experiments, we observed that these plasmids can be transferred to S. aureus, thus increasing its potential to resist drug therapy. Our findings show that S. nepalensis, an underestimated inhabitant of the cat microbiota, can be a reservoir of antimicrobial resistance genes for S. aureus and, like many other staphylococci, be an overlooked and silent threat to their animal hosts and humans living with them.

CRISPR tracking reveals global spreading of antimicrobial resistance genes by Staphylococcus of canine origin.

The close contact between pets and their owners is a potential source for microorganisms and genetic material exchange. Staphylococcus species considered as harmless inhabitants of animals' and humans' microbiota can act as reservoirs of antimicrobial resistance genes to more virulent species, thereby increasing their potential to resist drug therapy. This process could be inhibited by the antiplasmid immunity conferred by CRISPR systems. On the other hand, CRISPR spacer sequences can be explored as molecular clocks to track the history of genetic invasion suffered by a bacterial strain. To understand better the role of domestic dogs in human health as an antimicrobial resistance genes source, we analyzed 129 genomes of Staphylococcus strains of canine origin for the presence of CRISPR systems. Only 8% of the strains were positive for CRISPR, which is consistent with Staphylococcus role as gene reservoirs. The plasmidial origin or some spacers confirms the unsuccessful attempt of plasmid exchange in strains carrying CRISPRs. Some of these systems are within a staphylococcal cassette chromosome mec (SCCmec), sharing 98% of identity between their harboring strains. These CRISPRs' spacers reveal that this SCCmec was transferred between canine S. pseudintermedius strains, then to S. schleiferi and to Staphylococcus strains isolated from human beings. Our findings shows genetic evidence for the global spreading of pathogenic bacteria and the antimicrobial resistance genes carried by them and reinforce that, in the age of antimicrobial resistance, it is imperative that drug therapies consider the integrated nature of the relationship between pets and humans.