The role of coagulase-negative Staphylococci biofilms on late-onset sepsis: current challenges and emerging diagnostics and therapies

Infections are one of the most significant complications of neonates, especially those born preterm, with sepsis as one of the principal causes of mortality. Coagulase-negative staphylococci (CoNS), a group of staphylococcal species that naturally inhabit healthy human skin and mucosa, are the most common cause of late-onset sepsis, especially in preterms. One of the risk factors for the development of CoNS infections is the presence of implanted biomedical devices, which are frequently used for medications and/or nutrient delivery, as they serve as a scaffold for biofilm formation. The major concerns related to CoNS infections have to do with the increasing resistance to multiple antibiotics observed among this bacterial group and biofilm cells’ increased tolerance to antibiotics. As such, the treatment of CoNS biofilm-associated infections with antibiotics is increasingly challenging and considering that antibiotics remain the primary form of treatment, this issue will likely persist in upcoming years. For that reason, the development of innovative and efficient therapeutic measures is of utmost importance. This narrative review assesses the current challenges and emerging diagnostic tools and therapies for the treatment of CoNS biofilm-associated infections, with a special focus on late-onset sepsis.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the project with the reference EXPL/BIA-MIC/0032/2021.info:eu-repo/semantics/publishedVersio

High variability of gene expression in S. epidermidis biofilm population

In the past two decades S. epidermidis has emerged from a commensal microorganism into a predominant opportunistic pathogen associated with nosocomial infections due to the ability to adhere to abiotic surfaces and form biofilms. The increasing use of indwelling medical devices has influenced the rise of S. epidermidis to a major medical research topic. S. epidermidis biofilms are well known to be resistant to both the host immune response and antimicrobial therapy, making these infections hard to treat and often resulting in recurrent infections. To better understand why biofilms have evolved in this manner, many comparative studies have been performed using biofilms and planktonic cultures. However, since biofilm cultures are fundamentally different from planktonic cultures, some concerns have been raised in the past for such studies. While it seems reasonable to compare biofilm cultures with stationary planktonic cultures, recently it has been suggested that biofilms cultures could be compared directly to the bacteria in suspension, grown in the vicinity of the biofilms. Nevertheless and however interesting, this suggestion fails to accommodate the fact that mature biofilms will release bacteria from within the biofilm to the suspension. This phenomenon was suggested to be responsible for colonization of further niches. Therefore, such population can both contain biofilm outbound bacteria as well as plantkonic free floating bacteria. In an attempt to better understand the possible differences between cell populations, we selected 5 distinct bacterial isolates previously characterized for biofilm formation and compared the expression of some genes of interest, namely atlE (involved in initial adhesion) and icaA (involved immune evasion and biofilm maturation). Three populations were characterized: (1) late exponential planktonic cultures grown on Erlenmeyer flasks, (2) biofilm populations attached to polystyrene 24-well culture plates and (3) the bacteria grown in suspension on the same well of culture plates as the biofilms (non-adherent cells), all grown in TSB supplemented with 0,4% glucose. Differences in the gene expression profile were observed between S. epidermidis strains. The icaA expression values were generally higher in biofilms as compared with planktonic cultures. However, when comparing with the non-adherent cells grown in the vicinity of the biofilms, some strain to strain variation was observed, as in some cases the non-adherent cells has lower icaA expression but in other instances the opposite occurred. A similar effect occurred with atlE expression. A possible explanation for the higher variation on the non-adherent cells has to do with the washing step required before resuspending the biofilm: while more tenacious biofilm forming strains will withstand better the washing step, some weaker biofilm forming strains will be washed away. In the latter cases the bacterial population described as non-adherent cells will be very heterogeneous. Thus, with the variation found in the non-adherent bacteria, it seems that in order to study the physiological differences that occur when bacteria are living in a biofilm, planktonic cultures grown independently of biofilms should be used to better understand the pathophysiology of the biofilm-related infections

Characterization of the molecular interactions between Staphylococcus epidermidis biofilm infections and the host immune system

Tese de doutoramento em "Biomedical Engineering"Staphylococcus epidermidis ranks first among the causative agents of nosocomial infections associated with indwelling medical devices. This association is due to the microorganism’s ability to colonize the surface of these devices and form biofilms. The biofilm lifecycle is divided into initial adhesion, accumulation and maturation, and biofilm disassembly. The major clinical complications of biofilm formation is their high resistance to antimicrobials and to the host immune system, resulting in the development of chronic infections. To uncover the mechanisms by which biofilms evade the host immune system and cause chronic infections, a transcriptomic analysis of S. epidermidis biofilms exposed to human blood was performed. Our results revealed extensive changes in the transcriptome, suggesting that a quick adaptation to the new environment was made. Genes involved in amino acids biosynthesis and iron utilization were strongly affected, indicating that these mechanisms are important factors in S. epidermidis biofilm survival in human blood. The biofilm disassembly stage has been associated with the development of acute infections, however, despite its importance in the clinical setting, it is the less studied of the biofilm lifecycle stages. Hence, to comprehend the interactions between biofilm disassembly and the host immune system, biofilm-released cells were characterized with reference to several virulence parameters. Our results revealed that S. epidermidis biofilm-released cells are unique in their phenotype and virulence potential, sharing some features with planktonic cells, but simultaneously displaying features similar to biofilm cells. The phenotypic differences were also manifested as differences in the S. epidermidis transcriptome in response to immune cells. Thus, targeting the particular properties of biofilm-released cells could be important to prevent the serious acute infections associated with biofilm dissemination. As a preventive measure, the ability of a monoclonal antibody raised against PNAG to inhibit S. epidermidis biofilm accumulation was tested. Interestingly, it was observed that depending on the strain, the antibody present variable effect resulting, in some cases, in the enhancement of biofilm accumulation in vitro. In conclusion, the work described throughout this thesis has given an important contribution to the knowledge of biofilmsrelated infections, what will open new opportunities to effectively prevent the pathologic events associated with these serious and prevalent infections.A espécie Staphylococcus epidermidis é atualmente considerada uma das principais causas do desenvolvimento de infeções nosocomiais, com particular associação a pacientes com dispositivos médicos invasivos. Esta associação é devida à capacidade desta bactéria aderir e formar biofilmes na superfície desses dispositivos. A formação do biofilme é classicamente dividida em adesão inicial, acumulação e maturação e, finalmente, a libertação de células do biofilme para o meio envolvente, num processo designado por dispersão. As grandes implicações clinicas da formação de biofilmes são a sua elevada tolerância aos agentes antimicrobianos e à resposta do sistema imunitário do hospedeiro, o que leva ao desenvolvimento de infeções crónicas. De forma a desvendar quais os mecanismos usados pela bactéria para escapar à resposta do sistema imunitário e causar infeções crónicas, foi caracterizado o transcriptoma de biofilmes de S. epidermidis cultivados na presença de sangue humano. Os resultados revelaram que a presença de sangue humano estimula uma extensa e rápida remodelação do transcriptome da bactéria, provavelmente para promover a adaptação ao novo e complexo ambiente envolvente. Os genes envolvidos na síntese de aminoácidos e na utilização de ferro sofreram as alterações mais pronunciadas, sugerindo que estes dois mecanismos são importantes fatores na sobrevivência da bactéria no sangue humano. Outra das etapas com grandes implicações clinicas é a dispersão de células do biofilme para o meio envolvente, uma vez que está diretamente associada ao desenvolvimento de infeções agudas importantes no hospedeiro. No entanto, esta é a etapa do ciclo de vida dos biofilmes de S. epidermidis menos bem compreendida. Assim, de forma a explorar as interações que ocorrem entre o sistema imunitário do hospedeiro e as células libertadas dos biofilmes de S. epidermidis, estas foram caracterizadas em diversos e importantes parâmetros envolvidos na sua virulência. Os nossos resultados revelaram que as células libertadas pelos biofilmes apresentam um fenótipo único, exibindo características particulares das células planctónicas, bem como das células derivadas do biofilme resultando também num estímulo particular do sistema imunitário do hospedeiro. Assim, estes resultados demonstram que um estudo mais aprofundado destas células poderá revelar novas oportunidades no desenvolvimento de estratégias preventivas contra as consequências patológicas associados à dispersão de células do biofilme. Numa perspetiva mais preventiva, a capacidade de um anticorpo monoclonal, específico para a PNAG, de inibir a acumulação dos biofilmes foi testada num sistema in vitro. Curiosamente, dependendo da estirpe testada, o anticorpo apresentou eficácia variável resultando, em alguns casos particulares, no aumento da capacidade da estirpe de formar biofilme. O trabalho descrito nesta tese contribuiu, significativamente, para aumentar o conhecimento sobre as infeções-associadas à formação de biofilmes e a interação destes com o sistema imunitário do hospedeiro. Este conhecimento irá, seguramente, abrir novas oportunidades para prevenir os eventos patológicos associados com estas infeções, por vezes severas, altamente prevalentes na sociedade

Can commercial horse or sheep blood replace fresh human blood in an ex vivo model to study S. epidermidis virulence?

[Excerpt] Staphylococcus epidermidis, a commensal bacterium of healthy human skin and mucosae, can cause serious bloodstream infections such as bacteremia and sepsis. These infections are very hard to cure with current antimicrobial strategies and, thus, it is urgent to find new treatment options. To do so, the study of S. epidermidis virulence factors is of utmost importance. Therefore, the ex vivo human blood model has gained special interest because it enables the study of S. epidermidis behavior in the context of a bloodstream infection. However, this model presents limitations, mainly related to the availability of donors, complicating its implementation in the academic context. To overcome this limitation, the possibility of replacing fresh human blood by commercial blood from other mammals was evaluated. [...]This study was supported by FCT through the funded project PTDC/BIA-MOL/29553/2017, under the scope of COMPETE2020 (POCI-01-0145-FEDER-029553) and by the strategic funding of unit UID/BIO/04469/2019.info:eu-repo/semantics/publishedVersio

Transcriptome mining to identify molecular markers for the diagnosis of Staphylococcus epidermidis bloodstream infections

Bloodstream infections caused by Staphylococcus epidermidis are often misdiagnosed since no diagnostic marker found so far can unequivocally discriminate true infection from sample contamination. While attempts have been made to find genomic and/or phenotypic differences between invasive and commensal isolates, possible changes in the transcriptome of these isolates under in vivo-mimicking conditions have not been investigated. Herein, we characterized the transcriptome, by RNA sequencing, of three clinical and three commensal isolates after 2 h of exposure to whole human blood. Bioinformatics analysis was used to rank the genes with the highest potential to distinguish invasive from commensal isolates and among the ten genes identified as candidates, the gene SERP2441 showed the highest potential. A collection of 56 clinical and commensal isolates was then used to validate, by quantitative PCR, the discriminative power of the selected genes. A significant variation was observed among isolates, and the discriminative power of the selected genes was lost, undermining their potential use as markers. Nevertheless, future studies should include an RNA sequencing characterization of a larger collection of isolates, as well as a wider range of conditions to increase the chances of finding further candidate markers for the diagnosis of bloodstream infections caused by S. epidermidis.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the unit [UIDB/04469/2020]. S.B. was supported by a fellowship granted by a Doctoral Advanced Training [NORTE-69-2015-15] funded by the European Social Fund under the scope of NORTE2020.info:eu-repo/semantics/publishedVersio

Induced resistance to vancomycin impairs biofilm formation by S. epidermidis

Coagulase-negative staphylococci (CoNS), particularly Staphylococcus epidermidis, are one of the leading causes of sepsis in preterm neonates mostly due to their capacity to form biofilms on medical devices. A major concern regarding CoNS infections is associated with the increased antibiotic resistance that has been observed among this bacterial group. Hence, the treatment of these infections is becoming progressively challenging, which may lead to increased morbidity and mortality in preterm neonates. In that sense, we aim to explore the consequences of antibiotic resistance in biofilm formation by S. epidermidis. First, the susceptibility profile of S. epidermidis strains collected from Portuguese newborns with bloodstream infections was characterized. To induce resistance to vancomycin, the susceptible strains were exposed to increasing concentrations of the antibiotic and, after 36 passages, the MIC was re-evaluated and the capacity of vancomycin-induced resistance strains to form biofilms was assessed (Colony forming units (CFUs) and OD620nm readings). Overall, the strains with induced resistance to vancomycin formed biofilms with less biomass and CFUs than the isogenic strains. The acquisition of resistance to vancomycin in clinical strains of S. epidermidis seems to cause a decrease in the capacity of these strains to form biofilms. As such, in S. epidermidis, the resistance to vancomycin does not seem to be related to a stronger biofilm formation capacity.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the project EXPL/BIA-MIC/0032/2021.info:eu-repo/semantics/publishedVersio

Editorial: Women in biofilms vol. II

Editorial on the Research Topic Women in biofilms vol. II(undefined)info:eu-repo/semantics/publishedVersio

S. epidermidis response to human blood and its cellular and soluble components

Staphylococcus epidermidis, a normal inhabitant of healthy human skin and mucosae, can cause persistent and relapsing infections due to its ability to adhere to medical devices and form biofilms. Hence, S epidermidis is considered one of the most important medical device-associated nosocomial agents, being particularly associated with vascular catheters. Although the biofilms formed on these catheters are in constant contact with human blood, their mutual interaction is poorly understood. Here, we evaluated the expression of genes associated with biofilm formation (icaA, aap, bhp), immune evasion (icaA, mprF, sepA) and programmed cell death (lrgB), as well as biofilm structure and viability, upon bacterial interaction with human blood and its components. We observed that contact with human blood increased the transcription of icaA and bhp but decreased aap, sepA and lrgB gene expression, when compared with plasma. In contrast, no significant transcriptional alterations were detected upon contact with purified mononuclear cells, whereas purified polymorphonuclear cells lead to increased bhp and mprF gene expression. Furthermore, human blood reduced by 50% the number of viable cells within the biofilm and induced significant alterations in its structure, with the creation of a fibre-like matrix. In conclusion, our study reveals that S. epidermis biofilms adapt to particular environmental stress by changing the expression of specific genes and by altering their structure. Despite these overall observations, significant variability was found between different blood donors, suggesting that particularities of the host immune system may strongly affect the outcome of S. epidermidis infections

Developing an affordable but reliable human blood ex vivo model to analyse gene expression by Staphylococcus epidermidis

Microbiotec'17 - Congress of Microbiology and Biotechnology 2017Background: Staphylococcus epidermidis is a commensal inhabitant of healthy human skin and mucosae. However, when external barriers, such as the skin, are damaged, the bacterium gains accesses into the bloodstream and emerges as an opportunistic pathogen. S. epidermidis can originate important infections such as medical device-associated bloodstream infections. This is mainly due to its ability to attach and form biofilms on the surface of vascular catheters. Hence, due to the clinical relevance of S. epidermidis medical device-associated bloodstream infections, human blood is frequently used as an ex vivo model, to mimic the environment encountered by the bacterium and study its behavior. An important limitation in the use of human blood is the availability of donors and the considerable quantity of blood necessary. As any other biological resource, the use of blood shall be reduced to a minimum and, thus, our goal was to test the influence of different volumes of human blood on the stability of S. epidermidis gene expression and on bacterial culturability.This work was supported by national funds (FCT) by the strategic project of UID/BIO/04469/2013 and by BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020. SB had an individual fellowship (UMINHO/BD/15/2016) supported by the Doctoral Program in Chemical and Biological Engineering (NORTE-08-5369-FSE-000052 Operation) by NORTE 2020 Program and co-funded by European Social Fund.NC is an Investigador FCT.info:eu-repo/semantics/publishedVersio