Increased Proteolytic Activity of Serratia marcescens Clinical Isolate HU1848 Is Associated with Higher eepR Expression.

Serratia marcescens is a global opportunistic pathogen. In vitro cytotoxicity of this bacterium is mainly related to metalloprotease serralysin (PrtS) activity. Proteolytic capability varies among the different isolates. Here, we characterized protease production and transcriptional regulators at 37°C of two S. marcescens isolates from bronchial expectorations, HU1848 and SmUNAM836. As a reference strain the insect pathogen S. marcescens Db10 was included. Zymography of supernatant cultures revealed a single (SmUNAM836) or double proteolytic zones (HU1848 and Db10). Mass spectrometry confirmed the identity of PrtS and the serralysin-like protease SlpB from supernatant samples. Elevated proteolytic activity and prtS expression were evidenced in the HU1848 strain through azocasein degradation and qRT-PCR, respectively. Evaluation of transcriptional regulators revealed higher eepR expression in HU1848, whereas cpxR and hexS transcriptional levels were similar between studied strains. Higher eepR expression in HU1848 was further confirmed through an in vivo transcriptional assay. Moreover, two putative CpxR binding motifs were identified within the eepR regulatory region. EMSA validated the interaction of CpxR with both motifs. The evaluation of eepR transcription in a cpxR deletion strain indicated that CpxR negatively regulates eepR. Sequence conservation suggests that regulation of eepR by CpxR is common along S. marcescens species. Overall, our data incorporates CpxR to the complex regulatory mechanisms governing eepR expression and associates the increased proteolytic activity of the HU1848 strain with higher eepR transcription. Based on the global impact of EepR in secondary metabolites production, our work contributes to understanding virulence factors variances across S. marcescens isolates.

Candida albicans causes brain regional invasion and necrosis, and activation of microglia during lethal neonatal neurocandidiasis.

Neurocandidiasis is a fungal infection that primarily affects neonates, which is associated with 70% case fatality rates, while pediatric patients who survive infection often have long-term neurological sequelae, making it a clinical requirement to understand the pathogenesis of neonatal neurocandidiasis. Currently, the brain regions to Candida albicans invasion during the neonatal period are not characterized. In this study, 0-day-old mice were infected with C. albicans intravenously to determine dissemination and invasion into the brain at different times post-infection by fungal burden assay and histopathological analysis, additionally cellular death and microglial activation were evaluated by flow cytometry. The results evidenced the dissemination of C. albicans within the first hour of infection in the brain. The meninges were the initial site of invasion during the first 6 hours post infection and then filamentous structures into the brain parenchyma increases during infection, the anatomic regions most susceptible to invasion being the cerebral cortex, thalamus, hypothalamus, midbrain, pons, and medulla oblongata. Furthermore, C. albicans invasion of brain tissue results in cell necrosis and activation of microglia as a consequence of fungal invasion.

Serratia marcescens antibiotic resistance mechanisms of an opportunistic pathogen: a literature review.

Serratia marcescens is a ubiquitous bacterium from order Enterobacterales displaying a high genetic plasticity that allows it to adapt and persist in multiple niches including soil, water, plants, and nosocomial environments. Recently, S. marcescens has gained attention as an emerging pathogen worldwide, provoking infections and outbreaks in debilitated individuals, particularly newborns and patients in intensive care units. S. marcescens isolates recovered from clinical settings are frequently described as multidrug resistant. High levels of antibiotic resistance across Serratia species are a consequence of the combined activity of intrinsic, acquired, and adaptive resistance elements. In this review, we will discuss recent advances in the understanding of mechanisms guiding resistance in this opportunistic pathogen.

Evaluation of the synergistic antifungal activity of micafungin and voriconazole plus sertraline against Candida auris.

Candida auris is an emerging global public health threat. It is an opportunistic yeast that usually affects critically ill patients in healthcare settings and is characterized by reduced susceptibility to multiple antifungal classes. Combination therapy with antifungals and repurposed drugs is a feasible alternative to overcome this problem. The aim of this study was to examine the in vitro interactions and potential synergy of micafungin (MFG) and voriconazole (VRC) plus the antidepressant sertraline (SRT) against clinical isolates of C. auris. Conventional antifungal testing was first performed with the three drugs according to the CLSI methodology. Drug interactions were determined by the checkerboard microdilution assay using the fractional inhibitory concentration (FIC) index. Synergistic interactions were noted with the combination of MFG and SRT plus VRC with FIC values of 0.37 to 0.49 for some strains. Indifferent interactions were observed when MFG was combined with SRT with just one exception (FIC 0.53). No antagonism was observed for any combination. The combination of VRC with MCF or SRT may be relevant for treating C. auris infections.

A Participatory Sensing Study to Understand the Problems Older Adults Faced in Developing Medication-Taking Habits.

Past research has demonstrated that older adults tend to use daily activities as cues to remember to take medications. However, they may still experience medication non-adherence because they did not select adequate contextual cues or face situations that interfere with their medication routines. This work addresses two research questions: (1) How does the association that older adults establish between their daily routines and their medication taking enable them to perform it consistently? (2) What problems do they face in associating daily routines with medication taking? For 30 days, using a mixed-methods approach, we collected quantitative and qualitative data from four participants aged 70-73 years old about their medication taking. We confirm that older adults who matched their medication regimens to their habitual routines obtained better results on time-based consistency measures. The main constraints for using daily routines as contextual cues were the insertion of medication taking into broad daily routines, the association of multiple daily routines with medication taking, the lack of strict daily routines, and the disruption of daily routines. We argue that the strategies proposed by the literature for forming medication-taking habits should support their formulation by measuring patients' dosage patterns and generating logs of their daily activities.

Dissemination of Gram-positive bacteria to the lung of newborn mice increases local IL-6 and TNFα levels in lethal bacteremia.

Neonatal bacteremia remains the major cause of infectious diseases-related death, especially in preterm newborns. Gram-positive bacteria are the main causative agent of neonatal bacteremia and exhibit a high risk of causing pneumonia and/or meningitis. The pathogenesis of bacteremia in preterm newborn is poorly understood. Current neonatal models of bacterial infection have been used to study the disease mechanisms; however, these studies employed mice of several days of age that could be less comparable to the bacteremia in preterm infants. In this study, we infected intravenously 0-day-old BALB/c mice with different inocula of Staphylococcus aureus, Streptococcus agalactiae or Enterococcus faecalis. We found that the mortality of the newborn mice was inoculum-dependent and also bacterial species-dependent. We observed bacterial burden in the lung, liver, brain, kidney and spleen of the infected animals. The lung was the tissue with the greatest bacterial burden and cellular infiltration in animals infected with the three bacteria evaluated. We found increased production of IL-6 and TNFα in the lung from newborn mice at 3 days post-infection. This neonatal model shows bacterial dissemination to the lung and will be useful for promote a better understanding of the pathophysiology of neonatal pneumonia.

Dissemination of Candida auris to deep organs in neonatal murine invasive candidiasis.

Candida auris is an emerging multidrug resistant fungal pathogen, which represents a major challenge for newborns systemic infections worldwide. Management of C. auris infections is complicated due to its intrinsic antifungal resistance and the limited information available on its pathogenesis, particularly during neonatal period. In this study, we developed a murine model of C. auris neonatal invasive infection. C. auris dissemination was evaluated by fungal burden and histopathological analysis of lung, brain, liver, kidney, and spleen at different time intervals. We found fungal cells in all the analyzed tissues, neonatal liver and brain were the most susceptible tissues to fungal invasion. This model will help to better understand pathogenesis mechanisms and facilitate strategies for control and prevention of C. auris infections in newborns.

Stimulating the Involvement of Family Members in the Medication Management Activities of Older Adults Through Ambient Displays: Qualitative Study.

Little attention has been paid to how medication management technologies, designed for older adults, modify the participation of family caregivers. We developed a tablet-based ambient display that provides external cues to remind and motivate older adults to take their medications. This study aimed to understand the effect of ambient displays on the involvement of family members in the elderly's medication management. We conducted a 10-week study consisting of interviews administered weekly to nine elderly-caregivers. We identify that new involvement patterns of the family caregivers were provoked through external cues, which made them aware of older adults' medication adherence and encouraged younger relatives to help older adults.

CesL Regulates Type III Secretion Substrate Specificity of the Enteropathogenic E. coli Injectisome.

The type III secretion system (T3SS) is a complex molecular device used by several pathogenic bacteria to translocate effector proteins directly into eukaryotic host cells. One remarkable feature of the T3SS is its ability to secrete different categories of proteins in a hierarchical manner, to ensure proper assembly and timely delivery of effectors into target cells. In enteropathogenic Escherichia coli, the substrate specificity switch from translocator to effector secretion is regulated by a gatekeeper complex composed of SepL, SepD, and CesL proteins. Here, we report a characterization of the CesL protein using biochemical and genetic approaches. We investigated discrepancies in the phenotype among different cesL deletion mutants and showed that CesL is indeed essential for translocator secretion and to prevent premature effector secretion. We also demonstrated that CesL engages in pairwise interactions with both SepL and SepD. Furthermore, while association of SepL to the membrane does not depended on CesL, the absence of any of the proteins forming the heterotrimeric complex compromised the intracellular stability of each component. In addition, we found that CesL interacts with the cytoplasmic domains of the export gate components EscU and EscV. We propose a mechanism for substrate secretion regulation governed by the SepL/SepD/CesL complex.

Species distribution and antifungal susceptibility patterns of Aspergillus isolates from clinical specimens and soil samples in Mexico.

This study aimed to assess the species distribution and antifungal susceptibility patterns of 200 strains of Aspergillus isolated from clinical specimens (n = 146) and soil samples (n = 54) in Mexico. ITS, ß-tubulin, and calmodulin DNA sequencing was performed for species identification. Broth microdilution susceptibility testing for amphotericin B, voriconazole, posaconazole, itraconazole, isavuconazole, anidulafungin, caspofungin, and micafungin was done according to CLSI for all strains. A. fumigatus was most frequently recovered from clinical specimens, while A. niger was commonly encountered in soil, both followed by A. flavus in the second place. A total of 60 (30%) cryptic species were identified, with A. tubingensis and A. tamarii being the most commonly found. The decreased susceptibility to amphotericin B and azoles was 32% for both, and were mainly led by A. fumigatus, whereas this percentage decreased to 9% for caspofungin, particularly in A. terreus. More than 75% of cryptic species were susceptible in vitro to all antifungals. Multi-azole decreased susceptibility was detected only in seven isolates. Given that antifungal resistance in Aspergillus spp. is an increasing worldwide threat that causes major challenges in the clinical management of aspergillosis, these data highlight the need for continuous epidemiological surveillance of these pathogens for the implementation of locally adequate treatment strategies. LAY SUMMARY: This is an epidemiological study in Mexico. A. fumigatus was most frequent in clinical specimens and A. niger in soil samples. A. tubingensis and A. tamarii were the most common cryptic species. Resistance to amphotericin B and azoles was 32% each, and 9% for caspofungin.

Specialization of the Reiterated Copies of the Heterodimeric Integration Host Factor Genes in Geobacter sulfurreducens.

Integration host factor (IHF) is a widely distributed small heterodimeric protein member of the bacterial Nucleoid-Associated Proteins (NAPs), implicated in multiple DNA regulatory processes. IHF recognizes a specific DNA sequence and induces a large bend of the nucleic acid. IHF function has been mainly linked with the regulation of RpoN-dependent promoters, where IHF commonly recognizes a DNA sequence between the enhancer-binding region and the promoter, facilitating a close contact between the upstream bound activator and the promoter bound, RNA polymerase. In most proteobacteria, the genes encoding IHF subunits (ihfA and ihfB) are found in a single copy. However, in some Deltaproteobacteria, like Geobacter sulfurreducens, those genes are duplicated. To date, the functionality of IHF reiterated encoding genes is unknown. In this work, we achieved the functional characterization of the ihfA-1, ihfA-2, ihfB-1, and ihfB-2 from G. sulfurreducens. Unlike the ΔihfA-2 or ΔihfB-1 strains, single gene deletion in ihfA-1 or ihfB-2, provokes an impairment in fumarate and Fe(III) citrate reduction. Accordingly, sqRT-PCR experiments showed that ihfA-1 and ihfB-2 were expressed at higher levels than ihfA-2 and ihfB-1. In addition, RNA-Seq analysis of the ΔihfA-1 and ΔihfB-2 strains revealed a total of 89 and 122 differentially expressed genes, respectively. Furthermore, transcriptional changes in 25 genes were shared in both mutant strains. Among these genes, we confirmed the upregulation of the pilA-repressor, GSU1771, and downregulation of the triheme-cytochrome (pgcA) and the aconitate hydratase (acnA) genes by RT-qPCR. EMSA experiments also demonstrated the direct binding of IHF to the upstream promoter regions of GSU1771, pgcA and acnA. PilA changes in ΔihfA-1 and ΔihfB-2 strains were also verified by immunoblotting. Additionally, heme-staining of subcellular fractions in ΔihfA-1 and ΔihfB-2 strains revealed a remarkable deficit of c-type cytochromes. Overall, our data indicate that at least during fumarate and Fe(III) citrate reduction, the functional IHF regulator is likely assembled by the products of ihfA-1 and ihfB-2. Also, a role of IHF controlling expression of multiple genes (other than RpoN-dependent) affects G. sulfurreducens physiology and extracellular electron transfer.

Defining chaperone-usher fimbriae repertoire in Serratia marcescens.

Chaperone-usher (CU) fimbriae are surface organelles particularly prevalent among the Enterobacteriaceae. Mainly associated to their adhesive properties, CU fimbriae play key roles in biofilm formation and host cell interactions. Little is known about the fimbriome composition of the opportunistic human pathogen Serratia marcescens. Here, by using a search based on consensus fimbrial usher protein (FUP) sequences, we identified 421 FUPs across 39 S. marcescens genomes. Further analysis of the FUP-containing loci allowed us to classify them into 20 conserved CU operons, 6 of which form the S. marcescens core CU fimbriome. A new systematic nomenclature is proposed according to FUP sequence phylogeny. We also established an in vivo transcriptional assay comparing CU promoter expression between an environmental and a clinical isolate of S. marcescens, which revealed that promoters from 3 core CU operons (referred as fgov, fpo, and fps) are predominantly expressed in the two strains and might represent key core adhesion appendages contributing to S. marcescens pathogenesis.

Comparative Analysis of Virulence Profiles of Serratia marcescens Isolated from Diverse Clinical Origins in Mexican Patients.

Background: Serratia marcescens is an enteric bacterium with increasing incidence in clinical settings, attributed mainly to the opportune expression of diverse virulence determinants plus a wide intrinsic and acquired antibiotic resistance. Methods: The aim of this study was to compare the virulence factor profiles of 185 Serratia marcescens isolates from different clinical origins. In vitro proteolytic and hemolytic activities, biofilm formation, and motility were assessed in each strain. Additionally, the pathogenicity of four hypervirulent strains was analyzed in vivo in Galleria mellonella. Results: We found that bacterial isolates from wound/abscess and respiratory tract specimens exhibited the highest protease activity along with a strong biofilm production, while uropathogenic isolates showed the highest hemolytic activity. Swarming and swimming motilities were similar among all the strains. However, respiratory tract isolates showed the most efficient motility. Two hyperhemolytic and two hyperproteolytic strains were detected; the latter were more efficient killing Galleria mellonella with a 50%-60% larval mortality 48 hours after challenge. Conclusion: A correlation was found between biofilm formation and proteolytic and hemolytic activities in biopsy specimens and bloodstream isolates, respectively. Overall, it becomes critical to evaluate and compare the clinical strains virulence diversity in order to understand the underlying mechanisms that allow the establishment and persistence of opportunistic bacterial infections in the host.

Surveillance of antimicrobial resistance in Serratia marcescens in Mexico.

Antimicrobial resistance is a global public health threat. Therefore, surveillance studies are important tools to help direct antimicrobial use. The aim of this study was to investigate antimicrobial resistance in Serratia marcescens isolates collected in 2016-2017 at eight medical centers from two regions of Mexico. Selected S. marcescens isolates were further tested by polymerase chain reaction to detect the presence of genes encoding the ß-lactamases, SHV, TEM or CTX. Antimicrobial resistance continues to be high in Mexico, particularly to ciprofloxacin and aminoglycosides. Also, a widespread prevalence of blaTEM was detected in S. marcescens isolates.

Supporting the Medication Adherence of Older Mexican Adults Through External Cues Provided With Ambient Displays: Feasibility Randomized Controlled Trial.

BACKGROUND: Problems with prospective memory, which refers to the ability to remember future intentions, cause deficits in basic and instrumental activities of daily living, such as taking medications. Older adults show minimal deficits when they rely on mostly preserved and relatively automatic associative retrieval processes. On the basis of this, we propose to provide external cues to support the automatic retrieval of an intended action, that is, to take medicines. To reach this end, we developed the Medication Ambient Display (MAD), a system that unobtrusively presents relevant information (unless it requires the users' attention) and uses different abstract modalities to provide external cues that enable older adults to easily take their medications on time and be aware of their medication adherence. OBJECTIVE: This study aimed to assess the adoption and effect of external cues provided through ambient displays on medication adherence in older adults. METHODS: A total of 16 older adults, who took at least three medications and had mild cognitive impairment, participated in the study. We conducted a 12-week feasibility study in which we used a mixed methods approach to collect qualitative and quantitative evidence. The study included baseline, intervention, and postintervention phases. Half of the participants were randomly allocated to the treatment group (n=8), and the other half was assigned to the control group (n=8). During the study phases, research assistants measured medication adherence weekly through the pill counting technique. RESULTS: The treatment group improved their adherence behavior from 80.9% at baseline to 95.97% using the MAD in the intervention phase. This decreased to 76.71% in the postintervention phase when the MAD was no longer being used. Using a one-way repeated measures analysis of variance and a post hoc analysis using the Tukey honestly significant difference test, we identified a significant statistical difference between the preintervention and intervention phases (P=.02) and between the intervention and postintervention phases (P=.002). In addition, the medication adherence rate of the treatment group (95.97%) was greater than that of the control group (88.18%) during the intervention phase. Our qualitative results showed that the most useful cues were the auditory reminders, followed by the stylized representations of medication adherence. We also found that the MAD's external cues not only improved older adults' medication adherence but also mediated family caregivers' involvement. CONCLUSIONS: The findings of this study demonstrate that using ambient modalities for implementing external cues is useful for drawing the attention of older adults to remind them to take medications and to provide immediate awareness on adherence behavior. TRIAL REGISTRATION: ClinicalTrials.gov NCT04289246; https://tinyurl.com/ufjcz97.

The BPtpA protein from Burkholderia cenocepacia belongs to a new subclass of low molecular weight protein tyrosine phosphatases.

Low molecular weight protein tyrosine phosphatases (LMW-PTP) are ubiquitous enzymes found across a spectrum of genera from prokaryotes to higher eukaryotes. LMW-PTP belong to the Cys-based PTP class II protein family. Here, we show that LMW-PTP can be categorized into two different groups, referred as class II subdivision I (class II.I) and subdivision II (class II.II). Using BPtpA from the opportunistic pathogen Burkholderia cenocepacia, as a representative member of the LMW-PTP class II.I, we demonstrated that four conserved residues (W47, H48, D80, and F81) are required for enzyme function. Guided by an in silico model of BPtpA, we show that the conserved residues at α3-helix (D80 and F81) contribute to protein stability, while the other conserved residues in the W-loop (W47 and H48) likely play a role in substrate recognition. Overall, our results provide new information on LMW-PTP protein family and establish B. cenocepacia as a suitable model to investigate how substrates are recognized and sorted by these proteins.

Evaluation of the expanding spectrum of sertraline against uncommon fungal pathogens.

The emergence of non-Aspergillus mold pathogens has increased notoriously in the last decades with serious health consequences. The options of treatment for these microorganisms often resistant to a wide variety of antifungals is limited. Sertraline is an antidepressant with in vitro and in vivo antifungal properties which has been recently studied as an adjuvant in the treatment of invasive infections. In this study, we evaluated the in vitro interaction of sertraline with voriconazole and amphotericin B against Lomentospora prolificans, Scedosporium spp., Fusarium spp., Paecilomyces spp., Alternaria spp. and Curvularia spp. The minimum inhibitory concentration and minimum fungicidal concentration for sertraline were in the range of 8-32 µg/mL. Sertraline showed antifungal capacity against all fungi tested and synergism in combination with amphotericin B against some strains of Lomentospora prolificans, Scedosporium apiospermum and Alternaria alternata, antagonism with voriconazole against Purpureocillium lilacinum and indifference in both combinations for most of the other strains tested. These results suggest a potential role of sertraline as an adjuvant in the treatment of some of these serious mycoses.

First report of Candida bracarensis in Mexico: hydrolytic enzymes and antifungal susceptibility pattern.

Candida bracarensis is an emerging cryptic species within the Candida glabrata clade. To date, little is known about its epidemiology, virulence, and antifungal susceptibility. This study documents the occurrence of C. bracarensis for the first time in Mexico and focuses on its in vitro production of hydrolytic enzymes, as well as antifungal susceptibility to echinocandins. This strain was isolated from a vaginal swab of a female with vulvovaginal candidosis; exhibited a very strong activity of aspartyl proteinase, phospholipase, and hemolysin; and was susceptible to caspofungin, anidulafungin, and micafungin (MIC = 0.031 µg/mL). Data obtained could contribute to the knowledge of the epidemiology and virulence attributes of this yeast as a fungal opportunistic human pathogen.

The unphosphorylated form of the PilR two-component system regulates pilA gene expression in Geobacter sulfurreducens.

In Geobacter sulfurreducens, metal reduction and generation of bioelectricity require the participation of several elements, and among them, the type IV pili has an essential role. The pilus is composed of multiple PilA monomers. Expression of pilA gene depends mainly on the σ54 factor and the response regulator protein PilR. In this work, we characterized the role of the PilS-PilR two-component system in the regulation of the pilA gene expression. Experimental evidence indicates that PilS is autophosphorylated at the His-334 residue, which in turn is transferred to the conserved Asp-53 in PilR. Contrary to other PilS-PilR systems, substitution D53N in PilR resulted in higher activation of the pilA gene. By using a pilA::luxCDABE fusion with different promoter fragments and in vitro DNA-binding assays, we demonstrated the existence of multiple functional PilR binding sites. A regulatory model in which the non-phosphorylated PilR protein directs activation of pilA expression by binding to two sites in the promoter region of this gene is presented.