Population Analysis of O26 Shiga Toxin-Producing Escherichia coli Causing Hemolytic Uremic Syndrome in Italy, 1989-2020, Through Whole Genome Sequencing.

Shiga toxin-producing Escherichia coli (STEC) belonging to the O26 serogroup represent an important cause of Hemolitic Uremic Syndrome (HUS) in children worldwide. The localization of STEC virulence genes on mobile genetic elements allowed the emergence of clones showing different assets of this accessory genomic fraction. A novel O26 STEC clone belonging to Sequence Type (ST) 29 and harboring stx2a, ehxA and etpD plasmid-borne genes has emerged and spread in Europe since the mid-1990s, while another ST29 clone positive for stx2d and lacking plasmid-borne virulence genes was recently described as emerging in France. In Italy, O26 has been the most frequently detected STEC serogroup from HUS cases since the late 1990s. In this study we describe the genomic characterization and population structure of 144 O26 STEC strains isolated from human sources in Italy in the period 1989-2020. A total of 89 strains belonged to ST21, 52 to ST29, two to ST396 and one to ST4944. ST29 strains started to be isolated from 1999. 24 strains were shown to harbour stx1a, alone (n=20) or in combination with stx2a (n=4). The majority of the strains (n=118) harbored stx2a genes only and the two ST396 strains harbored stx2d. A Hierarchical Clustering on Principal Components (HCPC) analysis, based on the detection of accessory virulence genes, antimicrobial resistance (AMR) genes and plasmid replicons, classified the strains in seven clusters identified with numbers from 1 to 7, containing two, 13, 39, 63, 16, 10 and one strain, respectively. The majority of the genetic features defining the clusters corresponded to plasmid-borne virulence genes, AMR genes and plasmid replicons, highlighting specific assets of plasmid-borne features associated with different clusters. Core genome Multi Locus Sequence Typing grouped ST21 and ST29 strains in three clades each, with each ST29 clade exactly corresponding to one HCPC cluster. Our results showed high conservation of either the core or the accessory genomic fraction in populations of ST29 O26 STEC, differently from what observed in ST21 strains, suggesting that a different selective pressure could drive the evolution of different populations of these pathogens possibly involving different ecological niches.

Tracing Back the Evolutionary Route of Enteroinvasive Escherichia coli (EIEC) and Shigella Through the Example of the Highly Pathogenic O96:H19 EIEC Clone.

Enteroinvasive Escherichia coli (EIEC) cause intestinal illness through the same pathogenic mechanism used by Shigella spp. The latter species can be typed through genomic and phenotypic methods used for E. coli and have been proposed for reclassification within E. coli species. Recently the first appearance of a highly pathogenic EIEC O96:H19 was described in Europe as the causative agent of two large outbreaks that occurred in Italy and in the United Kingdom. In contrast to Shigella spp and to the majority of EIEC strains, EIEC O96:H19 fermented lactose, lacked pathoadaptive mutations, and showed good fitness in extracellular environment, similarly to non-pathogenic E. coli, suggesting they have emerged following acquisition of the invasion plasmid by a non-pathogenic E. coli. Here we describe the whole genome comparison of two EIEC O96:H19 strains isolated from severe cases of diarrhea in Uruguay in 2014 with the sequences of EIEC O96:H19 available in the public domain. The phylogenetic comparison grouped all the O96:H19 strains in a single cluster, while reference EIEC strains branched into different clades with Shigella strains occupying apical positions. The comparison of the virulence plasmids showed the presence of a complete conjugation region in at least one O96:H19 EIEC. Reverse Transcriptase Real Time PCR experiments confirmed in this strain the expression of the pilin-encoding gene and conjugation experiments suggested its ability to mobilize an accessory plasmid in a recipient strain. Noteworthy, the tra region was comprised between two reversely oriented IS600 elements, which were also found as remnants in another EIEC O96:H19 plasmid lacking the tra locus. We hypothesize that an IS-mediated recombination mechanism may have caused the loss of the conjugation region commonly observed in EIEC and Shigella virulence plasmids. The results of this study support the hypothesis of EIEC originating from non-pathogenic E. coli through the acquisition of the virulence plasmid via conjugation. Remarkably, this study showed the ability of a circulating EIEC strain to mobilize plasmids through conjugation, suggesting a mechanism for the emergence of novel EIEC clones.

Detection and isolation of Shiga Toxin-producing Escherichia coli (STEC) strains in caecal samples from pigs at slaughter in Italy.

Shiga toxin-producing Escherichia coli (STEC) strains are food-borne pathogens of public health concern. Despite ruminants are the most important reservoir, STEC human infections have also been attributed to pigs. We examined for the presence of STEC in 234 samples of swine caecal content collected during the year 2015 at Italian abattoirs in the framework of the harmonized monitoring of antimicrobial resistance (Decision 2013/652/EU). The presence of stx genes was detected in 122 (52.1%) samples, which were subsequently subjected to STEC isolation and characterization. The analysis of the 66 isolated STEC strains showed that the majority of the isolates (74.2%) possessed the stx2a gene subtype, in a few cases (16.7%) in combination with stx2b or stx2c. Only 25.8% of isolates possessed the stx2e subtype, typical of swine-adapted STEC. None of the isolates possessed the intimin-coding eae gene and the majority of them did not belong to serogroups commonly associated with human infections. The results of this study suggest that pigs can be considered as potential reservoir of certain STEC types.

Characterization of a novel plasmid encoding F4-like fimbriae present in a Shiga-toxin producing enterotoxigenic Escherichia coli isolated during the investigation on a case of hemolytic-uremic syndrome.

In February 2017 a case of Hemolytic-Uremic Syndrome (HUS) was reported to the National Registry of HUS in an adult living in Northern Italy. Stool specimens from the patient and his family contacts were collected and the analyses led to the isolation of a Locus of Enterocyte Effacement (LEE)-negative Shiga toxin 2 (Stx2)-producing Escherichia coli. The epidemiological investigations performed brought to collect fecal samples from the animals reared in a farm held by the case's family and a mixture of bovine and swine feces proved positive for Shiga toxin-producing E. coli (STEC) and yielded the isolation of a LEE-negative stx2-positive E. coli strain. Further characterization by whole genome sequencing led to identify the isolates as two identical O2:H27 hybrid Enterotoxigenic Shiga toxin-producing E. coli (ETEC-STEC). Sequencing of a high molecular weight plasmid present in the human isolate disclosed a peculiar plasmid harboring virulence genes characteristic for both pathotypes, including the enterohemolysin-coding gene and sta1, encoding the heat stable enterotoxin. Moreover, a complete fae locus encoding the ETEC F4 fimbriae could be identified, including a novel variant of faeG gene responsible for the production of the main structural subunit of the fimbriae. This novel faeG showed great diversity in the nucleotidic sequence when compared with the reference genes encoding the swine F4 allelic variants, whereas at the amino acid sequence level the predicted protein sequence showed some similarity with FaeG from E. coli strains of bovine origin. Further investigation on the plasmid region harboring the newly identified faeG allelic variant allowed to identify similar plasmids in NCBI sequence database, as part of the genome of other previously uncharacterized ETEC-STEC strains of bovine origin, suggesting that the novel F4-like fimbriae may play a role in bovine host specificity.

Use of SCW4 gene primers in PCR methods for the identification of six medically important Aspergillus species.

Aspergillus species are the cause of invasive mold infections in immunocompromised patients: Aspergillus fumigatus, A. flavus and A. terreus account for most cases of invasive aspergillosis (IA). As certain species are associated with higher mortality and vary in their resistance to antifungal therapy, diagnosis requires increasingly rapid molecular methods that enable sensitive detection and species discrimination. We have developed PCR and Multiplex PCR assays for the detection of six medically important Aspergillus spp. species DNA in bronchoalveolar lavage (BAL) specimens from hematology and intensive care unit (ICU) patients at risk of IA, using different species and genus-specific PCR primers, selected within the SCW4 gene, encoding a cell wall glucanase of A. fumigatus, similar to mannoprotein Mp65 of Candida albicans. The genus-specific PCR primers were able to amplify only Aspergillus DNAs but not that belonging to other fungal genera tested. The species-specific PCR primers allowed differentiation of each Aspergillus species by the amplicon length produced. The methods described in this study are rapid (less than 4 h), reproducible, simple and specific and demonstrate potential application in the clinical laboratory.

Studies of Immune Responses in Candida vaginitis.

The widespread occurrence of vaginal candidiasis and the development of resistance against anti-fungal agents has stimulated interest in understanding the pathogenesis of this disease. The aim of our work was to characterize, in an animal model of vaginal candidiasis, the mechanisms that play a role in the induction of mucosal immunity against C. albicans and the interaction between innate and adaptive immunity. Our studies evidenced the elicitation of cell-mediated immunity (CMIs) and antibody (Abs)-mediated immunity with a Th1 protective immunity. An immune response of this magnitude in the vagina was very encouraging to identify the proper targets for new strategies for vaccination or immunotherapy of vaginal candidiasis. Overall, our data provide clear evidence that it is possible to prevent C. albicans vaginal infection by active intravaginal immunization with aspartyl proteinase expressed as recombinant protein. This opens the way to a modality for anti-Candida protection at the mucosa. The recombinant protein Sap2 was assembled with virosomes, and a vaccine PEVION7 (PEV7) was obtained. The results have given evidence that the vaccine, constituted of virosomes and Secretory aspartyl proteinase 2 (Sap2) (PEV7), has an encouraging therapeutic potential for the treatment of recurrent vulvovaginal candidiasis.

Evaluation of efficacy, pharmacokinetics and tolerability of peptidomimetic aspartic proteinase inhibitors as cream formulation in experimental vaginal candidiasis.

OBJECTIVES: It has been previously shown that the treatment with the two protease inhibitors APG12 and APG19 confers protection in a rat model of mucosal candidiasis; in this study, we examined whether these peptidomimetic inhibitors are also effective as a cream formulation in reducing Candida albicans vaginal infection. METHODS: These efficacy studies were performed in a rat model of estrogen-dependent rat vaginitis by C. albicans on both azole-susceptible and azole-resistant C. albicans, and on both caspofungin-susceptible and caspofungin-resistant C. albicans strains. In vivo studies were also conducted in female albino rats and rabbits to obtain information about the safety, local tolerability and principal pharmacokinetics parameters of the two compounds. KEY FINDINGS AND CONCLUSIONS: Both hit compounds showed remarkable results within the 48-h range as effective inhibitors of the infection, particularly causing rapid decay of vaginal C. albicans burden. Importantly, the two compounds showed marked acceleration of fungus clearance in the rats challenged with the fluconazole-resistant as well as with the capsofungin-resistant strain of C. albicans. Both compounds showed fast elimination rates when given by the intravenous route, and poor systemic absorption after intravaginal cream administration. Test drugs were also well tolerated in 7-day local tolerability experiments in the rabbit.

Bicyclic peptidomimetics targeting secreted aspartic protease 2 (SAP2) from Candida albicans reveal a constrained inhibitory chemotype.

The in vitro screening of stereoisomeric bicyclic peptidomimetics towards SAP2 of Candida albicans revealed a constrained chemotype as aspartic protease inhibitor in the micromolar to nanomolar range. The results indicated that the acetal bridge may serve as a transition-state isostere, and that the right match between interactions with subsites and the orientation by hydrogen bonding with Gly85 is the main requisite for inhibitory activity. Molecular docking calculations suggested the bicyclic acetal scaffold to be capable of interacting with the two catalytic aspartic acids, thus resulting in good inhibitory activity with only two hydrophobic groups addressing the enzyme catalytic subsites.

A virosomal vaccine against candidal vaginitis: immunogenicity, efficacy and safety profile in animal models.

A novel vaccine (PEV7) consisting of a truncated, recombinant aspartyl proteinase-2 of Candida albicans incorporated into influenza virosomes was studied. This vaccine candidate generated a potent serum antibody response in mouse and rat following intramuscular immunization. Anti-Sap2 IgG and IgA were also detected in the vaginal fluid of rats following intravaginal or intramuscular plus intravaginal administration. In a rat model of candidal vaginitis, PEV7 induced significant, long-lasting, likely antibody-mediated, protection following intravaginal route of immunization. PEV7 was also found to be safe in a repeated-dose toxicological study in rats. Overall, these data provide a sound basis to envisage the clinical development of this new candidate vaccine against candidal vaginitis.

Peptides of the constant region of antibodies display fungicidal activity.

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.

The MP65 gene is required for cell wall integrity, adherence to epithelial cells and biofilm formation in Candida albicans.

BACKGROUND: The MP65 gene of Candida albicans (orf19.1779) encodes a putative ß-glucanase mannoprotein of 65 kDa, which plays a main role in a host-fungus relationship, morphogenesis and pathogenicity. In this study, we performed an extensive analysis of a mp65Δ mutant to assess the role of this protein in cell wall integrity, adherence to epithelial cells and biofilm formation. RESULTS: The mp65Δ mutant showed a high sensitivity to a range of cell wall-perturbing and degrading agents, especially Congo red, which induced morphological changes such as swelling, clumping and formation of hyphae. The mp65Δ mutant showed an activation of two MAPKs (Mkc1p and Cek1p), a high level of expression of two stress-related genes (DDR48 and SOD5), and a modulated expression of ß-glucan epitopes, but no gross changes in cell wall polysaccharide composition. Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type. All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion. CONCLUSIONS: We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus.

Rapid, simple, and low-cost identification of Candida species using high-resolution melting analysis.

The feasibility of using high-resolution melting analysis (HRMA) was examined for its rapid and simple detection of 5 clinically relevant Candida species (C. albicans, C. glabrata, C. kefyr, C. parapsilosis, and C. guilliermondii). HRMA was able to differentiate clinical Candida species and resulted in being sensitive, reproducible, and inexpensive.

Outcome of experimental rat vaginitis by Candida albicans isolates with different karyotypes.

Candida albicans isolates with different genomic background, designed as b and c karyotypes, have been previously shown to differentially modulate their response to macrophage candidacidal activity. While b-type isolates were susceptible to intracellular killing, strains with c karyotype survived upon internalization and were able to replicate inside macrophages. Furthermore, it was also shown that c type strains escape microglial cell mediated growth inhibition, suggesting that these strains form a more virulent cluster. In this report, the pathogenicity exerted by C. albicans isolates with b and c karyotypes was analyzed in vivo using a model of experimental rat vaginitis. Although both types induced infection, c-type-infected animals suffered from more persistent vaginitis, confirming the higher virulence potential the c karyotype exerted in vivo. The analysis of fungal cells recovered from vaginal fluids of infected animals indicated that c-type was more prone to undergo morphogenesis and to express SAP2 than b-type; these different traits may account for the differences observed in the outcome of experimental rodent vaginitis induced by the two C. albicans karyotypes.

Protection against rat vaginal candidiasis by adoptive transfer of vaginal B lymphocytes.

Vulvovaginal candidiasis is a mucosal infection affecting many women, but the immune mechanisms operating against Candida albicans at the mucosal level remain unknown. A rat model was employed to further characterize the contribution of B and T cells to anti-Candida vaginal protection. Particularly, the protective role of vaginal B cells was studied by means of adoptive transfer of vaginal CD3(-) CD5(+) IgM(+) cells from Candida-immunized rats to naïve animals. This passive transfer of B cells resulted into a number of vaginal C. albicans CFU approximately 50% lower than their controls. Sorted CD3(-) CD5(+) IgM(+) vaginal B lymphocytes from Candida-infected rats proliferated in response to stimulation with an immunodominant mannoprotein (MP) antigen of the fungus. Importantly, anti-MP antibodies and antibody-secreting B cells were detected in the supernatant and cell cultures, respectively, of vaginal B lymphocytes from infected rats incubated in vitro with vaginal T cells and stimulated with MP. No such specific antibodies were found when using vaginal B cells from uninfected rats. Furthermore, inflammatory and anti-inflammatory cytokines, such as interleukin-2 (IL-2), IL-6 and IL-10, were found in the supernatant of vaginal B cells from infected rats. These data are evidence of a partial anti-Candida protective role of CD3(-) CD5(+) IgM(+) vaginal B lymphocytes in our experimental model.

Identification of inhibitors of drug-resistant Candida albicans strains from a library of bicyclic peptidomimetic compounds.

The screening of a library of small molecule peptidomimetics toward secreted aspartic proteinase-2 (SAP2) of Candida albicans allowed us to identify two compounds that showed in vitro inhibitory potency comparable to pepstatin A. In an experimental model of vaginal candidiasis, the two candidate compounds were as active as a therapeutic dose of fluconazole. Importantly, this activity was fully preserved when the challenger was a fluconazole-resistant strain of the fungus. Altogether, our data demonstrate SAP2 as a valid C. albicans target for the development of new drugs against this important human pathogen.

Phenotypic and functional characterization of vaginal dendritic cells in a rat model of Candida albicans vaginitis.

This study analyzes the phenotype of vaginal dendritic cells (VDCs), their antigenic presentation and activation of T-cell cytokine secretion, and their protective role in a rat model of Candida vaginitis. Histological observation demonstrated a significant accumulation of OX62(+) VDCs in the mucosal epithelium of Candida albicans-infected rats at the third round of infection. We identified two subsets of OX62(+) VDCs differing in the expression of CD4 molecule in both noninfected and Candida-infected rats. The OX62(+) CD4(+) subset of VDCs displayed a lymphoid cell-like morphology and expressed the T-cell antigen CD5, whereas the OX62(+) CD4(-) VDC subset exhibited a myeloid morphology and was CD5 negative. Candida infection resulted in VDC maturation with enhanced expression of CD80 and CD134L on both CD4(+) and CD4(-) VDC subsets at 2 and 6 weeks after Candida infection. CD5(-) CD4(-) CD86(-) CD80(-) CD134L(+) VDCs from infected, but not noninfected, rats spontaneously released large amounts of interleukin-12 (IL-12) and tumor necrosis factor alpha, whereas all VDC subsets released comparable levels of IL-10 and IL-2 cytokines. Furthermore, OX62(+) VDCs from infected rats primed naïve CD4(+) T-cell proliferation and release of cytokines, including gamma interferon, IL-2, IL-6, and IL-10, in response to staphylococcal enterotoxin B stimulation in vitro. Adoptive transfer of highly purified OX62(+) VDCs from infected rats induced a significant acceleration of fungal clearance compared with that in rats receiving naive VDCs, suggesting a protective role of VDCs in the anti-Candida mucosal immunity. Finally, VDC-mediated protection was associated with their ability to rapidly migrate to the vaginal mucosa and lymph nodes, as assessed by adoptive transfer of OX62(+) VDCs labeled with 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester.

Use of 65 kDa mannoprotein gene primers in Real Time PCR identification of Candida albicans in biological samples.

A method for the detection and quantification of Candida albicans in biological samples (blood, urine and serum) was developed with the use of Real-Time PCR utilizing CaMP65-specific primers. Two different systems were used for the detection in the LightCycler platform (Roche): the SYBR green fluorescent dye with melting peak analysis and the 5'nuclease fluorescent-probe detection. The amplification was highly specific for C. albicans, providing no cross-reaction on genomic DNA extracted from other Candida species or Aspergillus. The sensitivity in simulated biological samples was especially high (1 genome) when applied to sera and urine, and in blood samples the limit of detection was higher by ten-fold. Finally, the real-time PCR was employed in order to detect and quantify C. albicans in the sera from patients with invasive candidiasis.

Anti-retroviral therapy with protease inhibitors decreases virulence enzyme expression in vivo by Candida albicans without selection of avirulent fungus strains or decreasing their anti-mycotic susceptibility.

Highly active anti-retroviral therapies (HAART) with human immunodeficiency virus (HIV) protease inhibitors (PIs) or nonnucleoside reverse-transcriptase inhibitors (NNRTI) were compared for their effect on prevalence, aspartyl proteinase (Sap) production and the biotypes and anti-mycotic sequential susceptibility of Candida spp. isolates from the oral cavity in a longitudinal prospective study. HAART-PI, but not HAART-NNRTI strongly inhibited Sap expression in the oral cavity without exerting any consistent effect on the role of Candida spp. isolation or selection of low virulence or anti-mycotic resistant fungus biotype. More importantly, the sequential isolates of Candida albicans from HAART-PI, but not those from suspended HAART-NNRTI, showed an increased Sap production in vitro. While further demonstrating that HIV-PI inhibit Sap expressions, our results do not support the view that the mentioned inhibition could eliminate Candida or its selection of the oral cavity.