Correlates of postnatal human cytomegalovirus transmission in term babies in the first year.

Postnatal human cytomegalovirus (HCMV) infection in newborns is well characterized for preterm infants but less so for term infants. We sought to analyze the rates and routes of HCMV transmission in full-term infants during the first year of life. A cohort of 120 HCMV seropositive mothers and their 122 newborns were tested after delivery for HCMV-DNA shedding in different bodily fluids. Postnatal HCMV infection was defined as the detection of >2.5 × 102 HCMV-DNA copies/mL in infants' saliva swabs. Maternal neutralizing antibody serum titer, HCMV-specific T-cell response, and HCMV glycoprotein B immunoglobulin G on breastmilk were analyzed. HCMV shedding was detected in 67 of 120 mothers (55.8%), and 20 of 122 infants (16.4%) developed HCMV infection within the first 3 months of life. Six additional infants were infected during the first year, for a postnatal infection rate of 21.3%. Viral shedding was more frequent in breastmilk than saliva, urine, and vaginal secretions, and the mothers of infected infants showed higher levels of HCMV-DNA in milk. No association was found between the antibody levels in serum or milk and maternal viral shedding, whereas a slightly lower frequency of HCMV-specific CD4+ T-cells with long-term memory phenotype was observed in women with HCM-DNA-positive milk. About one out of five infants develop HCMV infection within the first year of life. Breastmilk appears the major route of transmission of the infection, maternal saliva has a minor role whereas the role of vaginal secretions is negligible.

Antifungal activity of Myriocin on clinically relevant Aspergillus fumigatus strains producing biofilm.

BACKGROUND: The human pathogenic mold Aspergillus fumigatus is able to form a complex biofilm embedded in extracellular matrix. Biofilms confer antimicrobial resistance and it is well known that aspergillosis is often refractory to the conventional antifungal therapy. The treatment of biofilm-related infections poses a significant clinical challenge on a daily basis, promoting the search for new therapeutic agents. Our aim was to exploit the modulation of sphingolipid mediators as new therapeutic target to overcome antifungal resistance in biofilm-related infections. RESULTS: Antifungal susceptibility testing was performed on 20 clinical isolates of Aspergillus fumigatus and one reference strain (A. fumigatus Af293) according the EUCAST protocol. Sessile MICs were assessed on 24-h preformed-biofilm by means of XTT-reduction assay. Myriocin (0.25-64 mg/L), a commercial sphingolipid synthesis inhibitor, was used. The MEC50 value (mg/L) of Myriocin was 8 (range 4-16) for both planktonic and sessile cells. Drug-induced morphological alterations were analyzed by optical and electron microscopy (TEM) on 24h preformed A. fumigatus Af293 biofilms. An evident hyphal damage, resulting in short, stubby, and highly branched hyphae was observed by optical microscopy. At 24h, TEM studies showed important morphological alterations, such as invaginations of the cell membrane, modification in the vacuolar system and presence of multilamellar bodies, in some cases within vacuoles. CONCLUSIONS: The direct antifungal activity, observed on both planktonic and sessile fungi, suggests that inhibition of sphingolipid synthesis could represent a new target to fight biofilm-related A. fumigatus resistance.

Evaluation of reduced susceptibility to quaternary ammonium compounds and bisbiguanides in clinical isolates and laboratory-generated mutants of Staphylococcus aureus.

The MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates of Staphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened for qac genes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence of qacA, qacB, qacC, and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride, while only qacA and qacB increased the chlorhexidine mode MIC. Isolates with a wild-type norA promoter or mutations in the norA promoter had similar biocide MIC distributions; notably, not all clinical isolates with norA mutations were resistant to fluoroquinolones. In vitro efflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct from this region of the clinical isolates. Still, none of the in vitro mutants displayed fitness defects in a killing assay in Galleria mellonella larvae. In conclusion, our data provide an in-depth comparative overview on efflux in S. aureus mutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitro tests appear not to be suitable for predicting levels of resistance that are clinically relevant.

Human cytomegalovirus non-primary infection during pregnancy: antibody response, risk factors and newborn outcome.

OBJECTIVES: Human cytomegalovirus (HCMV) non-primary infections can occur in pregnant women and may result in congenital infection. Comprehensive studies investigating the frequency, characteristics, risk factors and immune response of non-primary infection in pregnancy are missing, while the rate of vertical transmission is not known. METHODS: HCMV non-primary infection was investigated prospectively in 250 pregnant women. Blood and urine samples as well as saliva and vaginal swabs were collected at 13, 21 and 31 weeks of gestation and at delivery. HCMV-DNA and specific IgG and IgM levels were determined. RESULTS: Overall, 105/250 pregnant women (42.0%) developed non-primary infection. HCMV-DNA was detected more frequently in vaginal secretions (84/250 of the women, 33.6%) than in urine (35/250, 14.0%), saliva (26/250, 10.4%) and blood (7/250, 3.0%). The rate of HCMV non-primary infection increased significantly with the progression of pregnancy (from 12.9% in the first trimesters of gestation to 21.9% at delivery, p < 0.01). IgM was detected in 25/250 of the women (10.0%), with no association with non-primary infection, while anti-gB IgG was significantly higher (p < 0.01) in women with non-primary infection. Age and close contact with children were not associated with non-primary infection. No woman with non-primary infection transmitted the infection to the fetus (95% confidence interval of transmission rate: 0-3.5%). DISCUSSION: Although HCMV non-primary infection is frequent during pregnancy, the rate of congenital infection as a consequence of non-primary infection is likely to be ≤ 3.5%.

Detection of Genotype-Specific Antibody Responses to Glycoproteins B and H in Primary and Non-Primary Human Cytomegalovirus Infections by Peptide-Based ELISA.

BACKGROUND: Strain-specific antibodies to human cytomegalovirus (HCMV) glycoproteins B and H (gB and gH) have been proposed as a potential diagnostic tool for identifying reinfection. We investigated genotype-specific IgG antibody responses in parallel with defining the gB and gH genotypes of the infecting viral strains. METHODS: Subjects with primary (n = 20) or non-primary (n = 25) HCMV infection were studied. The seven gB (gB1-7) and two gH (gH1-2) genotypes were determined by real-time PCR and whole viral genome sequencing, and genotype-specific IgG antibodies were measured by a peptide-based enzyme-linked immunosorbent assay (ELISA). RESULTS: Among subjects with primary infection, 73% (n = 8) infected by gB1-HCMV and 63% (n = 5) infected by gB2/3-HCMV had genotype-specific IgG antibodies to gB (gB2 and gB3 are similar in the region tested). Peptides from the rarer gB4-gB7 genotypes had nonspecific antibody responses. All subjects infected by gH1-HCMV and 86% (n = 6) infected by gH2-HCMV developed genotype-specific responses. Among women with non-primary infection, gB and gH genotype-specific IgG antibodies were detected in 40% (n = 10) and 80% (n = 20) of subjects, respectively. CONCLUSIONS: Peptide-based ELISA is capable of detecting primary genotype-specific IgG responses to HCMV gB and gH, and could be adopted for identifying reinfections. However, about half of the subjects did not have genotype-specific IgG antibodies to gB.

Repurposing Pilocarpine Hydrochloride for Treatment of Candida albicans Infections.

Acetylcholine modulates the virulence of Candidaalbicans and regulates an appropriate immune response to infection in a Galleria mellonella infection model. Indeed, the evidence suggests that C. albicans possesses a functional cholinergic receptor that can regulate filamentous growth and biofilm formation. Furthermore, G. mellonella immune cell subsets possess repertories of cholinergic receptors which regulate an effective and appropriate cellular immune response to C. albicans infection. This study aimed to investigate the cholinergic receptor subtype involved in regulation of filamentous growth and biofilm formation by C. albicans and determine the roles of cholinergic receptors in modulation of G. mellonella immune cell subsets. The general muscarinic receptor agonist, pilocarpine hydrochloride, inhibited C. albicans biofilm formation and pathogenicity, a phenomenon that could be reversed using the general muscarinic receptor antagonist, scopolamine. Pilocarpine hydrochloride protected G. mellonella larvae from C. albicans infection via inhibition of C. albicans filamentation and appropriate regulation of cellular immunity. However, scopolamine abrogated the capacity of pilocarpine hydrochloride to protect G. mellonella larvae from C. albicans infection. Furthermore, acetylcholine and pilocarpine hydrochloride exhibited differential modulatory capabilities on Galleria mellonella hemocyte responses to C. albicans The data in this article demonstrate that a muscarinic receptor modulates C. albicans filamentation and biofilm formation. Furthermore, the results suggest that G. mellonella hemocyte subsets possess unique repertoires of cholinergic receptors that regulate their differentiation, activation, and function in contrasting manners. Therefore, targeting cholinergic receptors by repurposing currently licensed cholinergic drugs may offer novel therapeutic solutions for the prevention or treatment of fungal infections.IMPORTANCECandida albicans is the most common human fungal pathogen with an estimated crude mortality rate of 40%. The ability of the organism to switch from the yeast to hyphal form and produce biofilms are important virulence factors. C. albicans infections are combatted by the host immune system. However, Candida triggers a strong inflammatory response that, if not appropriately regulated, can damage host tissues. Therefore, it is important that the host immune response eliminates the fungus but limits tissue damage. This study provides evidence that targeting cholinergic receptors cannot only curb the virulence of C. albicans by inhibiting filamentous growth and biofilm formation but can also appropriately regulate the host immune response to induce rapid clearance with limited damage to vital tissues. This article provides evidence that repurposing licensed drugs that target cholinergic receptors may offer novel therapeutic solutions for the prevention or treatment of fungal infections.

Molecular Identification and Echinocandin Susceptibility of Candida parapsilosis Complex Bloodstream Isolates in Italy, 2007-2014.

The Candida parapsilosis group encompasses three species: C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Here, we describe the incidence and echinocandin susceptibility profile of bloodstream isolates of these three species collected from patients admitted to an Italian university hospital from 2007 to 2014. Molecular identification of cryptic species of the C. parapsilosis complex was performed using polymerase chain reaction amplification of the gene encoding secondary alcohol dehydrogenase, followed by digestion with the restriction enzyme BanI. Minimum inhibitory concentrations were determined using the broth microdilution method according to European Committee for Antimicrobial Susceptibility Testing (EUCAST EDef 7.2) and Clinical Laboratory Standards Institute (CLSI M27-A3) guidelines, and the results were compared with those obtained using the E-test and Sensititre methods. Of the 163 C. parapsilosis complex isolates, 136 (83.4%) were identified as C. parapsilosis, and 27 (16.6%) as C. orthopsilosis. The species-specific incidences were 2.9/10,000 admissions for C. parapsilosis and 0.6/10,000 admissions for C. orthopsilosis. No resistance to echinocandins was detected with any of the methods. The percent essential agreement (EA) between the EUCAST and E-test/Sensititre methods for anidulafungin, caspofungin, and micafungin susceptibility was, respectively, as follows: C. parapsilosis, 95.6/97.8, 98.5/88.2, and 93.4/96.3; C. orthopsilosis, 92.6/92.6, 96.3/77.8, and 63.0/66.7. The EA between the CLSI and E-test/Sensititre methods was, respectively, as follows: C. parapsilosis, 99.3/100, 98.5/89.0, and 96.3/98.5; C. orthopsilosis, 96.3/92.6, 100/81.5, and 92.6/88.9. Only minor discrepancies, ranging from 16.9% (C. parapsilosis) to 11.1% (C. orthopsilosis), were observed between the CLSI and E-test/Sensititre methods. In conclusion, this epidemiologic study shows a typical C. parapsilosis complex species distribution, no echinocandin resistance, and it reinforces the relevance of using commercially available microbiological methods to assess antifungal susceptibility. These data improve our knowledge of the national distribution of species of the psilosis group, as there are very few studies of these species in Italy.

Methicillin-Resistant Staphylococcus aureus in Raw Milk: Prevalence, SCCmec Typing, Enterotoxin Characterization, and Antimicrobial Resistance Patterns.

Staphylococcus aureus is a known major cause of foodborne illnesses, and raw milk and dairy products are often contaminated by enterotoxigenic and antimicrobial-resistant S. aureus strains. In the present study, 35 S. aureus strains were isolated from 383 raw milk samples collected from various dairy herds in the province of Milan (northern Italy). The isolates were characterized based on their antimicrobial susceptibility patterns and the presence of genes encoding staphylococcal enterotoxins (sea, seb, sec, sed, and see). About half (45.7%) of the strains were enterotoxigenic, and 37.1% were resistant to at least one of the antimicrobial drugs tested. Seven (20%) of 35 isolates were identified as methicillin-resistant S. aureus (MRSA), and SCCmec typing performed with a multiplex PCR assay revealed the presence of gene cassettes IV and V, typical of community-acquired MRSA, and I and II, characteristic of health care-associated MRSA. The MRSA strains were evaluated for the presence of the Panton-Valentine leukocidin gene, but this gene was not found. The results of the present study revealed the presence of toxin-producing S. aureus and MRSA strains in raw milk. MRSA and enterotoxigenic S. aureus in dairy farms are an important risk factor for the spread of staphylococcal infections; therefore, further studies are needed to find strategies for monitoring and controlling the presence of S. aureus, especially MRSA, in dairy products.

Antifungal resistance does not necessarily affect Candida glabrata fitness.

Although there has been an overall good coverage of Candida glabrata infections by the echinocandins, emergence of antifungal resistance during therapy has been reported. We investigated, by using an invertebrate host model, the fitness of sequential C. glabrata isolates with different echinocandins susceptibility patterns. The studied strains were isolated from a case of recurrent fungemia with a fatal outcome due to C. glabrata that developed cross-resistance to echinocandins during caspofungin therapy. The sequential strains isolated post-therapy showed a S663P mutation in the Fks2p hot spot 1. In vivo study in the invertebrate host Galleria mellonella did not suggest a fitness cost related to the acquired antifungal resistance, the three isolates displayed a similar rate of killing (P  =  0.54). We observed a clear correlation between emergence of antifungal resistance and persistence of the causal agent, probably aided by the unchanged fitness and unresponsiveness in vivo to the adopted therapy.

Correlation between Candida albicans biofilm formation and invasion of the invertebrate host Galleria mellonella.

AIM: The aim of our study was to investigate whether biofilm production by Candida albicans clinical isolates could be a hallmark of virulence in vivo. MATERIALS & METHODS: Twenty clinical isolates of C. albicans were examined via histological studies on larvae infected with various fungal doses (from 10(3) to 10(5) CFU/larva) of biofilm producer and nonproducer strains. RESULTS: The poor prognostic role of infection due to a biofilm-producing isolate was confirmed by the Wald test (hazard ratio: 2.63; 95% CI: 2.03-3.41). Histological examinations at 24 h showed a strong innate immune response, with evidence of melanization for both infection groups. However, at 48 h, we found huge differences in filamentation and tissue invasion capability between biofilm nonproducing and producing isolates, the latter being highly organized into biofilm and invading the larval intestinal tract. Invasion corroborated survival data. CONCLUSION: The histological results demonstrate that the production of biofilm could enhance the invasiveness of C. albicans.

Experimental biofilm-related Candida infections.

AIM: We investigated the pathogenic role of biofilm and the therapeutic efficacy of anidulafungin in experimental infections of the wax moth Galleria mellonella by Candida albicans clinical strains. MATERIALS & METHODS: On the basis of the in vitro propensity to form biofilm, five biofilm-producer (BP) and four nonproducer (NP) C. albicans clinical strains were used in this study. For each strain, we assessed the virulence by infecting G. mellonella larvae and observing survival. Anidulafungin was administered 2 h after yeast inoculum at 0.6 µg, according to the therapeutic dose recommended for humans. RESULTS: Biofilm-forming ability highly influenced the larva-killing rate. A significant (p < 0.0001) survival decrease was observed in the BP group, with 80% of the infected larvae dying within 72 h. NP isolates did not reach the same killing rate, even at the end of experiments (216 h). Larval survival was enhanced (p < 0.0001) by anidulafungin administration in both groups. Survival rate at 72 h was similar in both groups (BP 78.5% and NP 87.5%); whereas there were still differences at the end of the experiments, with a higher survival in the NP group (75 vs 48%). CONCLUSION: Our data confirm the pathogenic role of biofilm in C. albicans infections. Its importance was further enhanced by a lack of contribution from extracellular enzymes, detected in both NP and BP strains. In addition, we demonstrated anidulafungin efficacy in treating biofilm-related invasive candidiasis.