Antimicrobial properties of Lactobacillus cell-free supernatants against multidrug-resistant urogenital pathogens.

The healthy vaginal microbiota is dominated by Lactobacillus spp., which provide an important critical line of defense against pathogens, as well as giving beneficial effects to the host. We characterized L. gasseri 1A-TV, L. fermentum 18A-TV, and L. crispatus 35A-TV, from the vaginal microbiota of healthy premenopausal women, for their potential probiotic activities. The antimicrobial effects of the 3 strains and their combination against clinical urogenital bacteria were evaluated together with the activities of their metabolites produced by cell-free supernatants (CFSs). Their beneficial properties in terms of ability to interfere with vaginal pathogens (co-aggregation, adhesion to HeLa cells, biofilm formation) and antimicrobial activity mediated by CFSs were assessed against multidrug urogenital pathogens (S. agalactiae, E. coli, KPC-producing K. pneumoniae, S. aureus, E. faecium VRE, E. faecalis, P. aeruginosa, P. mirabilis, P. vulgaris, C. albicans, C. glabrata). The Lactobacilli tested exhibited an extraordinary ability to interfere and co-aggregate with urogenital pathogens, except for Candida spp., as well as to adhere to HeLa cells and to produce biofilm in the Lactobacillus combination. Lactobacillus CFSs and their combination revealed a strong bactericidal effect on the multidrug resistant indicator strains tested, except for E. faecium and E. faecalis. The antimicrobial activity was maintained after heat treatment but decreased after enzymatic treatment. All Lactobacilli showed lactic dehydrogenase activity and production of D- and L-lactic acid isomers on Lactobacillus CFSs, while only 1A-TV and 35A-TV released hydrogen peroxide and carried helveticin J and acidocin A bacteriocins. These results suggest that they can be employed as a new vaginal probiotic formulation and bio-therapeutic preparation against urogenital infections. Further, in vivo studies are needed to evaluate human health benefits in clinical situations.

Potential Associations Among Alteration of Salivary miRNAs, Saliva Microbiome Structure, and Cognitive Impairments in Autistic Children.

Recent evidence has demonstrated that salivary molecules, as well as bacterial populations, can be perturbed by several pathological conditions, including neuro-psychiatric diseases. This relationship between brain functionality and saliva composition could be exploited to unveil new pathological mechanisms of elusive diseases, such as Autistic Spectrum Disorder (ASD). We performed a combined approach of miRNA expression profiling by NanoString technology, followed by validation experiments in qPCR, and 16S rRNA microbiome analysis on saliva from 53 ASD and 27 neurologically unaffected control (NUC) children. MiR-29a-3p and miR-141-3p were upregulated, while miR-16-5p, let-7b-5p, and miR-451a were downregulated in ASD compared to NUCs. Microbiome analysis on the same subjects revealed that Rothia, Filifactor, Actinobacillus, Weeksellaceae, Ralstonia, Pasteurellaceae, and Aggregatibacter increased their abundance in ASD patients, while Tannerella, Moryella and TM7-3 decreased. Variations of both miRNAs and microbes were statistically associated to different neuropsychological scores related to anomalies in social interaction and communication. Among miRNA/bacteria associations, the most relevant was the negative correlation between salivary miR-141-3p expression and Tannerella abundance. MiRNA and microbiome dysregulations found in the saliva of ASD children are potentially associated with cognitive impairments of the subjects. Furthermore, a potential cross-talking between circulating miRNAs and resident bacteria could occur in saliva of ASD.

Detection of methicillin-resistant Staphylococcus aureus persistence in osteoblasts using imaging flow cytometry.

Methicillin-resistant S. aureus has been reported as the main pathogen involved in chronic infections, osteomyelitis, and prosthetic joint infections. The host/pathogen interaction is dynamic and requires several changes to promote bacterial survival. Here, we focused on the internalization and persistence behavior of well-characterized Staphylococcus aureus invasive strains belonging to the main ST-MRSA-SCCmec clones. To overcome the limitations of the cell culture method, we comparatively analyzed the ability of internalization within human MG-63 osteoblasts with imaging flow cytometry (IFC). After evaluation by cell culture assay, the MRSA clones in the study were all able to readily internalize at 3h postinfection, the persistence of intracellular bacteria was evaluated at 24h both by routine cell culture and IFC assay, after vancomycin-BODIPY staining. A statistical difference of persistence was found in ST5-SCCmecII (26.59%), ST228-SCCmecI (20.25%), ST8-SCCmecIV (19.52%), ST239-SCCmecIII (47.82%), and ST22-SCCmecIVh (50.55%) showing the same ability to internalize as ATCC12598 (51%), the invasive isolate used as control strain for invasion and persistence assays. We demonstrated that the intracellular persistence process depends on the total number of infected cells. Comparing our data obtained by IFC with those of the cell culture assay, we obtained greater reproducibility rates and a number of intracellular bacteria, with the advantage of analyzing live host cells. Moreover, with some limitations related to the lack of whole-genome sequencing analysis, we validated the different proclivities to persist in the main Italian HA-MRSA invasive isolates and our results highlighted the heterogeneity of the different clones to persist during cell infection.

Genotypic analysis of Italian MRSA strains exhibiting low-level ceftaroline and ceftobiprole resistance.

The aim of this study was to address the involvement of PBP mutations in the bactericidal activity to novel cephalosporins, alone and in combination with daptomycin, in not-related multidrug-resistant methicillin-resistant Staphylococcus aureus strains isolated during a nationwide Italian survey. MICs determination and time-killing assays were performed and mecA, pbp1, pbp2, pbp3, pbp4, and gdpP genes were sequenced. Ten strains showed low-level resistance to ceftaroline and ceftobiprole. PBP2a sequence analysis identified four different mutations (N146K; N204K; T235I; E239K) uniquely present in the non-penicillin-binding domain (nPBD). Epidemiologically, this resistance was associated with the most widespread MDR Italian clone ST228-SCCmecI-t001/t041, confirming its proclivity to accumulate mutations, and it is also associated to substitutions in the GdpP signaling protein, involved in the maintenance of di-AMP balance, recently associated with resistance to beta-lactams. Despite these mutations, both drugs retained their potent in vitro bactericidal activity and showed a synergistic effect towards difficult-to-treat isolates.

Burden of Rifampicin- and Methicillin-Resistant Staphylococcus aureus in Italy.

Rifampicin is one of the major drugs used on its own and also in combination to treat numerous infections sustained by methicillin-resistant Staphylococcus aureus (MRSA). In Italy, rifampicin resistance (RIF-R) is increasing in multidrug-resistant-MRSA isolates (16.4%), with respect to Europe (5.7%). In our study, the relationship between clones, rpoB mutations, and susceptibility profiles in 50 RIF-R MRSA isolated from hospitalized patients was evaluated. Antimicrobial susceptibility testing was performed by the broth microdilution method. Isolates were typed by MLST/SCCmec/spa-typing. The rpoB gene was analyzed by PCR and sequence analysis. RIF-R isolates were 60% heterogeneous vancomycin-intermediate S. aureus (hVISA) and 22% daptomycin nonsusceptible and belonged to the major MRSA clones: ST228-SCCmec I (44%), ST8-SCCmec IV (18%), ST239-SCCmec III (16%), ST5-SCCmec II (14%), and ST22-SCCmec IVh (4%). Thirteen diverse RpoB amino acid substitutions were identified. Half of the strains harbored the H481N substitution, conferring low-level resistance. Different single mutations at the equivalent locus (H481D; H481Y) or in other loci, and multiple mutations conferred high-level resistance. In conclusion, this study investigated the nature of RIF-R in Italy among RIF-R-MRSA strains, finding a prevalence of ST228, strongly associated with reduced susceptibility to glycopeptides (hVISA). The spread of RIF-R strains in clinical settings represents a serious threat, due to their complex resistance nature even to new anti-Gram-positive drugs, making these infections particularly difficult to treat.

High Resolution Melting-Typing (HRMT) of methicillin-resistant Staphylococcus aureus (MRSA): The new frontier to replace multi-locus sequence typing (MLST) for epidemiological surveillance studies.

We report an implemented molecular-typing-method based on HRMA to detect SNPs within MLST loci, characterizing 100 clinical MRSA and 11 control strains, representative of Italian clones. The results provide solid evidence that HRMT could be a fast, cost-effective and reliable alternative to MLST, for MRSA molecular epidemiology.

Added value of multi-pathogen probe-based real-time PCR SeptiFast in the rapid diagnosis of bloodstream infections in patients with bacteraemia.

The commercial multi-pathogen probe-based real-time PCR SeptiFast (SF) was evaluated as a rapid and complementing tool for the microbiological diagnosis of bloodstream infections (BSIs) in a series of 138 matched blood samples from 65 patients with bacteraemia, hospitalized in an intensive care unit, when antibiotics had already been administered. SF was positive in 32.6 % of the samples, whereas blood culture (BC) was positive in 21.7 % (P < 0.05). SF identified more pathogens (11 versus 5; specificity, 90.7 %) and reduced the time of aetiological diagnosis, with a mean of 16.3 versus 55.4 h needed for BC (P < 0.05). SF enabled appropriate pathogen-oriented therapy in 72 % (36/50) of the BSI group of patients on the basis of epidemiological data. According to our data, the use of SF provided important added value to BC, in terms of earlier aetiological diagnosis of BSIs, enabling pathogen-oriented therapy in patients receiving empirical antibiotic treatment.

Epidemiology of Staphylococcus aureus in Italy: First nationwide survey, 2012.

A 3-month epidemiological study to determine the prevalence and antibiotic resistance of Staphylococcus aureus nosocomial infections was performed in 52 centres throughout Italy in 2012. A total of 21,873 pathogens were analysed. The prevalence of S. aureus among all nosocomial pathogens isolated in that period was 11.6% (n=2541), whilst the prevalence of methicillin-resistant S. aureus (MRSA) among the S. aureus was 35.8% (n=910). All tested antimicrobials demonstrated ≥92.2% susceptibility against methicillin-susceptible S. aureus, with the exception of clindamycin (89.7%) and erythromycin (84.2%). Among MRSA, percentages of resistance ranged from 12.6% to >39% for tetracycline, rifampicin, clindamycin and gentamicin; higher percentages were found for erythromycin (65.4%) and fluoroquinolones (72.3-85.8%). Overall, the glycopeptide minimum inhibitory concentration (MIC) distribution showed that 58.3% of strains possessed MICs of 1-2mg/L and few strains were linezolid- or daptomycin-resistant. Molecular characterisation was performed on 102 MRSA selected from Northern, Central and Southern regions. Five major clones were found: Italian/ST228-I (t001-t023-t041-t1686-t3217), 33.3%; USA500/ST8-IV (t008), 17.6%; E-MRSA15/ST22-IVh (t020-t025-t032-t223), 16.7%; USA100/ST5-II (t002-t653-t1349-t2164-t3217-t388), 14.7%; and Brazilian/ST239/241-III (t030-t037), 3.9%. Five PVL-positive CA-MRSA isolates, belonging to USA300 and minor clones, were also identified. In conclusion, this first nationwide surveillance study showed that in Italy, S. aureus infections accounted for 11.6% of all nosocomial infections; MRSA accounted for approximately one-third of the S. aureus isolates and these were multidrug-resistant organisms. Five major MRSA epidemic clones were observed and were inter-regionally distributed, with ST228-SCCmecI becoming predominant.

Worrisome trend of new multiple mechanisms of linezolid resistance in staphylococcal clones diffused in Italy.

In order to assess the frequency of clinically relevant linezolid-resistant staphylococcal isolates, and the role of linezolid in maintaining and coselecting multiple resistance mechanisms (cfr, 23S rRNA, L3/L4 mutations), a prospective Italian study was performed from 2010 to 2011 to confirm the diffusion of three major multidrug-resistant clones (ST2, ST5, ST23).

DNA methylase modifications and other linezolid resistance mutations in coagulase-negative staphylococci in Italy.

BACKGROUND: Despite 10 years of clinical use, linezolid resistance in Staphylococcus aureus and coagulase-negative staphylococci (CoNS) is still a rare phenomenon. This study reports the mechanisms of resistance and strain types seen in clusters of linezolid-resistant CoNS from two different hospitals in Italy during the period 2008-09. METHODS: Genes associated with linezolid resistance were subjected to molecular analysis and isolates were characterized by PFGE macrorestriction analysis using SmaI. RESULTS: Thirty-three linezolid-resistant isolates of methicillin-resistant CoNS comprising Staphylococcus epidermidis (24), Staphylococcus hominis (5) and Staphylococcus simulans (4) were studied. The isolates showed varying levels of linezolid resistance. Almost all isolates for which linezolid MICs were 64 mg/L possessed point mutations in domain V of 23S rRNA, while isolates for which the MICs were 256 mg/L expressed methylase activity at position A2503 mediated by the cfr gene. Overall, the isolates showed reduced susceptibility to vancomycin (MICs 1-2 mg/L) and 11 of the 33 isolates showed no susceptibility to teicoplanin. These strains were also resistant to chloramphenicol (28 of 33), lincomycin (24 of 33), erythromycin (17 of 33) and quinupristin/dalfopristin (13 of 33). S. epidermidis isolates, showing mutations or methylase modifications, belonged to different PFGE profiles and to two different sequence types (ST2 and ST23), in which the cfr gene was carried on a plasmid of ∼50 kb. CONCLUSIONS: Clinical CoNS strains with resistance to linezolid and other second-line antibiotics, as well as reduced susceptibility to glycopeptides, have emerged in Italy.

Linezolid Resistance in Staphylococci.

Linezolid, the first oxazolidinone to be used clinically, is effective in the treatment of infections caused by various Gram-positive pathogens, including multidrug resistant enterococci and methicillin-resistant Staphylococus aureus. It has been used successfully for the treatment of patients with endocarditis and bacteraemia, osteomyelitis, joint infections and tuberculosis and it is often used for treatment of complicated infections when other therapies have failed. Linezolid resistance in Gram-positive cocci has been encountered clinically as well as in vitro, but it is still a rare phenomenon. The resistance to this antibiotic has been, until now, entirely associated with distinct nucleotide substitutions in domain V of the 23S rRNA genes. The number of mutated rRNA genes depends on the dose and duration of linezolid exposure and has been shown to influence the level of linezolid resistance. Mutations in associated ribosomal proteins also affect linezolid activity. A new phenicol and clindamycin resistance phenotype has recently been found to be caused by an RNA methyltransferase designated Cfr. This gene confers resistance to lincosamides, oxazolidinones, streptogramin A, phenicols and pleuromutilins, decrease the susceptibility of S. aureus to tylosin, to josamycin and spiramycin and thus differs from erm rRNA methylase genes. Research into new oxazolidinones with improved characteristics is ongoing. Data reported in patent applications demonstrated that some oxazolidinone derivatives, also with improved characteristics with respect to linezolid, are presently under study: at least three of them are in an advanced phase of development.