GHEP-ISFG collaborative exercise on mixture profiles (GHEP-MIX06). Reporting conclusions: Results and evaluation.

One of the main goals of the Spanish and Portuguese-Speaking Group of the International Society for Forensic Genetics (GHEP-ISFG) is to promote and contribute to the development and dissemination of scientific knowledge in the field of forensic genetics. Due to this fact, GHEP-ISFG holds different working commissions that are set up to develop activities in scientific aspects of general interest. One of them, the Mixture Commission of GHEP-ISFG, has organized annually, since 2009, a collaborative exercise on analysis and interpretation of autosomal short tandem repeat (STR) mixture profiles. Until now, six exercises have been organized. At the present edition (GHEP-MIX06), with 25 participant laboratories, the exercise main aim was to assess mixture profiles results by issuing a report, from the proposal of a complex mock case. One of the conclusions obtained from this exercise is the increasing tendency of participating laboratories to validate DNA mixture profiles analysis following international recommendations. However, the results have shown some differences among them regarding the edition and also the interpretation of mixture profiles. Besides, although the last revision of ISO/IEC 17025:2017 gives indications of how results should be reported, not all laboratories strictly follow their recommendations. Regarding the statistical aspect, all those laboratories that have performed statistical evaluation of the data have employed the likelihood ratio (LR) as a parameter to evaluate the statistical compatibility. However, LR values obtained show a wide range of variation. This fact could not be attributed to the software employed, since the vast majority of laboratories that performed LR calculation employed the same software (LRmixStudio). Thus, the final allelic composition of the edited mixture profile and the parameters employed in the software could explain this data dispersion. This highlights the need, for each laboratory, to define through internal validations its criteria for editing and interpreting mixtures, and to continuous train in software handling.

Analysis of uni and bi-parental markers in mixture samples: Lessons from the 22nd GHEP-ISFG Intercomparison Exercise.

Since 1992, the Spanish and Portuguese-Speaking Working Group of the ISFG (GHEP-ISFG) has been organizing annual Intercomparison Exercises (IEs) coordinated by the Quality Service at the National Institute of Toxicology and Forensic Sciences (INTCF) from Madrid, aiming to provide proficiency tests for forensic DNA laboratories. Each annual exercise comprises a Basic (recently accredited under ISO/IEC 17043: 2010) and an Advanced Level, both including a kinship and a forensic module. Here, we show the results for both autosomal and sex-chromosomal STRs, and for mitochondrial DNA (mtDNA) in two samples included in the forensic modules, namely a mixture 2:1 (v/v) saliva/blood (M4) and a mixture 4:1 (v/v) saliva/semen (M8) out of the five items provided in the 2014 GHEP-ISFG IE. Discrepancies, other than typos or nomenclature errors (over the total allele calls), represented 6.5% (M4) and 4.7% (M8) for autosomal STRs, 15.4% (M4) and 7.8% (M8) for X-STRs, and 1.2% (M4) and 0.0% (M8) for Y-STRs. Drop-out and drop-in alleles were the main cause of errors, with laboratories using different criteria regarding inclusion of minor peaks and stutter bands. Commonly used commercial kits yielded different results for a micro-variant detected at locus D12S391. In addition, the analysis of electropherograms revealed that the proportions of the contributors detected in the mixtures varied among the participants. In regards to mtDNA analysis, besides important discrepancies in reporting heteroplasmies, there was no agreement for the results of sample M4. Thus, while some laboratories documented a single control region haplotype, a few reported unexpected profiles (suggesting contamination problems). For M8, most laboratories detected only the haplotype corresponding to the saliva. Although the GHEP-ISFG has already a large experience in IEs, the present multi-centric study revealed challenges that still exist related to DNA mixtures interpretation. Overall, the results emphasize the need for further research and training actions in order to improve the analysis of mixtures among the forensic practitioners.

Nanomedicines for antimicrobial interventions.

The development of new antimicrobial therapeutic tools addresses the emergence of multidrug-resistant micro-organisms or clones and the need for more effective antimicrobial strategies. Overcoming the hurdles in providing early diagnosis and intervention on hard-to-reach and/or resting bacteria (i.e. biofilm-embedded cells) represents a challenging task. In this review, we identify a set of organic, inorganic, and hybrid materials that might be used for prevention and control of healthcare-associated infections. We report the current knowledge on nano- and microparticle-based antimicrobial agents and describe the possible mode of their action.

Prevention of group B streptococcal neonatal disease revisited. The DEVANI European project.

The purpose of this paper was to present the current knowledge on the prevention of group B streptococcus (GBS) neonatal infections and the status of prevention policies in European countries and to present the DEVANI pan-European program, launched in 2008. The aim of this program was to assess the GBS neonatal infection burden in Europe, to design a new vaccine to immunize neonates against GBS infections, to improve the laboratory performance for the diagnosis of GBS colonization and infection, and to improve the methods for the typing of GBS strains. The current guidelines for GBS prevention in different countries were ascertained and a picture of the burden before and after the instauration of prevention policies has been drawn. After the issue of the Centers for Disease Control and Prevention (CDC) guidelines, many European countries have adopted universal screening for the GBS colonization of pregnant women and intrapartum prophylaxis to colonized mothers. Nevertheless, some European countries continue advocating the risk factor approach to GBS prevention. Most European countries have implemented policies to prevent GBS neonatal infections and the burden of the disease has decreased during the last several years. Nevertheless, further steps are necessary in order to develop new strategies of prevention, to improve microbiological techniques to detect GBS colonization and infection, and to coordinate the prevention policies in the EU.

Identification and molecular characterization of a S. agalactiae strain lacking the capsular locus.

During a national surveillance program on Group B streptococci (GBS) maternal carriage and neonatal infections, a GBS strain isolated from a pregnant woman's vagino-rectal swab was non typable by either serological or molecular methods. Further molecular characterization demonstrated that the strain lacked the entire capsular locus, possibly by a recombination event that excised a 14,1 Kbase pairs genomic fragment extending from the regulatory protein cpsX gene to the neuA gene. The natural loss of the capsular locus by GBS isolated from a human has never been described so far. Such an event, while possibly a dead-end from the evolutionary point of view, leaves a still able-to-colonize organism unrecognizable by the vaccines currently under development.

Invasive neonatal GBS infections from an area-based surveillance study in Italy.

During an area-based study, 75 group B streptococcus (GBS) strains isolated both from early-onset disease (EOD, 37 strains) and from late-onset disease (LOD, 38 strains) were analysed for serotype, pulsed field gel electrophoresis (PFGE) and multilocus sequence typing profiles, protein markers and antibiotic resistance. Serotype III, possessing the rib gene, was the most frequent (54 strains, 72%) and responsible for 89.5% and 54% of LOD and EOD, respectively. Forty-six serotype III strains belonged to the same PFGE type and clonal complex 17, already described as an over-represented clone in neonatal invasive GBS infections. Other serotypes were Ia (9.3%), II (6.7%), Ib (5.3%), V (5.3%) and IV (1.3%). Seventeen PFGE groups were identified comprising strains with related sequence types; conversely, strains displaying the same sequence type could belong to different PFGE groups. When both neonate and maternal strains from vaginorectal swabs and/or milk were available (eight cases), they were indistinguishable. Resistance to erythromycin (12%) was associated with a constitutive resistance to clindamycin in five cases (four carrying the erm(B) gene and one both the erm(B) and mef(E) genes) and with an inducible clindamycin resistance in two cases (one possessing the erm(A) gene, the other the erm(T) gene). Two isolates displayed the M phenotype (mef(E) gene). All strains but five were resistant to tetracycline, mostly mediated by the tet(M) gene (97.1%). The study underlined the importance of an active surveillance system for the elucidation of a GBS population structure causing neonatal infections and allowed the detection of rare antibiotic resistance determinants [erm(T)].

Human milk as a vector and an indicator of exposure to PCBs and PBDEs: temporal trend of samples collected in Rome.

Thirty-seven polychlorobiphenyl (PCB) congeners and seven polybromodiphenylether (PBDE) congeners were measured in human milk samples collected in Rome between 2005 and 2007. The comparison of results with two previous studies performed in Rome in 1984 and in 2000-2001 indicates a 64% decrease of PCB levels, still in progress; profile differences with time were also evident as lighter congeners are less relevant now; data are in good agreement with recent European studies. PBDE contamination profiles were different in individual samples and a similar variability was observed in data from different countries, suggesting different exposure pathways and profiles.

Contribution of serotype and genetic background to biofilm formation by Streptococcus pneumoniae.

Streptococcus pneumoniae is the main cause of a variety of infections in children and the elderly ranging from otitis media to pneumonia. In recent years, biofilm formed by S. pneumoniae has begun to attract attention for a possible role in strains fitness and/or virulence. We evaluated the ability to form biofilm in a collection of clinical isolates, including antibiotic-resistant isolates whose genetic background had been previously ascertained. It appears that biofilm formation is a rather common feature among pneumococci, an observation which would fit with some types of infections caused by this microorganism (i.e. otitis, meningitis), which have often been associated with the ability to form biofilm. Antibiotic-susceptible isolates were able to form thicker biofilms compared to resistant strains, although no specific association could be observed with either serotypes or clones. This lack of association between the ability to form biofilm and any of the characters examined, while being a very common feature of pneumococci, may be suggestive of an important role for biofilm in pneumococcal ecology.

The Alpha-like surface proteins: an example of an expanding family of adhesins.

The Alpha-like protein (Alp) family, repeat-containing surface proteins once thought to be important adhesion factors confined to pathogenic streptococci and enterococci, is broader than previously known. Analysis of the annotated microbial genomes has identified new potential members of the Alp family not only in other Gram- positive opportunistic pathogens but also in commensal microflora of the human gut and the skin. This finding has highlighted the importance of genome sequencing projects for unraveling in greater detail lateral gene transfer events involving virulence factors between pathogens and commensals. These should receive constant attention not only as part of infectious disease prevention programs, but also in the food and biotechnology industries.

Combating implant infections. Remarks by a women's team.

Research on implant infections requires cooperative efforts and integration between basic and clinical expertises. An international group of women scientists is acting together in this field. The main research topics of the participants of this group are described. Formation of bacterial biofilms, antibiotic resistance and production of virulence factors like adhesins and toxins are investigated. New biomaterials, coatings and drugs designed to inhibit microbial adhesion are evaluated, and infection-resistant biomaterials are under study, such as a novel heparinizable polycarbonate-urethane (Bionate) or incorporation of diamino-diamide-diol (PIME) to reduce bacterial attachment. The correlation between biofilm production and the accessory-gene-regulator (agr) is investigated in Staphylococcus aureus. The ability to form biofilm has also been shown to be one of the important virulence factors of Enterococcus faecalis, favouring colonization of inert and biological surfaces. The study of quorum sensing has led to the discovery of a quorum sensing inhibitor termed RIP that suppresses staphylococcal biofilm and infections. The immune response and the local defence mechanisms of the host against implant-associated infections, activation and infiltration of immunocompetent cells into the sites of infection have been studied in patients with implant-associated osteomyelitis. Production of monoclonal antibodies (mAbs) as possible vaccines against the staphylococcal collagen-binding MSCRAMMs is in progress.

Underestimated collateral effects of antibiotic therapy in prosthesis-associated bacterial infections.

Antibiotic treatment of infections associated with the use of indwelling medical devices in ageing and/or severely ill patients represents a significant healthcare problem due to the difficulty of treating such infections and to the various collateral effects that may be observed following the often aggressive therapy. We summarize some effects of antibiotics on the expression of virulence factors of the microorganisms which cause such infections. These effects, particularly those resulting in a stimulation of bacterial virulence, might be usefully included among the other well-known collateral effects of antibiotic therapy.

Prevalence of genes encoding for staphylococcal leukocidal toxins among clinical isolates of Staphylococcus aureus from implant orthopedic infections.

Staphylococcus aureus has emerged as a major cause of implant infections. It is known that it is able to produce several toxins that contribute to its armory of virulent weapons, but there are still no data on their prevalence among isolates recovered from biomaterial-centered infections. In this study, 200 Staphylococcus aureus isolates from infections related to different types of orthopedic implants (hip and knee arthroprostheses, internal and external fixation devices) were tested by polymerase chain reaction for the prevalence of genes encoding for leukotoxins. Although almost all isolates were positive for the ã-hemolysin gene (99%), none was positive for lukM. The leukotoxin genes lukE/lukD were found in 67% of isolates. The presence of lukE/lukD was significantly associated with that of Accessory Gene Regulatory locus agr II. The lukE/lukD-positive isolates were significantly more prevalent in the staphylococcal isolates from knee arthroprostheses than in the isolates from the other implant types. The genes encoding Panton-Valentine leukocidin components were detected in only one isolate that, curiously enough, was taken solely from a knee arthroprosthesis infection.

Prevalence and antibiotic resistance of 15 minor staphylococcal species colonizing orthopedic implants.

Several species belonging to Staphylococcus genus (non Sau/ non Sep species) exhibit increasing abilities as opportunistic pathogens in colonisation of periprosthesis tissues. Here we report on antibiotic resistance of 193 strains, belonging to non Sau/ non Sep species, consecutively collected from orthopedic implant infections in a period of about 40 months. The 193 strains (representing 17% of all staphylococci isolated) were analysed for their antibiotic resistance to 16 different drugs. Five species turned out more prevalent, ranging from 1 to 5%: S. hominis (4.2%), S. haemolyticus (3.7%), S. capitis (2.7%), S. warneri (2.6%), and S. cohnii (1.6%). Among these, the prevalence of antibiotic resistance to penicillins was similar, ranging from 51% to 66%. Conversely, significant differences were observed for all the remaining antibiotics. For S. haemolyticus the resistances to oxacillin and imipenem, the four aminoglycosides and erythromycin were at least twice that of the other three species which were compared. S. warneri was on the contrary the species with the lowest occurrence of resistant strains. Ten species appeared only rarely at the infection sites: S. lugdunensis, S. caprae, S. equorum, S. intermedius, S. xylosus, S. simulans, S. saprophyticus, S. pasteuri, S. sciuri, and S. schleiferi. The behaviours of these species, often resistant to penicillins, were individually analysed. Differences in both the frequencies and the panels of antibiotic resistances observed among the non Sau/ non Sep species: i) suggest that horizontal spreading of resistance factors, if acting, was not sufficient per se to level their bio-diversities; ii) highlight and confirm the worrisome appearance within the Staphylococcus genus of emerging ""new pathogens"", not homogeneous for their virulence and antibiotic resistance prevalence, which deserve to be recognised and treated individually.

Virulence factors in enterococcal infections of orthopedic devices.

Enterococci are opportunistic pathogens which today represent one of the leading causes of nosocomial infections. We have examined a collection of 52 Enterococcus faecalis isolated from orthopedic infections to determine if they were characterized by a specific pattern of virulence factors. The isolates were evaluated for biofilm formation, presence of genes coding the enterococcal surface protein (esp) and gelatinase (gelE), as well as for gelatinase production. While the rate of esp-positive isolates was comparable to that found among strains from other clinical sources, we found a significantly higher rate of strong biofilm formers and gelatinase producers. Particularly high was the rate of gelE-carrying strains expressing the gene. Data suggest that these two factors in particular may play an important role in enterococcal infections associated with biomaterials.

Automated ribotyping to distinguish the different non Sau/ non Sep staphylococcal emerging pathogens in orthopedic implant infections.

Several species belonging to Staphylococcus genus, other than Staphylococcus aureus and Staphylococcus epidermidis (non Sau/ non Sep species), exhibit increasing abilities as opportunistic pathogens in the colonisation of periprosthetic tissues. Consequently, the availability of means for accurate identification is crucial to assess the pathogenic characteristics and to clarify clinical relevance of the individual species. Here, 146 clinical staphylococcal isolates belonging to non Sau/ non Sep species from prosthesis-associated orthopedic infections were analyzed by conventional enzymatic galleries and by automated ribotyping. Twelve different species were recognised: S. capitis, S. caprae, S. cohnii, S. equorum, S. haemolyticus, S. hominis, S. lugdunensis, S. pasteuri, S. sciuri, S. simulans, S. warneri, S. xylosus. Ribotype identifications were compared with the phenotypes obtained by the Api 20 Staph system and/or ID 32 Staph system. ID 32 Staph profiles were more consistent with ribotyping results than Api Staph profiles. Across the different staphylococcal species investigated, correct identifications with Api Staph were 45%, while with ID 32 Staph they were 59%. It has, however, to be mentioned that ID 32 Staph was mostly applied to discriminate unmatched ribotyping and Api Staph identifications, thus to a subpopulation of strains with ""atypical"" metabolic profile. Automated ribotyping provided a correct identification for 91% of the isolates. These results confirm automated ribotyping as a convenient rapid technique, still subject to improvements, which will accurately and rapidly recognise the newly emerging staphylococcal pathogens in implant-related orthopedic infections.

Distribution of four capsular serotypes of Enterococcus faecalis among clinical isolates from different geographical origins and infection sites.

BACKGROUND: Enterococci possess capsular polysaccharide antigens that are the targets of opsonic antibodies. These antibodies are potential candidates for development as immunotherapy. MATERIAL AND METHODS: The present study analyzes the distribution of four capsular serotypes within a collection of 157 isolates of Enterococcus faecalis from four countries with different sites of clinical infection. RESULTS: By using a capsular polysaccharide-specific ELISA, 42% of the isolates were grouped into one of four serogroups, and another 9% showed cross-reactivity between two serotype-specific sera. Heterogeneity of serotype distribution by both geographical origin and infection site was observed. CONCLUSION: Half of the strain collection could be typed with four serotype-specific sera. No serotype from a given country or infection site clearly predominated.

Pathogenesis of implant infections by enterococci.

Enterococci are commensals of human and animal intestinal tract that have emerged in the last decades as a major cause of nosocomial infections of bloodstream, urinary tract and in infected surgical sites. Enterococcus faecalis is responsible for ca. 80% of all enterococcal infections while Enterococcus faecium accounts for most of the others; among the most relevant risk factors for development of enterococcal infections is the presence of implanted devices. The pathogenesis of such infections is poorly understood, but several virulence factors have been proposed. Among them, the ability to form biofilm has recently been shown to be one of the most prominent features of this microorganism, allowing colonization of inert and biological surfaces, while protecting against antimicrobial substances, and mediating adhesion and invasion of host cells and survival within professional phagocytes. Biofilm formation has been shown to be particularly important in the development of prosthetic valve enterococcal endocarditis and stent occlusion. Enterococci are also able to express other surface factors that may support colonization of both inert and biological surfaces, and that may be involved in the invasion of, and survival within, the host cell.

Antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis clinical isolates from implant orthopedic infections.

In the last decade the rising phenomenon of resistance to most common antibiotic drugs among staphylococcal clinical isolates has been a reason for serious concern and alarm. The present study investigated the prevalence of antibiotic resistance within a large microbial collection including 530 clinical strains of S. aureus and 408 strains of S. epidermidis to a panel of 16 different drugs. All strains were isolated from orthopedic infections, either associated or non-associated with implant materials. Interestingly, our data show that the profile of the prevalence of antibiotic resistance within the two species of pathogens is extremely similar for the vast majority of the drugs screened. The only statistically significant variations in prevalence concerned, in order of relevance, the following 5 out of 16 antibiotics: sulfamethoxazole (in combination with trimethoprim), erythromycin, and, to a lesser extent, oxacillin, imipenem, and clindamycin. In the case of Staphylococcus aureus, the isolates associated to implant materials were found more frequently resistant to all 4 aminoglycosides screened as well as to ciprofloxacin.

Clonality among Enterococcus faecium clinical isolates.

In the course of a survey to determine the epidemiology of enterococcal infections in Italy, a sudden increment, in a 1-year time, was noted in the number of glycopeptide resistant Enterococcus faecium isolated from different wards of the University Hospital in Rome, Italy. The isolates were characterized for clonal relatedness by comparing SmaI gel electropherotypes, presence of vancomycin-resistance genes, and expression of virulence factors. PFGE identified in a single pulsed type all the glycopeptide-resistant isolates but one. Resistance to high levels of aminoglycosides was expressed by these same isolates, which also included a majority of non biofilm-forming strains. Two esp gene-carrying strains were also identified in different PFGE types. Data indicates that a specific clone acquired, in the clinical setting, the genetic determinant for glycopeptide resistance, thus improving environmental adaptation and favoring its persistence and spread.