Typing methods for discriminating different bacterial isolates of the same species are essential epidemiological tools in infection prevention and control. Traditional typing systems based on phenotypes, such as serotype, biotype, phage-type, or antibiogram, have been used for many years. However, more recent methods that examine the relatedness of isolates at a molecular level have revolutionised our ability to differentiate among bacterial types and subtypes. Importantly, the development of molecular methods has provided new tools for enhanced surveillance and outbreak detection. This has resulted in better implementation of rational infection control programmes and efficient allocation of resources across Europe. The emergence of benchtop sequencers using next generation sequencing technology makes bacterial whole genome sequencing (WGS) feasible even in small research and clinical laboratories. WGS has already been used for the characterisation of bacterial isolates in several large outbreaks in Europe and, in the near future, is likely to replace currently used typing methodologies due to its ultimate resolution. However, WGS is still too laborious and time-consuming to obtain useful data in routine surveillance. Also, a largely unresolved question is how genome sequences must be examined for epidemiological characterisation. In the coming years, the lessons learnt from currently used molecular methods will allow us to condense the WGS data into epidemiologically useful information. On this basis, we have reviewed current and new molecular typing methods for outbreak detection and epidemiological surveillance of bacterial pathogens in clinical practice, aiming to give an overview of their specific advantages and disadvantages.

The multidrug-resistant Streptococcus pneumoniae Taiwan19F-14, or PMEN14, clone was first observed with a 19F serotype, which is targeted by the heptavalent polysaccharide conjugate vaccine (PCV7). However, “vaccine escape” PMEN14 isolates with a 19A serotype became an increasingly important cause of disease post-PCV7. Whole genome sequencing was used to characterize the recent evolution of 173 pneumococci of, or related to, PMEN14. This suggested that PMEN14 is a single lineage that originated in the late 1980s in parallel with the acquisition of multiple resistances by close relatives. One of the four detected serotype switches to 19A generated representatives of the sequence type (ST) 320 isolates that have been highly successful post-PCV7. A second produced an ST236 19A genotype with reduced resistance to β-lactams owing to alteration of pbp1a and pbp2x sequences through the same recombination that caused the change in serotype. A third, which generated a mosaic capsule biosynthesis locus, resulted in serotype 19A ST271 isolates. The rapid diversification through homologous recombination seen in the global collection was similarly observed in the absence of vaccination in a set of isolates from the Maela refugee camp in Thailand, a collection that also allowed variation to be observed within carriage through longitudinal sampling. This suggests that some pneumococcal genotypes generate a pool of standing variation that is sufficiently extensive to result in “soft” selective sweeps: The emergence of multiple mutants in parallel upon a change in selection pressure, such as vaccine introduction. The subsequent competition between these mutants makes this phenomenon difficult to detect without deep sampling of individual lineages.

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Real-time PCR targeting lytA (the major autolysin gene) and piaB (permease gene of the pia ABC transporter) are currently used as the gold-standard culture-independent assays for Streptococcus pneumoniae identification. We evaluated the performance of a new real-time PCR assay – targeting SP2020 (putative transcriptional regulator gene) – and compared its performance with the assays previously described. A collection of 150 pneumococci, 433 non-pneumococci and 240 polymicrobial samples (obtained from nasopharynx, oropharynx, and saliva; 80 from each site) was tested. SP2020 and lytA -CDC assays had the best performance (sensitivity of 100% for each compared to 95.3% for piaB ). The specificity for lytA and piaB was 99.5% and for SP2020 was 99.8%. Misidentifications occurred for the three genes: lytA , piaB and SP2020 were found in non-pneumococcal strains; piaB was absent in some pneumococci including a serotype 6B strain. Combining lytA and SP2020 assays resulted in no misidentifications. Most polymicrobial samples (88.8%) yielded concordant results for the three molecular targets. The remaining samples seemed to contain non-typeable pneumococci (0.8%), and non-pneumococci positive for lytA (1.7%) or SP2020 (8.7%). We propose that combined detection of both lytA -CDC and SP2020 is a powerful strategy for the identification of pneumococcus either in pure cultures or in polymicrobial samples.

Recent reports suggest that methicillin-resistant Staphylococcus aureus (MRSA) may be emerging as a community pathogen. In Portuguese hospitals, the incidence of MRSA among disease causing isolates is extremely high (48-50%). To determine the prevalence of MRSA in the Portuguese community, nasal samples were obtained from 823 draftees, 484 nonmedical university students, and 107 high-school students. In addition, throat samples were obtained from the 823 draftees and S. aureus isolates were also recovered from 283 (13%) nasopharyngeal samples obtained from 2,111 children attending day-care centers. The rate of nasal colonization of S. aureus was 34%, 25%, and 46% for draftees, nonmedical university students, and high-school students, respectively. The rate of pharyngeal colonization of the draftees was 33%. Of the 1,001 S. aureus isolates obtained, seven were MRSA and eight were borderline oxacillin-resistant S. aureus (BORSA). By molecular typing techniques, five of the seven MRSA were identified as belonging to one of three highly epidemic clones, the Brazilian, Iberian, and Pediatric clones of MRSA, which were identified as endemic in Portuguese hospitals. The eight BORSA were all members of clones previously identified in international samples. In spite of the extremely high prevalence of MRSA in Portuguese hospitals, the carriage rate of MRSA in healthy and young individuals remains low.

Background Limited information is available on pneumococcal colonization among adults. We studied pneumococcal carriage dynamics in healthy adults using high-sensitivity approaches Methods Eighty-seven adults (25-50 years old), were followed for six months in Portugal. Nasopharyngeal, oropharyngeal and saliva samples were obtained monthly; pneumococcal carriers were also sampled weekly. Carriage was investigated by real-time PCR (targeting lytA and piaB) and culture. Positive samples were serotyped Results Approximately 20% of the adults were intermittent carriers; 10% were persistent carriers (>4 months). Pneumococcal acquisition and clearance rates were 16.5 (95% CI 11.2-24.2) and 95.9 (95% CI 62.3-145.0) cases/1000 persons-week, respectively. Living with children increased pneumococcal acquisition (HR:9.7, 95% CI 2.6-20.5; p<0.001). Median duration of carriage was seven weeks and did not depend on regular contact with children Conclusions The pneumococcal carrier state in healthy adults is more dynamic than generally assumed: acquisition is frequent and duration of carriage is often long. This suggests that some adults may act as reservoirs of pneumococci and hence, depending on the social structure of a community, the magnitude of herd effects potentially attainable through children vaccination may vary. These findings are important when designing strategies to prevent pneumococcal disease in adults

We previously characterized over 100 Staphylococcus sciuri isolates, mainly of animal origin, and found that they all carried a genetic element (S. sciuri mecA) closely related to the mecA gene of methicillin-resistant Staphylococcus aureus (MRSA) strains. We also found a few isolates that carried a second copy of the gene, identical to MRSA mecA. In this work, we analyzed a collection of 28 S. sciuri strains isolated from both healthy and hospitalized individuals. This was a relatively heterogeneous group, as inferred from the different sources, places, and dates of isolation and as confirmed by pulsed-field gel electrophoresis analysis. All strains carried the S. sciuri mecA copy, sustaining our previous proposal that this element belongs to the genetic background of S. sciuri. Moreover, 46% of the strains also carried the MRSA mecA copy. Only these strains showed significant levels of resistance to beta-lactams. Strikingly, the majority of the strains carrying the additional MRSA mecA copy were obtained from healthy individuals in an antibiotic-free environment. Most of the 28 strains were resistant to penicillin, intermediately resistant to clindamycin, and susceptible to tetracycline, erythromycin, and gentamicin. Resistance to these last three antibiotics was found in some strains only. The findings reported in this work confirmed the role of S. sciuri in the evolution of the mechanism of resistance to methicillin in staphylococci and suggested that this species (like the pathogenic staphylococci) may accumulate resistance markers for several classes of antibiotics.

Statistical analysis. The geometric mean was used to summarize the distribution of Ct values from lytA and piaB of nasopharyngeal and oropharyngeal samples. To look for associations between Ct values from nursing home vs family home and nasopharynx vs oropharynx an adjusted generalized linear model (GLM) using a Gaussian distribution and a log link function was used. The McNemar's test was used to compare culture and real-time PCR methods based on paired individuals. The Chi-square test was used to compare prevalence of S. pneumoniae between nasopharyngeal and oropharyngeal samples and between nursing home and family home by real-time PCR or culture-based methods. A p-value of <0.05 was considered statistically significant for all the tests used. The Gini-Simpson index of diversity (GSID) was used to calculate serotype diversity. All analyses were performed using R version 3.2.3 41. ethics statement. This study was conducted in accordance with the European Statements for Good Clinical Practice and the declaration of Helsinki of the World Health Medical Association. In addition, it was registered and approved at health care centers of Oeiras and Montemor-o-Novo that report to Administração Regional de Saúde (ARS, "Regional Health Administration") of Lisboa e Vale do Tejo, and Alentejo, respectively, from the Ministry of Health. Informed written consent was obtained from all participants. All samples and questionnaires were attributed a numeric code and were processed anonymously.