Brucella spp. are Gram-negative, non-motile, non-spore-forming, slow-growing, facultative intracellular bacteria causing brucellosis. Brucellosis is an endemic of specific geographic areas and, although underreported, represents the most common zoonotic infection, with an annual global incidence of 500,000 cases among humans. Humans represent an occasional host where the infection is mainly caused by B. melitensis, which is the most virulent; B. abortus; B. suis; and B. canis. A microbiological analysis is crucial to identifying human cases because clinical symptoms of human brucellosis are variable and aspecific. The laboratory diagnosis is based on three different microbiological approaches: (i) direct diagnosis by culture, (ii) indirect diagnosis by serological tests, and (iii) direct rapid diagnosis by molecular PCR-based methods. Despite the established experience with serological tests and highly sensitive nucleic acid amplification tests (NAATs), a culture is still considered the “gold standard” in the laboratory diagnosis of brucellosis due to its clinical and epidemiological relevance. Moreover, the automated BC systems now available have increased the sensitivity of BCs and shortened the time to detection of Brucella species. The main limitations of serological tests are the lack of common interpretative criteria, the suboptimal specificity due to interspecies cross-reactivity, and the low sensitivity during the early stage of disease. Despite that, serological tests remain the main diagnostic tool, especially in endemic areas because they are inexpensive, user friendly, and have high negative predictive value. Promising serological tests based on new synthetic antigens have been recently developed together with novel point-of-care tests without the need for dedicated equipment and expertise. NAATs are rapid tests that can help diagnose brucellosis in a few hours with high sensitivity and specificity. Nevertheless, the interpretation of NAAT-positive results requires attention because it may not necessarily indicate an active infection but rather a low bacterial inoculum, DNA from dead bacteria, or a patient that has recovered. Refined NAATs should be developed, and their performances should be compared with those of commercial and home-made molecular tests before being commercialized for the diagnosis of brucellosis. Here, we review and report the most common and updated microbiological diagnostic methods currently available for the laboratory diagnosis of brucellosis.

Background: Stenotrophomonas maltophiliais emerging as one of the most frequently found bacteria in cystic fibrosis (CF) patients. In the present study, phenotypic and genotypic traits of a set of 98 isolates ofS. maltophiliaobtained from clinical (CF and non-CF patients) and environmental sources were comparatively evaluated.Results: S. maltophiliaexhibited a high level of genomic diversity in both CF and non-CF group, thus possibly allowing this bacterium to expand its pathogenic potentials. Strains sharing the same pulsotype infected different patients, thus likely indicating the occurrence of clonal spread or acquisition by a common source. CF isolates differed greatly in some phenotypic traits among each other and also when compared with non-CF isolates, demonstrating increased mean generation time and susceptibility to oxidative stress, but reduced ability in forming biofilm. Furthermore, in CF isolates flagella- and type IV pili-based motilities were critical for biofilm development, although not required for its initiation. Sequential isogenic strains isolated from the same CF patient displayed heterogeneity in biofilm and other phenotypic traits during the course of chronic infection. CF and non-CF isolates showed comparable virulence in a mouse model of lung infection.Conclusions: Overall, the phenotypic differences observed between CF and non-CF isolates may imply different selective conditions and persistence (adaptation) mechanisms in a hostile and heterogeneous environment such as CF lung. Molecular elucidation of these mechanisms will be essential to better understand the selective adaptation in CF airways in order to design improved strategies useful to counteract and eradicateS. maltophiliainfection.

Group B streptococci (GBS) comprising three different sets of isolates (31 invasive, 36 noninvasive, and 24 colonizing isolates) were collected in Italy during the years 2002 to 2005. Clonal groups were established by pulsed-field gel electrophoresis (PFGE), and selected isolates were studied by multilocus sequence typing (MLST). GBS isolates were also characterized by classical and molecular techniques for serotyping and protein gene and antibiotic resistance profiling. Some serotypes were significantly associated with a particular isolate population: serotype Ia more frequently corresponded to invasive strains than other strains, serotype V was more frequently encountered among noninvasive strains, and nontypeable strains were more common among isolates from carriers. Four major clonal groups accounted for 52.7% of all isolates: PFGE type 1/clonal complex 1 (CC1) comprised mainly serotype V isolates carrying the alp3 gene, PFGE type 2/CC23 encompassed serotype Ia isolates with the alp1 or alpha gene, PFGE type 3/CC17 comprised serotype III isolates carrying the rib gene, and PFGE type 4/CC19 consisted mainly of serotype II isolates possessing the rib gene. The same serotypes were shared by isolates of different clonal groups, and conversely, isolates belonging to the same clonal groups were found to be of different serotypes, presumably due to capsular switching by the horizontal transfer of capsular genes. Erythromycin resistance (prevalence, 16.5%; 15 resistant isolates of 91) was restricted to strains isolated from patients with noninvasive infections and carriers, while tetracycline resistance was evenly distributed (prevalence, 68.1%; 62 resistant isolates of 91). Most erythromycin-resistant GBS strains were of serotype V, were erm (B) positive, and belonged to the PFGE type 1/CC1 group, suggesting that macrolide resistance may have arisen both by clonal dissemination and by the horizontal transfer of resistance genes.

Background: Streptococcus pyogenes or group A streptococcus (GAS) is an important human pathogen responsible for a broad range of infections, from uncomplicated to more severe and invasive diseases with high mortality and morbidity. Epidemiological surveillance has been crucial to detect changes in the geographical and temporal variation of the disease pattern; for this purpose the M protein gene (emm) gene typing is the most widely used genotyping method, with more than 200 emm types recognized. Molecular epidemiological data have been also used for the development of GAS M protein-based vaccines. Methods: The aim of this paper was to provide an updated scenario of the most prevalent GAS emm types responsible for invasive infections in developed countries as Europe and North America (US and Canada), from 1st January 2000 to 31st May 2017. The search, performed in PubMed by the combined use of the terms (“emm”) and (“invasive”) retrieved 264 articles, of which 38 articles (31 from Europe and 7 from North America) met the inclusion criteria and were selected for this study. Additional five papers cited in the European articles but not retrieved by the search were included. Results: emm1 represented the dominant type in both Europe and North America, replaced by other emm types in only few occasions. The seven major emm types identified (emm1, emm28, emm89, emm3, emm12, emm4, and emm6) accounted for approximately 50–70% of the total isolates; less common emm types accounted for the remaining 30–50% of the cases. Most of the common emm types are included in either one or both the 26-valent and 30-valent vaccines, though some well-represented emm types found in Europe are not. Conclusion: This study provided a picture of the prevalent emm types among invasive GAS (iGAS) in Europe and North America since the year 2000 onward. Continuous surveillance on the emm-type distribution among iGAS infections is strongly encouraged also to determine the potential coverage of the developing multivalent vaccines.

Approximately 40–60% of group A streptococcal (GAS) isolates are capable of opacifying sera, due to the expression of the sof (serum opacity factor) gene. The emm (M protein gene) and sof 5′ sequences were obtained from a diverse set of GAS reference strains and clinical isolates, and correlated with M serotyping and anti-opacity-factor testing results. Attempts to amplify sof from strains with M serotypes or emm types historically associated with the opacity-factor-negative phenotype were negative, except for emm12 strains, which were found to contain a highly conserved sof sequence. There was a strong correlation of certain M serotypes with specific emm sequences regardless of strain background, and likewise a strong association of specific anti-opacity-factor (AOF) types to sof gene sequence types. In several examples, M type identity, or partial identity shared between strains with differing emm types, was correlated with short, highly conserved 5′ emm sequences likely to encode M-type-specific epitopes. Additionally, each of three pairs of historically distinct M type reference strains found to share the same 5′ emm sequence, were also found to share M serotype specificity. Based upon sof sequence comparisons between strains of the same and of differing AOF types, an approximately 450 residue domain was determined likely to contain key epitopes required for AOF type specificity. Analysis of two Sof sequences that were not highly homologous, yet shared a common AOF type, further implicated a 107 aa portion of this 450-residue domain in putatively containing AOF-specific epitopes. Taken together, the serological data suggest that AOF-specific epitopes for all Sof proteins may reside within a region corresponding to this 107-residue sequence. The presence of specific, hypervariable emm/sof pairs within multiple isolates appears likely to be a reliable indicator of their overall genetic relatedness, and to be very useful for accurate subtyping of GAS isolates by an approach that has relevance to decades of past M-type-based epidemiological data.