Liposomal Rifabutin-A Promising Antibiotic Repurposing Strategy against Methicillin-Resistant Staphylococcus aureus Infections.

Methicillin-resistant Staphylococcus aureus (M RSA) infections, in particular biofilm-organized bacteria, remain a clinical challenge and a serious health problem. Rifabutin (RFB), an antibiotic of the rifamycins class, has shown in previous work excellent anti-staphylococcal activity. Here, we proposed to load RFB in liposomes aiming to promote the accumulation of RFB at infected sites and consequently enhance the therapeutic potency. Two clinical isolates of MRSA, MRSA-C1 and MRSA-C2, were used to test the developed formulations, as well as the positive control, vancomycin (VCM). RFB in free and liposomal forms displayed high antibacterial activity, with similar potency between tested formulations. In MRSA-C1, minimal inhibitory concentrations (MIC) for Free RFB and liposomal RFB were 0.009 and 0.013 µg/mL, respectively. Minimum biofilm inhibitory concentrations able to inhibit 50% biofilm growth (MBIC50) for Free RFB and liposomal RFB against MRSA-C1 were 0.012 and 0.008 µg/mL, respectively. Confocal microscopy studies demonstrated the rapid internalization of unloaded and RFB-loaded liposomes in the bacterial biofilm matrix. In murine models of systemic MRSA-C1 infection, Balb/c mice were treated with RFB formulations and VCM at 20 and 40 mg/kg of body weight, respectively. The in vivo results demonstrated a significant reduction in bacterial burden and growth index in major organs of mice treated with RFB formulations, as compared to Control and VCM (positive control) groups. Furthermore, the VCM therapeutic dose was two fold higher than the one used for RFB formulations, reinforcing the therapeutic potency of the proposed strategy. In addition, RFB formulations were the only formulations associated with 100% survival. Globally, this study emphasizes the potential of RFB nanoformulations as an effective and safe approach against MRSA infections.

Panobinostat-loaded folate targeted liposomes as a promising drug delivery system for treatment of canine B-cell lymphoma.

Introduction: Cancer is a major public health problem with over 19 million cases reported in 2020. Similarly to humans, dogs are also largely affected by cancer, with non-Hodgkin's lymphoma (NHL) among the most common cancers in both species. Comparative medicine has the potential to accelerate the development of new therapeutic options in oncology by leveraging commonalities between diseases affecting both humans and animals. Within this context, in the present study, we investigated the potential of panobinostat (Pan)-loaded folate-targeted PEGylated liposomes (FA-PEG-Pan-Lip) for the treatment of canine B-cell lymphoma, while contributing to new perspectives in comparative oncology. Methods and results: Two formulations were developed, namely: PEG-Pan-Lip and FA-PEG-Pan-Lip. Firstly, folate receptor expression in the CLBL-1 canine B-cell lymphoma cell line was assessed. After confirming receptor expression, both Pan-loaded formulations (PEG-Pan-Lip, FA-PEG-Pan-Lip) demonstrated dose-dependent inhibitory effects on CLBL-1 cell proliferation. The FA-PEG-Pan-Lip formulation (IC50 = 10.9 ± 0.03 nM) showed higher cytotoxicity than the non-targeted PEG-Pan-Lip formulation (IC50 = 12.9 ± 0.03 nM) and the free panobinostat (Pan) compound (IC50 = 18.32±0.03 nM). Moreover, mechanistically, both Pan-containing formulations induced acetylation of H3 histone and apoptosis. Flow cytometry and immunofluorescence analysis of intracellular uptake of rhodamine-labeled liposome formulations in CLBL-1 cells confirmed cellular internalization of PEG-Lip and FA-PEG-Lip formulations and higher uptake profile for the latter. Biodistribution studies of both radiolabeled formulations in CD1 and SCID mice revealed a rapid clearance from the major organs and a 1.6-fold enhancement of tumor uptake at 24 h for 111In-FA-PEG-Pan-Lip (2.2 ± 0.1 %ID/g of tumor) compared to 111In-PEG-Pan-Lip formulation (1.2±0.2 %ID/g of tumor). Discussion: In summary, our results provide new data validating Pan-loaded folate liposomes as a promising targeted drug delivery system for the treatment of canine B-cell lymphoma and open innovative perspectives for comparative oncology.

Characterization of the canine CD20 as a therapeutic target for comparative passive immunotherapy.

Anti-CD20 therapies have revolutionized the treatment of B-cell malignancies. Despite these advances, relapsed and refractory disease remains a major treatment challenge. The optimization of CD20-targeted immunotherapies is considered a promising strategy to improve current therapies. However, research has been limited by the scarcity of preclinical models that recapitulate the complex interaction between the immune system and cancers. The addition of the canine lymphoma (cNHL) model in the development of anti-CD20 therapies may provide a clinically relevant approach for the translation of improved immunotherapies. Still, an anti-CD20 therapy for cNHL has not been established stressing the need of a comprehensive target characterization. Herein, we performed an in-depth characterization on canine CD20 mRNA transcript and protein expression in a cNHL biobank and demonstrated a canine CD20 overexpression in B-cell lymphoma samples. Moreover, CD20 gene sequencing analysis identified six amino acid differences in patient samples (C77Y, L147F, I159M, L198V, A201T and G273E). Finally, we reported the use of a novel strategy for the generation of anti-CD20 mAbs, with human and canine cross-reactivity, by exploring our rabbit derived single-domain antibody platform. Overall, these results support the rationale of using CD20 as a target for veterinary settings and the development of novel therapeutics and immunodiagnostics.

Diazaborines Are a Versatile Platform to Develop ROS-Responsive Antibody Drug Conjugates*.

Antibody-drug conjugates (ADCs) are a new class of therapeutics that combine the lethality of potent cytotoxic drugs with the targeting ability of antibodies to selectively deliver drugs to cancer cells. In this study we show for the first time the synthesis of a reactive-oxygen-species (ROS)-responsive ADC (VL-DAB31-SN-38) that is highly selective and cytotoxic to B-cell lymphoma (CLBL-1 cell line, IC50 value of 54.1 nM). The synthesis of this ADC was possible due to the discovery that diazaborines (DABs) are a very effective ROS-responsive unit that are also very stable in buffer and in plasma. DFT calculations performed on this system revealed a favorable energetic profile (ΔGR=-74.3 kcal mol-1 ) similar to the oxidation mechanism of aromatic boronic acids. DABs' very fast formation rate and modularity enabled the construction of different ROS-responsive linkers featuring self-immolative modules, bioorthogonal functions, and bioconjugation handles. These structures were used in the site-selective functionalization of a VL antibody domain and in the construction of the homogeneous ADC.

Immunotherapeutic Strategies for Canine Lymphoma: Changing the Odds Against Non-Hodgkin Lymphoma.

The new era of immune-oncology has brought complexities and challenges that emphasize the need to identify new strategies and models to develop successful and cost-effective therapies. The inclusion of a canine model in the drug development of cancer immunotherapies is being widely recognized as a valid solution to overcome several hurdles associated with conventional preclinical models. Driven by the success of immunotherapies in the treatment of human non-Hodgkin lymphoma (NHL) and by the remarkable similarities of canine NHL to its human counterpart, canine NHL has been one of the main focus of comparative research. Under the present review, we summarize a general overview of the challenges and prospects of today's cancer immunotherapies and the role that comparative medicine might play in solving the limitations brought by this rapidly expanding field. The state of art of both human and canine NHL and the rationale behind the use of the canine model to bridge the translational gap between murine preclinical studies and human clinical trials are addressed. Finally, a review of currently available immunotherapies for canine NHL is described, highlighting the potential of these therapeutic options.

Liposomes as a Nanoplatform to Improve the Delivery of Antibiotics into Staphylococcus aureus Biofilms.

Staphylococcus aureus biofilm-associated infections are a major public health concern. Current therapies are hampered by reduced penetration of antibiotics through biofilm and low accumulation levels at infected sites, requiring prolonged usage. To overcome these, repurposing antibiotics in combination with nanotechnological platforms is one of the most appealing fast-track and cost-effective approaches. In the present work, we assessed the potential therapeutic benefit of three antibiotics, vancomycin, levofloxacin and rifabutin (RFB), through their incorporation in liposomes. Free RFB displayed the utmost antibacterial effect with MIC and MBIC50 below 0.006 µg/mL towards a methicillin susceptible S. aureus (MSSA). RFB was selected for further in vitro studies and the influence of different lipid compositions on bacterial biofilm interactions was evaluated. Although positively charged RFB liposomes displayed the highest interaction with MSSA biofilms, RFB incorporated in negatively charged liposomes displayed lower MBIC50 values in comparison to the antibiotic in the free form. Preliminary safety assessment on all RFB formulations towards osteoblast and fibroblast cell lines demonstrated that a reduction on cell viability was only observed for the positively charged liposomes. Overall, negatively charged RFB liposomes are a promising approach against biofilm S. aureus infections and further in vivo studies should be performed.

Dominance of vaccine serotypes in pediatric invasive pneumococcal infections in Portugal (2012-2015).

We evaluated the impact of continued 13-valent pneumococcal conjugate vaccine (PCV13) use in the private market (uptake of 61%) in pediatric invasive pneumococcal disease (pIPD) in Portugal (2012-2015). The most frequently detected serotypes were: 3 (n = 32, 13.8%), 14 (n = 23, 9.9%), 1 (n = 23, 9.9%), 7F (n = 15, 6.4%), 19A (n = 13, 5.6%), 6B and 15B/C (both n = 12, 5.2%), and 24F, 10A and 12B (all with n = 10, 4.3%). Taken together, non-PCV13 serotypes were responsible for 42.2% of pIPD with a known serotype. The use of PCR to detect and serotype pneumococci in both pleural and cerebrospinal fluid samples contributed to 18.1% (n = 47) of all pIPD. Serotype 3 was mostly detected by PCR (n = 21/32, 65.6%) and resulted from a relevant number of vaccine failures. The incidence of pIPD varied in the different age groups but without a clear trend. There were no obvious declines of the incidence of pIPD due to serotypes included in any of the PCVs, and PCV13 serotypes still accounted for the majority of pIPD (57.8%). Our study indicates that a higher vaccination uptake may be necessary to realize the full benefits of PCVs, even after 15 years of moderate use, and highlights the importance of using molecular methods in pIPD surveillance, since these can lead to substantially increased case ascertainment and identification of particular serotypes as causes of pIPD.

Establishment of a bioluminescent canine B-cell lymphoma xenograft model for monitoring tumor progression and treatment response in preclinical studies.

Canine diffuse large B-cell lymphoma (DLBCL) is one of the most common cancers in dogs which shares remarkable similarities with its human counterpart, making the dog an excellent model for the investigation of novel therapeutic agents. However, the integration of canine lymphoma in comparative studies has been limited due in part to the lack of suitable xenograft mouse models for preclinical studies. To overcome these limitations, we established and characterized a localized subcutaneous bioluminescent canine DLBCL xenograft mouse model. The canine CLBL-1 cell line stably expressing the luciferase and green fluorescent protein reporters was generated and used to establish the xenograft tumor model. A pilot study was first conducted with three different cell densities (0.1×10(6), 0.5×10(6) and 1×10(6) cells) in SCID mice. All mice presented homogeneous tumor induction within eight days after subcutaneous injection, with a 100% engraftment efficiency and no significant differences were observed among groups. The tumors were highly aggressive and localized at the site of inoculation and reproduced histological features and immunophenotype consistent with canine DLBCL. Importantly, xenograft tumors were detected and quantified by bioluminescent imaging. To assess response to therapy, a therapeutic study with a histone deacetylase inhibitor, panobinostat, was performed. The results demonstrated that panobinostat (20 mg/kg) efficiently inhibited tumor growth and that bioluminescent imaging allowed the monitorization and quantification of tumor response to therapy. In summary, this study provides a bioluminescence canine DLBCL model that offers high engraftment efficiency, preservation of tumor features, and noninvasive monitoring of tumor progression, validating the model as a promising preclinical tool for both veterinary and human medicine.

The histone deacetylase inhibitor panobinostat is a potent antitumor agent in canine diffuse large B-cell lymphoma.

Non-Hodgkin lymphoma (NHL) is one of the most common causes of cancer-related death in the United States and Europe. Although the outcome of NHL patients has improved over the last years with current therapies, the rate of mortality is still high. A plethora of new drugs is entering clinical development for NHL treatment; however, the approval of new treatments remains low due in part to the paucity of clinically relevant models for validation. Canine lymphoma shares remarkable similarities with its human counterpart, making the dog an excellent animal model to explore novel therapeutic molecules and approaches. Histone deacetylase inhibitors (HDACis) have emerged as a powerful new class of anti-cancer drugs for human therapy. To investigate HDACi antitumor properties on canine diffuse large B-cell lymphoma, a panel of seven HDACi compounds (CI-994, panobinostat, SBHA, SAHA, scriptaid, trichostatin A and tubacin) was screened on CLBL-1 canine B-cell lymphoma cell line. Our results demonstrated that all HDACis tested exhibited dose-dependent inhibitory effects on proliferation of CLBL-1 cells, while promoting increased H3 histone acetylation. Amongst all HDACis studied, panobinostat proved to be the most promising compound and was selected for further in vitro and in vivo evaluation. Panobinostat cytotoxicity was linked to H3 histone and α-tubulin acetylation, and to apoptosis induction. Importantly, panobinostat efficiently inhibited CLBL-1 xenograft tumor growth, and strongly induced acetylation of H3 histone and apoptosis in vivo. In conclusion, these results provide new data validating HDACis and, especially, panobinostat as a novel anti-cancer therapy for veterinary applications, while contributing to comparative oncology.

Pediatric Complicated Pneumonia Caused by Streptococcus pneumoniae Serotype 3 in 13-Valent Pneumococcal Conjugate Vaccinees, Portugal, 2010-2015.

Despite use of 7-valent pneumococcal conjugate vaccine, incidence of pleural effusion and empyema (pediatric complicated pneumococcal pneumonia [PCPP]) is reportedly increasing globally. We cultured and performed PCR on 152 pleural fluid samples recovered from pediatric patients in Portugal during 2010-2015 to identify and serotype Streptococcus pneumoniae. We identified only 17 cases by culture, but molecular methods identified S. pneumoniae in 68% (92/135) of culture-negative samples. The most frequent serotypes were 3, 1, and 19A, together accounting for 62% (68/109) of cases. Nineteen cases attributable to 13-valent pneumococcal conjugate vaccine (PCV13) serotypes (mostly serotype 3) were detected among 22 children age-appropriately vaccinated with PCV13. The dominance of the additional serotypes included in PCV13 among PCPP cases in Portugal continues, even with PCV13 available on the private market (without reimbursement) since 2010 and with average annual coverage of 61% among age-eligible children. Our data suggest reduced effectiveness of PCV13 against serotype 3 PCPP.

miR-363-5p regulates endothelial cell properties and their communication with hematopoietic precursor cells.

Recent findings have shown that the blood vessels of different organs exert an active role in regulating organ function. In detail, the endothelium that aligns the vasculature of most organs is fundamental in maintaining organ homeostasis and in promoting organ recovery following injury. Mechanistically, endothelial cells (EC) of tissues such as the liver, lungs or the bone marrow (BM) have been shown to produce "angiocrine" factors that promote organ recovery and restore normal organ function. Controlled production of angiocrine factors following organ injury is therefore essential to promote organ regeneration and to restore organ function. However, the molecular mechanisms underlying the coordinated production and function of such "angiocrine" factors are largely undisclosed and were the subject of the present study. In detail, we identified for the first time a microRNA (miRNA) expressed by BM EC that regulates the expression of angiocrine genes involved in BM recovery following irradiation. Using a microarray-based approach, we identified several miRNA expressed by irradiated BMEC. After validating the variations in miRNA expression by semi-quantitative PCR, we chose to study further the ones showing consistent variations between experiments, and those predicted to regulate (directly or indirectly) angiogenic and angiocrine factors. Of the mi-RNA that were chosen, miR-363-5p (previously termed miR-363*) was subsequently shown to modulate the expression of numerous EC-specific genes including some angiocrine factors. By luciferase reporter assays, miR-363-5p is shown to regulate the expression of angiocrine factors tissue inhibitor of metalloproteinases-1 (Timp-1) and thrombospondin 3 (THBS3) at post-transcriptional level. Moreover, miR-363-5p reduction using anti-miR is shown to affect EC angiogenic properties (such as the response to angiogenic factors stimulation) and the interaction between EC and hematopoietic precursors (particularly relevant in a BM setting). miR-363-5p reduction resulted in a significant decrease in EC tube formation on matrigel, but increased hematopoietic precursor cells adhesion onto EC, a mechanism that is shown to involve kit ligand-mediated cell adhesion. Taken together, we have identified a miRNA induced by irradiation that regulates angiocrine factors expression on EC and as such modulates EC properties. Further studies on the importance of miR-363-5p on normal BM function and in disease are warranted.

The majority of adult pneumococcal invasive infections in Portugal are still potentially vaccine preventable in spite of significant declines of serotypes 1 and 5.

In Portugal, pneumococcal conjugate vaccines have been administered to children outside of the national immunization plan since 2001. We determined the serotype and antimicrobial susceptibility of 1265 isolates responsible for adult invasive pneumococcal infections (IPD) between 2009 and 2011 and compared the results with previously published data from 1999 to 2008. Serotypes 3 (12.6%), 7F (10.0%), 19A (9.1%), 14 (8.4%), 1 (6.9%) and 8 (6.2%) were the most frequent and together accounted for 53.2% of adult IPD. Serotypes 1 and 5 declined significantly while serotype 34, not included in any vaccine, increased. Taken together, the serotypes included in the 13-valent conjugate vaccine (PCV13) peaked among adult IPD isolates in 2008 (70.2%) and declined since then reaching 53.5% in 2011. The decline in the serotypes included in the 23-valent polysaccharide vaccine since 2007 was also significant but much more modest with 79.2% of the isolates causing IPD in 2011 expressing these serotypes. Since the changes in serotypes causing IPD in adults coincided with the 10-valent and PCV13 introduction in children, it is unlikely that vaccination triggered these changes although it may have accelerated them. The proportion of IPD caused by serotypes included in the 7-valent conjugate vaccine remained stable (19.0%). Both penicillin non-susceptibility and erythromycin resistance increased in the study period, with serotypes 14 and 19A accounting for the majority of resistant isolates.

Use of the 13-valent conjugate vaccine has the potential to eliminate pilus carrying isolates as causes of invasive pneumococcal disease.

We examined the distribution of the two pilus island loci (PI-1 and PI-2) in all pediatric invasive pneumococcal disease (IPD) isolates recovered in Portugal over the ten year period 1999-2008 (n=575). While 199 isolates (35%) harbored PI-2, only 98 isolates (17%) harbored PI-1. A decline of isolates carrying PI-1 was observed in the post 7-valent conjugate vaccine (PCV7) period since most PI-1 carrying isolates expressed PCV7 serotypes. On the other hand, the PI-2 increase in the post-PCV7 period was associated mostly with serotypes 1 and 7F. The overall proportion of piliated isolates declined in the period following PCV7 but quickly recovered to pre-PCV7 levels (50%). Only serotypes 19F and 19A contained isolates carrying simultaneously PI-1 and PI-2 and the proportion of isolates carrying both pilus islands remained stable and low. All PI-1 clades were represented in our collection and were associated with multiple serotypes. Over 99% of pilus island carrying isolates belonged to the 13-valent conjugate vaccine serotypes.

Serotype changes in adult invasive pneumococcal infections in Portugal did not reduce the high fraction of potentially vaccine preventable infections.

We determined the serotype and antimicrobial susceptibility of 1100 isolates responsible for adult invasive pneumococcal infections (IPD) in Portugal between 2006 and 2008. Serotypes 3 (13%), 1 (12%), 7F (11%), 19A (10%) and 14 (7%) were the most frequent causes of IPD and the two later serotypes accounted for the majority of erythromycin and penicillin nonsusceptible isolates. Serotype 1 was associated with younger adults whereas serotype 3 was associated with older adults. Despite the availability of the 23-valent polysaccharide vaccine (PPV23) in Portugal since 1996, the proportion of PPV23 preventable IPD remained stable and above 80%. Comparing with previous data from Portugal, we showed a continued decline of the serotypes included in the 7-valent conjugate vaccine (PCV7) in adult IPD and a rise of serotypes included in the 13-valent conjugate vaccine, increasing its potential coverage of adult IPD to 70% in 2008. Penicillin non-susceptibility remained stable (17%) whereas erythromycin resistance (18%) has continued to rise in the post-PCV7 years.

Serotypes 1, 7F and 19A became the leading causes of pediatric invasive pneumococcal infections in Portugal after 7 years of heptavalent conjugate vaccine use.

We characterized 353 isolates responsible for pediatric invasive pneumococcal infections (IPD) in Portugal between 2006 and 2008. Serotypes included in the seven-valent conjugate vaccine (PCV7) accounted for 17% of IPD. Serotypes 1, 7F and 19A were the most frequent causes of IPD and the later consolidated as the most frequent serotype among erythromycin and penicillin non-susceptible isolates. Serotype 1 was associated with older children and empyemas, while serotype 19A was associated with IPD in younger (<2 years) children. The higher valency vaccines PCV10 and PCV13 have a potentially superior coverage, 55% and 83% respectively, but non-vaccine serotypes are emerging as important causes of IPD. A decline of resistance with patient age was noted. Comparing with previous data from Portugal, this study showed a continued decline of PCV7 serotypes and that overall resistance has stabilized following the initial decline of the first post-PCV7 years.

Denmark14-230 clone as an increasing cause of pneumococcal infection in Portugal within a background of diverse serotype 19A lineages.

Pneumococci of serotype 19A are increasingly found to be the cause of infection in various geographic regions. We have characterized the serotype 19A isolates (n = 288) found among pneumococci responsible for infections (n = 1,925) and pneumococci recovered from asymptomatic carriers (n = 1,973) in Portugal between 2001 and 2006. We show that despite the existence of serotype 19A clones that have a greater potential to cause invasive disease or an enhanced colonization capacity, the lineage that is increasing as a cause of infection in Portugal is a multiresistant clone that is competent at both. The expanding Denmark(14)-230 clone found in Portugal is disseminated in other Mediterranean countries, where it is also increasingly responsible for invasive infections in both children and adults. The lineages driving the rise of serotype 19A infections in Asia and the United States (sequence type 320 [ST320] and ST199) are either absent or account for only a small proportion of isolates in Portugal. These data highlight the importance of locally circulating clones with the ability to compete in the nasopharyngeal niche in the emergence of the serotype 19A lineages which are an increasing cause of infection in various geographic regions.

Optimal control and analysis of two-color genomotyping experiments using bacterial multistrain arrays.

BACKGROUND: Microarray comparative genomic hybridization (aCGH) evaluates the distribution of genes of sequenced bacterial strains among unsequenced strains of the same or related species. As genomic sequences from multiple strains of the same species become available, multistrain microarrays are designed, containing spots for every unique gene in all sequenced strains. To perform two-color aCGH experiments with multistrain microarrays, the choice of control sample can be the genomic DNA of one strain or a mixture of all the strains used in the array design. This important problem has no universally accepted solution. RESULTS: We performed a comparative study of the two control sample options with a Streptococcus pneumoniae microarray designed with three fully sequenced strains. We separately hybridized two of these strains (R6 and G54) as test samples using the third strain alone (TIGR4) or a mixture of the three strains as control. We show that for both types of control it is advantageous to analyze spots in separate sets according to their expected control channel signal (5-15% AUC increase). Following this analysis, the use of a mix control leads to higher accuracies (5% increase). This enhanced performance is due to gains in sensitivity (21% increase, p = 0.001) that compensate minor losses in specificity (5% decrease, p = 0.014). CONCLUSION: The use of a single strain control increases the error rate in genes that are part of the accessory genome, where more variation across unsequenced strains is expected, further justifying the use of the mix control.

The presence of the pilus locus is a clonal property among pneumococcal invasive isolates.

BACKGROUND: Pili were recently recognized in Streptococcus pneumoniae and implicated in the virulence of this bacterium, which led to the proposal of using these antigens in a future pneumococcal vaccine. However, pili were found to be encoded by the rlrA islet that was not universally distributed in the species. We examined the distribution of the pilus islet, using the presence of the rlrA gene as a marker for the locus, among a collection of invasive isolates recovered in Portugal and analyzed its association with capsular serotypes, clusters defined by the pulsed-field gel electrophoretic profiles (PFGE) and multilocus sequence types. RESULTS: Only a minority of the isolates were positive for the presence of the rlrA gene (27%). There was a high correspondence between the serotype and the presence or absence of rlrA (Wallace coefficient, W = 0.778). In particular, there was an association between the presence of rlrA and the vaccine serotypes 4, 6B, 9V and 14 whereas the gene was significantly absent from other serotypes, namely 1, 7F, 8, 12B and 23F, a group that included a vaccine serotype (23F) and serotype 1 associated with enhanced invasiveness. Even within serotypes, there was variation in the presence of the pilus islet between PFGE clones and a higher Wallace coefficient (W = 0.939) indicates that carriage of the islet is a clonal property of pneumococci. Analysis of rlrA negative isolates revealed heterogeneity in the genomic region downstream of the rfl gene, the region where the islet is found in other isolates, compatible with recent loss of the islet in some lineages. CONCLUSION: The pilus islet is present in a minority of pneumococcal isolates recovered from human invasive infections and is therefore not an essential virulence factor in these infections. Carriage of the pilus islet is a clonal property of pneumococci that may vary between isolates expressing the same serotype and loss and acquisition of the islet may be ongoing.

Emergence of optochin resistance among Streptococcus pneumoniae in Portugal.

In most clinical microbiology laboratories optochin susceptibility is used in the screening and identification of Streptococcus pneumoniae. We report the characterization of 32 optochin-resistant S. pneumoniae strains from 10 laboratories that constituted 3.2% of all isolates recovered in 2005 in 30 laboratories in Portugal. Resistant isolates consisted of bile-soluble optochin-susceptible and optochin-resistant subpopulations with identical antimicrobial susceptibility patterns, capsular types and pulsed-field gel electrophoresis (PFGE) profiles. The most frequent serotypes--1, 19A, 11A, 3, 8, and 15A--were all common serotypes present in infection and colonization isolates in the country. The PFGE profiles of the 32 isolates corresponded to those of previously identified clones and confirmed that the emergence of these strains could not be attributed to clonal expansion. Clinical laboratories must be aware that optochin-resistant pneumococci are presently circulating in the community. Because accurate identification of S. pneumoniae is essential for correct diagnosis and adequate therapy of patients, we recommend that at least the bile solubility test should be routinely performed in cases of suspected pneumococcal etiology, even if the isolates are optochin-resistant.