CRISPR-Cas systems in enterococci.

Enterococci are members of the microbiota of humans and other animals. They can also be found in the environment, associated with food, healthcare infections, and hospital settings. Due to their wide distribution, they are inserted in the One Health context. The selective pressure caused by the extensive use of antimicrobial agents in humans, animals, and agriculture has increased the frequency of resistance to various drugs among enterococcal species. CRISPR-Cas system, an important prokaryotic defense mechanism against the entry of mobile genetic elements, may prevent the acquisition of genes involved in antimicrobial resistance and virulence. This system has been increasingly used as a gene editing tool, which can be used as a way to recognize and inactivate genes of interest. Here, we conduct a review on CRISPR systems found in enterococci, considering their occurrence, structure and organization, mechanisms of action and use as a genetic engineering technology. Type II-A CRISPR-Cas systems were shown to be the most frequent among enterococcal species, and the orphan CRISPR2 was the most commonly found system (54.1%) among enterococcal species, especially in Enterococcus faecalis. Distribution of CRISPR systems varied among species. CRISPR systems had 1 to 20 spacers, with size between 23 and 37 bp and direct repeat sequences from 25 to 37 bp. Several applications of the CRISPR-Cas biotechnology have been described in enterococci, mostly in vitro, using this editing tool to target resistance- and virulence-related genes.

Detection of Mycoplasma spp. in free-living seabirds.

This study aimed to investigate the presence of Mycoplasma spp. and identify the species of mycoplasma isolates obtained from seabirds found on Brazilian coastal beaches. Tracheal and cloacal swab samples were collected from 50 seabirds rescued by three conservation and marine animal rehabilitation centers located in Brazil. The tracheal and cloacal samples were subjected to mycoplasma culture and the isolates were identified through PCR. A "Mollicutes-specific" 16S rRNA PCR reaction was employed for triage. Four species-specific PCR reactions were used to detect Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis, or M. gallinarum. The Mollicutes positive and species negative samples were submitted do 16S rRNA sequencing. Eighteen (36%) of 50 seabirds tested positive for mycoplasma by culture. In the PCR for the genus, 28 (56%) of 50 seabirds were positive for Mycoplasma spp., with 13 (26%) detected in the trachea, one (2%) in the cloaca, and 14 (28%) in both sites. In the species-specific PCR, M. gallisepticum was detected in 17.8%, and M. meleagridis in 17.8%. Both species were detected in 14.3%. Of the isolates not characterized at species level, we obtained ten sequences and they were divided into three clusters. The first cluster was closely related to M. meleagridis, the second to M. synoviae, and the third grouped M. tully, M. gallisepticum, and M. imitans. Four and five of nine species of seabirds studied had mycoplasma detected by culture or PCR, respectively. Mycoplasmas were found in the majority of the animals studied, with the highest prevalence proportionally found in Sula leucogaster, and the lowest in Fregata magnificens. The phylogenetic analysis identified Mycoplasma spp. adapted to aquatic birds.

Antimicrobial Resistance in Streptococcus pneumoniae before and after the Introduction of Pneumococcal Conjugate Vaccines in Brazil: A Systematic Review.

Streptococcus pneumoniae causes serious illnesses, such as pneumonia, bacteremia, and meningitis, mainly in immunocompromised individuals and those of extreme ages. Currently, pneumococcal conjugate vaccines (PCVs) are the best allies against pneumococcal diseases. In Brazil, the 10-valent and 13-valent PCVs have been available since 2010, but the threat of antimicrobial resistance persists and has been changing over time. We conducted a systematic review of the literature with works published since 2000, generating a parallel between susceptibility data on isolates recovered from colonization and invasive diseases before and after the implementation of PCVs for routine childhood use in Brazil. This systematic review was based on the Cochrane Handbook for Systematic Reviews of Interventions and Preferred Reporting Items for Systematic Literature Reviews and Meta-Analyses (PRISMA) guidelines. Despite the inclusion of PCVs at a large scale in the national territory, high frequencies of non-susceptibility to important drugs used in pneumococcal diseases are still observed, especially penicillin, as well as increasing resistance to macrolides. However, there are still drugs for which pneumococci have a comprehensive sensitivity profile.

Carriage prevalence, serotype distribution, antimicrobial resistance, pspA typing and pilus islets of Streptococcus pneumoniae isolated from adults living in a Brazilian urban slum.

INTRODUCTION: For Brazilian adults, pneumococcal vaccines have been usually taken only by those who are at higher risk for development of pneumococcal diseases. Since populations from lower socioeconomic status are at high risk of acquiring pneumococcal infections, we investigated the carriage prevalence, colonization risk factors, capsular and surface protein types, and antimicrobial resistance among pneumococcal isolates recovered from adults living in a Brazilian urban slum. METHODS: Between September-December 2016, we conducted a cross-sectional study among individuals aged ≥ 18 years who attended a public primary clinic in Niterói/RJ, Brazil. Pneumococci were isolated by culture on sheep blood agar plates with and without gentamicin. Antimicrobial susceptibility was determined for all isolates. We used PCR to determine capsular types, PspA families (Fam) and pilus islets (PI). RESULTS: Of 385 adults, 32 (8.3 %) were pneumococcal carriers. Three carriers had two different pneumococci, totaling 35 isolates. After multivariate analysis, smoking, previous hospitalization, alcohol consumption and co-habitation with children aged < 6 years increased the odds of pneumococcal carriage, but antibiotic use in the previous 2 weeks was found to be a protective factor. Fourteen different serogroups/serotypes were detected and the prevalent ones were 9 N/L, 10A, 15B/C and 35F/47F (n = 3; 8.6 % each). Non-typeable (NT) isolates made up 31.4 %. All isolates were susceptible to chloramphenicol, levofloxacin and vancomycin. We found eight (22.9 %) penicillin non-susceptible pneumococci (PNSP) with minimum inhibitory concentrations (MICs) of 0.38-1.5 µg/mL. The two (5.7 %) erythromycin-resistant isolates had MIC > 256 µg/mL, cMLSB phenotype and the erm(B) gene. Twelve (34.3 %) and 17 (48.6 %) isolates had PspA Fam1 and Fam2, respectively. Three (8.6 %) isolates had genes for pilitwo PI-1 and one PI-2. CONCLUSION: We detected a low frequency of pneumococcal carriage among the adult population, but a high diversity of serotypes. Frequencies of PNSP and NT isolates resistant to antimicrobial agents are concerning.

Virulence of Lactobacillus spp. misidentified as Enterococcus faecalis from children's carious dentine.

Objective: This study aimed to search for Enterococcus faecalis in children's deep carious dentine and characterize their virulence traits.Material and Methods: Eight isolates from 15 carious molars identified by 16S rDNA species-specific PCR as E. faecalis were included. These eight isolates were subject to identification by MALDI-TOF and characterized regarding: (i) bacterial aggregation and biofilm formation on polystyrene and glass, with/without saliva, as single or dual-species (associated to Streptococcus mutans); (ii) environmental pH measurement before and after 24 h incubation; (iii) acidogenicity; (iv) gelatinase production; (v) macrophage adherence; and (vi) toxicity towards Caenorhabditis elegans. Statistical analyses were performed using two-way ANOVA/Tukey or Fisher's exact tests.Results: All isolates initially identified as E. faecalis by PCR were correctly identified as Lactobacillus by MALDI-TOF, being designated as Lactobacillus misidentified as Enterococcus (LME). These isolates produced biofilm in the presence of saliva and in the dual-species assays. Bacterial aggregation was only observed in the dual-species model. After 24 h, environmental pH dropped from 7.5 to 4.5 for seven of eight isolates, and to 4.0 in all dual-species models. LME isolates were acidogenic, none of them produced gelatinase or adhered to macrophages, but all presented toxicity towards C. elegans.Conclusions: No E. faecalis were identified in the children's caries lesions. All LME isolates presented important virulence traits, including biofilm formation and high acidogenicity, which cause enamel demineralization, that might increase the risk of dental caries in children carrying LME. Thus, the correct identification and in-depth virulence characterization of microorganisms isolated from dental caries are important to understand the dynamics of this disease.

Going Online in Pandemic Time: A DivulgaMicro Workshop Experience.

DivulgaMicro is a Brazilian science communication and outreach project run by three young female scientists. In 2020, due to the COVID-19 pandemic, we promoted the first virtual edition of the DivulgaMicro Workshop. Here, we describe adaptations implemented to deliver the virtual edition and its assessment by the participants. We offered a 3-day workshop addressing manuscript writing and publishing, poster design and oral presentations, and strategies for effectively communicating with the lay public on Days 1, 2, and 3, respectively. Each daily session was divided into short lectures and active learning through interactive and cooperative activities. We applied pre- and postsession surveys daily to assess participants' learning, with an opinion questionnaire at the end of the workshop. We selected 50 of 221 applicants, but only 38 of those selected participated in the workshop. Correct answers were significantly higher (p < 0.01) in the postsession surveys each day. Most of the 34 participants who answered the opinion questionnaire (97%) would recommend the workshop to their colleagues. Participants stated that the workshop content was transmitted in a clear and straightforward manner, and they considered the online format suitable for knowledge acquisition. Participants were satisfied with the organization, dynamics, and interactivity. Topics addressed on Day 3 (61.8%) and Day 1 (23.5%) were most liked and were considered most useful in the participants' careers. We believe that the overall success of the workshop is due to the combination of short lectures with active-learning activities, the use of virtual platforms that enabled effective communication between participants and instructors, and the support of our collaborators.

CRISPR elements and their association with antimicrobial resistance and virulence genes among vancomycin-resistant and vancomycin-susceptible enterococci recovered from human and food sources.

We aimed to investigate the occurrence of CRISPR elements in the genomes of vancomycin-resistant (VRE) and vancomycin-susceptible (VSE) enterococci and their association with the presence of antimicrobial resistance and virulence genes. We analyzed 180 isolates, including 91 VRE and 89 VSE. Isolates were identified by PCR or MALDI-TOF. Antimicrobial susceptibility and MICs for vancomycin were determined by the disk-diffusion method and E-test®, respectively. The presence of resistance and virulence genes, as well as CRISPR elements, was investigated by PCR. We identified 95 (53%) E. faecalis, 78 (43%) E. faecium, five (2.8%) E. gallinarum, and one (0.6% each) E. casseliflavus and E. durans. The highest and the lowest non-susceptibility frequencies were observed for erythromycin (n = 152; 84.4%) and fosfomycin (n = 5; 2.8%), respectively. Most erythromycin-resistant isolates had the erm(B) gene (106/152; 69.7%). Of 118 (65.6%) isolates with high-level resistance to aminoglycoside, 69 (58.5%) had at least one aminoglycoside resistance gene, mostly ant(6)-Ia and aac(6')-Ie + aph(2″)-Ia. We found at least one virulence gene among 135 (75%) isolates, mostly gelE (79/180; 43.9%). Ninety-two (51.1%) isolates had at least one CRISPR element, especially CRISPR3 (62/92; 67.4%). CRISPR elements were more common among E. faecalis, in which we observed a relationship between the absence of CRISPR and the presence of the vanA resistance gene, and the hyl and esp virulence genes. Among VRE. faecium, a relationship was found between the absence of CRISPR and the hyl gene. In conclusion, we found evident associations between the lack of CRISPR elements with species, multidrug resistance, and major resistance- and virulence-associated genes.

Prevalence of PspA families and pilus islets among Streptococcus pneumoniae colonizing children before and after universal use of pneumococcal conjugate vaccines in Brazil.

In 2010, the 10-valent (PCV10) and 13-valent (PCV13) pneumococcal conjugate vaccines were introduced in Brazil to immunize children, resulting in serotype replacement. We analyzed 253 carriage isolates recovered from children aged <6 years in Brazil, including 124 and 129 isolates from the pre-PCV10/13 (December 2009-July 2010) and post-PCV10/13 (September-December 2014) periods, respectively, to investigate the prevalence of PspA families and pilus islets, potential vaccine candidates. Serotypes and resistance profiles were previously characterized. We used PCR to type PspA families (Fam1-3) and pilus islets (PI-1 and PI-2). We identified the PspA family of 130 (51.4%) isolates. PspA families 1, 2, and 3 were identified in 12.2%, 38.7%, and 0.4% of the isolates, respectively. Eighteen (58.1%) Fam1 isolates were serogroup 6. Nine (81.8%) of 11 serotype 14 isolates were Fam2. Fam1 isolates resistant to penicillin (50%), erythromycin (43.7%), clindamycin (31.2%), and chloramphenicol (6.2%) were only found after PCV10/13 introduction. Resistance among Fam2 isolates was higher in the post-PCV10/13 period to erythromycin (1.8% vs. 18.6%), clindamycin (0 vs. 13.9%), and tetracycline (10.9% vs. 16.3%). PI-I was detected in 42 (16.6%) isolates. Fourteen (56%) of 25 serotype 15B/C and nine (81.8%) of 11 serotype 14 isolates had PI-1 (p < 0.01). Eight (3.2%) isolates had PI-2, and six (75%) were serogroup 19. Five (2%) serogroup 19 isolates had both PI-1 and PI-2. We found associations between serogroups/serotypes, PspA families, and pilus islets, but distribution of PspA families and pilus islets was similar in both periods. After universal vaccination, we observed higher antimicrobial resistance frequencies, regardless PspA or pilus types.

Differences in gram-positive bacterial colonization and antimicrobial resistance among children in a high income inequality setting.

BACKGROUND: Staphylococcus aureus and beta-hemolytic streptococci (BHS) diseases disproportionately affect populations in middle/low-income countries. To assess if this disparity is reflected in colonization by these organisms, we compared their colonization frequency among children from different socioeconomic status (SES) communities in a city with high income inequality. METHODS: Between May-August 2014, we collected nasal and throat swabs to investigate S. aureus and BHS colonization among children who attended private and public pediatric clinics. Patients were classified as high SES, middle/low SES, and slum residents. We investigated the antimicrobial resistance profile, the SCCmec types and the presence of PVL genes among methicillin-resistant S. aureus (MRSA). We also examined the antimicrobial resistance profile and serogroups of BHS. RESULTS: Of 598 children, 221 (37%) were colonized with S. aureus, of which 49 (22%) were MRSA. MRSA colonization was higher in middle/low SES (n = 18; 14%) compared with high SES (n = 17; 6%) and slum (n = 14; 8%) residents (p = 0.01). All MRSA strains were susceptible to clindamycin, nitrofurantoin, and rifampin. The highest non-susceptibility frequency (42.9%) was observed to erythromycin. SCCmec type V was only found in isolates from high SES children; types I and II were found only in middle/low SES children. Ten (20%) MRSA isolates carried PVL genes. Twenty-four (4%) children were BHS carriers. All BHS (n = 8) found in high SES children and six (67%) isolates from slum patients belonged to group A. All group B streptococci were from middle/low SES children, corresponding to five (71%) of the seven BHS isolated in this group. BHS isolates were susceptible to all drugs tested. CONCLUSIONS: Children from different SES communities had distinct bacterial colonization profiles, including MRSA carriage. Public health officials/researchers should consider SES when assessing disease transmission and control measures.

Evolution of Penicillin Non-susceptibility Among Streptococcus pneumoniae Isolates Recovered From Asymptomatic Carriage and Invasive Disease Over 25 years in Brazil, 1990-2014.

Streptococcus pneumoniae is a major cause of community-acquired pneumonia and meningitis, and it is also found as a commensal, colonizing the human upper respiratory tract of a portion of the human population. Its polysaccharide capsule allows the recognition of more than 90 capsular types and represents the target of the currently available pneumococcal conjugate vaccines (PCVs), such as the 10-valent (PCV10) and the 13-valent (PCV13). Penicillin non-susceptible pneumococci (PNSP) have been listed as one of the current major antimicrobial-resistant pathogen threats. In Brazil, the emergence of PNSP was initially detected in the mid 1990s and PCV10 has been part of the National Immunization Program since 2010. Here, we investigated the distribution of capsular types and penicillin susceptibility profiles of 783 pneumococcal strains isolated in Brazil between 1990 and 2014 to assess the evolution of penicillin non-susceptibility among pneumococci associated with asymptomatic carriage and invasive pneumococcal disease (IPD). The most common serotypes among carriage isolates were 19F, 6B, 6C, 23F, and 14. Among IPD isolates, the most frequent types were 14, 3, 6B, 5, 19F, and 4. We detected 21 types exclusively associated with IPD isolates, whereas non-typeable (NT) isolates were only detected in carriage. Nearly half of the isolates belonged to PCV10 serotypes, which remarkably decreased in occurrence (by nearly 50%) after PCV10 introduction (2011-2014), while non-PCV10 serotypes increased. PNSP frequency and levels were much higher among carriage isolates, but PNSP belonging to PCV10 serotypes were more common in IPD. While the occurrence of PNSP has decreased significantly among IPD isolates since 2011, it kept increasing among carriage strains. Such a difference can be attributed to the serotypes that emerged in each clinical source after PCV10 usage. PNSP with multidrug resistance profiles that emerged within carriage isolates comprised mostly serotypes 6C and 35B, as well as NT isolates. In turn, penicillin-susceptible capsular types 3, 20, and 8 have risen among IPD. Overall, our results reinforce the relevance of PNSP surveillance over a long period of time to better understand the dynamics of antimicrobial resistance in response to PCV introduction and may also contribute to improve control measures toward drug-resistant pneumococci.

Papillomavirus infections in the oral and genital mucosa of asymptomatic women

Abstract Human papillomavirus (HPV) has been found in several regions of the body, including the oral cavity. Recently, this virus has been associated with oropharyngeal cancer, but little is known about HPV transmission to the oral cavity. We carried out a study to investigate concurrent oral and cervical infections in 76 asymptomatic women attending a healthcare program. Demographic and behavior data were obtained through a structured questionnaire. Oral and cervical mucosa scrapings were collected and stored for DNA extraction. HPV DNA amplification was performed by polymerase chain reaction assay (PCR) using both primers My09/My11 and FAP59/64, followed by HPV typing with restriction fragment length polymorphism analysis (RFLP) and sequencing. The data collected revealed no risk factors for HPV infection in these 76 women. HPV prevalence of 9.2 and 5.3% was found in cervical and oral mucosa, respectively. Concurrent infections by discordant types were detected in one case only. Sequencing procedures allowed us to detect a new putative HPV 17 subtype from the Betapapillomavirus genus. Our results support the view that cervical and oral HPV infections are independent events. The observed low prevalence of both oral and cervical HPV infections could be associated with attendance in a healthcare program.

Serotypes, antimicrobial resistance and genotypes of Streptococcus pneumoniae associated with infections in cancer patients in Brazil.

We sought to characterize pneumococcal isolates associated with bacteremia, pneumonia and meningitis in cancer patients and to estimate the coverage of the available pneumococcal vaccines. Fifty isolates recovered from 49 patients attending a cancer reference center over a 1-year period were analyzed. The prevalent serotypes were: 23F (12%), 6A (8%), 3, 4, 20, and 23A (6% each). All isolates were susceptible to chloramphenicol, levofloxacin, rifampicin, and vancomycin. Resistance or reduced susceptibility to penicillin made up 14%, and one isolate was also intermediately resistant to ceftriaxone. The three (6%) erythromycin-resistant isolates presented the M or cMLSB phenotypes and harbored the mef(A/E) gene exclusively or along with the erm(B) gene. Twenty-two (44%) isolates were closely related to 11 international clones, being strongly associated with penicillin non-susceptibility. Combined immunization with the 13-valent conjugate and the 23-valent polysaccharide vaccines might contribute to reduce (76%) the burden of the pneumococcal infections in the population investigated.

Papillomavirus infections in the oral and genital mucosa of asymptomatic women.

Human papillomavirus (HPV) has been found in several regions of the body, including the oral cavity. Recently, this virus has been associated with oropharyngeal cancer, but little is known about HPV transmission to the oral cavity. We carried out a study to investigate concurrent oral and cervical infections in 76 asymptomatic women attending a healthcare program. Demographic and behavior data were obtained through a structured questionnaire. Oral and cervical mucosa scrapings were collected and stored for DNA extraction. HPV DNA amplification was performed by polymerase chain reaction assay (PCR) using both primers My09/My11 and FAP59/64, followed by HPV typing with restriction fragment length polymorphism analysis (RFLP) and sequencing. The data collected revealed no risk factors for HPV infection in these 76 women. HPV prevalence of 9.2 and 5.3% was found in cervical and oral mucosa, respectively. Concurrent infections by discordant types were detected in one case only. Sequencing procedures allowed us to detect a new putative HPV 17 subtype from the Betapapillomavirus genus. Our results support the view that cervical and oral HPV infections are independent events. The observed low prevalence of both oral and cervical HPV infections could be associated with attendance in a healthcare program.

Genetic diversity of Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil.

The aim of this study was to characterize Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil. In addition, all the clinical and hematological findings observed in these dogs were reported. PCR targeting the 16S rRNA gene was used for diagnostic purposes, and the TRP19 and TRP36 genes were sequenced to evaluate the genetic diversity. Fifteen samples were positive for E. canis. The polymerase chain reaction for the TRP19 gene resulted in 11 amplicons (11/15), which were cloned into the pGEM-T easy vector for sequencing. The complete sequence of TRP19 gene was compared to those in the GenBank, revealing high identicalness. Phylogenetic analysis on the TRP36 gene sequences demonstrated two distinct strains from two dogs, named 56C and 70C. The 56C strain was grouped with the strain Cuiaba 16, which is a hybrid strain formed by Brazilian and US genogroups; and the 70C strain was grouped with other strains of the US genogroup, thus suggesting that there are at least two genogroups of E. canis in Rio de Janeiro (US and Brazilian). Those animals, in which the 70C and 56C strains were isolated, showed distinct clinical and hematological manifestations of the disease. The appearance of different genotypes may express new phenotypes, thus resulting in different forms of presentation of the disease and making its diagnosis more complex.

Genetic diversity of Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil / Diversidade genética de cepas de Ehrlichia canis encontradas em cães naturalmente infectados no Rio de Janeiro, Brasil

The aim of this study was to characterize Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil. In addition, all the clinical and hematological findings observed in these dogs were reported. PCR targeting the 16S rRNA gene was used for diagnostic purposes, and the TRP19 and TRP36 genes were sequenced to evaluate the genetic diversity. Fifteen samples were positive for E. canis. The polymerase chain reaction for the TRP19 gene resulted in 11 amplicons (11/15), which were cloned into the pGEM-T easy vector for sequencing. The complete sequence of TRP19 gene was compared to those in the GenBank, revealing high identicalness. Phylogenetic analysis on the TRP36 gene sequences demonstrated two distinct strains from two dogs, named 56C and 70C. The 56C strain was grouped with the strain Cuiaba 16, which is a hybrid strain formed by Brazilian and US genogroups; and the 70C strain was grouped with other strains of the US genogroup, thus suggesting that there are at least two genogroups of E. canis in Rio de Janeiro (US and Brazilian). Those animals, in which the 70C and 56C strains were isolated, showed distinct clinical and hematological manifestations of 1the disease. The appearance of different genotypes may express new phenotypes, thus resulting in different forms of presentation of the disease and making its diagnosis more complex.

O objetivo deste estudo foi caracterizar as cepas de Ehrlichia canis em cães naturalmente infectados no Rio de Janeiro, Brasil. Além disso, os achados clínicos e hematológicos observados nos cães foram relatados. O gene 16S rRNA foi utilizado como alvo da PCR para fins diagnósticos, e os genes TRP19 e TRP36 para avaliar a diversidade genética. Quinze amostras foram positivas para E. canis. PCR para o gene TRP19 produziu 11 amplicons (11/15) que foram clonados no pGEM-T easy vector para sequenciamento. A comparação das sequências completas do gene TRP19 com outras sequências depositadas no GenBank revelou uma alta identidade. Duas amostras (56C e 70C) após o ensaio da PCR, tendo como alvo o gene TRP36, geraram sequências, e a análise filogenética mostrou que a cepa 56C foi agrupada com a cepa Cuiabá 16, que é uma cepa híbrida, formada pelo genogrupo Brasileiro e o genogrupo US; e a cepa 70C agrupou com as outras cepas do genogrupo US, sugerindo a existência de pelo menos dois genogrupos de E. canis no Rio de Janeiro (US e Brasileiro). Esses animais apresentaram manifestações clínicas e hematológicas distintas, e diferentes genótipos podem expressar novos fenótipos, resultando em diferentes formas de apresentação da doença e fazendo com que o diagnóstico seja mais complexo.

Multidrug-resistant nontuberculous mycobacteria isolated from cystic fibrosis patients.

Worldwide, nontuberculous mycobacteria (NTM) have become emergent pathogens of pulmonary infections in cystic fibrosis (CF) patients, with an estimated prevalence ranging from 5 to 20%. This work investigated the presence of NTM in sputum samples of 129 CF patients (2 to 18 years old) submitted to longitudinal clinical supervision at a regional reference center in Rio de Janeiro, Brazil. From June 2009 to March 2012, 36 NTM isolates recovered from 10 (7.75%) out of 129 children were obtained. Molecular identification of NTM was performed by using PCR restriction analysis targeting the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene, and susceptibility tests were performed that followed Clinical and Laboratory Standards Institute recommendations. For evaluating the genotypic diversity, pulsed-field gel electrophoresis (PFGE) and/or enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) was performed. The species identified were Mycobacterium abscessus subsp. bolletii (n = 24), M. abscessus subsp. abscessus (n = 6), Mycobacterium fortuitum (n = 3), Mycobacterium marseillense (n = 2), and Mycobacterium timonense (n = 1). Most of the isolates presented resistance to five or more of the antimicrobials tested. Typing profiles were mainly patient specific. The PFGE profiles indicated the presence of two clonal groups for M. abscessus subsp. abscessus and five clonal groups for M. abscesssus subsp. bolletii, with just one clone detected in two patients. Given the observed multidrug resistance patterns and the possibility of transmission between patients, we suggest the implementation of continuous and routine investigation of NTM infection or colonization in CF patients, including countries with a high burden of tuberculosis disease.

Nasopharyngeal carriage, serotype distribution and antimicrobial resistance of Streptococcus pneumoniae among children from Brazil before the introduction of the 10-valent conjugate vaccine.

BACKGROUND: Streptococcus pneumoniae remains a major cause of childhood morbidity and mortality worldwide. Nasopharyngeal colonization plays an important role in the development and transmission of pneumococcal diseases, and infants and young children are considered to be the main reservoir of this pathogen. The aim of this study was to evaluate the rates and characteristics associated with nasopharyngeal carriage, the distribution of serotypes and the antimicrobial resistance profiles of Streptococcus pneumoniae among children in a large metropolitan area in Brazil before the introduction of the 10-valent pneumococcal conjugate vaccine. METHODS: Between March and June 2010, nasopharyngeal swabs were collected from 242 children aged <6 years attending one day care center and the emergency room of a pediatric hospital. Pneumococcal isolates were identified by conventional methods and serotypes were determined by a sequential multiplex PCR assay and/or the Quellung reaction. The antimicrobial susceptibilities of the pneumococci were assessed by the disk diffusion method. MICs for erythromycin and penicillin were also performed. Erythromycin resistance genes were investigated by PCR. RESULTS: The overall colonization rate was 49.2% and it was considerably higher among children in the day care center. Pneumococcal carriage was more common among day care attenders and cohabitants with young siblings. The most prevalent serotypes were 6B, 19F, 6A, 14, 15C and 23F, which accounted for 61.2% of the isolates. All isolates were susceptible to clindamycin, levofloxacin, rifampicin and vancomycin. The highest rate of non-susceptibility was observed for sulphamethoxazole-trimethoprim (51.2%). Penicillin non-susceptible pneumococci (PNSP) accounted for 27.3% of the isolates (MICs of 0.12-4 µg/ml). Penicillin non-susceptibility was strongly associated with serotypes 14 and 23F. Hospital attendance and the presence of respiratory or general symptoms were frequently associated with PNSP carriage. The two erythromycin-resistant isolates (MICs of 2 and 4 µg/ml) belonged to serotype 6A, presented the M phenotype and harbored the mef(A/E) gene. CONCLUSIONS: Correlations between serotypes, settings and penicillin non-susceptibility were observed. Serotypes coverage projected for the 10-valent pneumococcal conjugate vaccine was low (45.5%), but pointed out the potential reduction of PNSP nasopharyngeal colonization by nearly 20%.

Application of DNA sequence analysis based on five different conserved genes (16S rDNA, rpoB, gdh, est and pgm) for intra-species discrimination of Bacteroides fragilis.

In the past few years, many studies revealed a remarkable genetic variability in Bacteroides fragilis species, and the existence of two divisions was proposed according to presence or absence of the cfiA (metallo-ß-lactamase/carbapenemase) gene. The aim of this study was to evaluate the use of DNA sequence analysis for glutamate dehydrogenase (gdh), phosphoglucomutase (pgm) and esterase (est) metabolic genes, in comparison to RNA polymerase ß subunit (rpoB) and 16S ribosomal RNA (rrs) gene sequencing, to identify the presence of these two groups in seventeen B. fragilis strains. Based on phylogenetic trees, only the est gene sequences generated a classification similar to rrs- and rpoB-genes. On the other hand, the genes pgm and gdh did not allow the discrimination of these divisions. The est gene sequence can be suggested as an additional tool for differentiation of the two groups in B. fragilis, providing highly reproducible and reliable data in B. fragilis taxonomy.