Trends in serotype distribution and antimicrobial susceptibility pattern of invasive Haemophilus influenzae isolates from Brazil, 2009-2021.

INTRODUCTION: Invasive Haemophilus influenzae (Hi) disease poses a significant global health challenge. With the relaxation of COVID-19 pandemic measures and declining H. influenzae serotype b (Hib) vaccination coverage, there is concern about a potential increase in Hi cases worldwide. METHODOLOGY: This study analyzed 1437 invasive Hi isolates in Brazil over 13 years, determining capsular serotypes, antimicrobial susceptibility, and genetic relatedness through multilocus sequence typing. RESULTS: The primary source of isolation for these invasive H. influenzae isolates was blood (54.4%), followed by cerebrospinal fluid (37.1%) and lung specimens (8.5%), respectively. Consequently, bacteremia (47%) was the most common clinical presentation, followed by meningitis (39.6%) and pneumonia (13.4%). Non-encapsulated Hi (NTHi) predominated among the isolates (51.4%), along with serotype a (22%) and serotype b (21.5%) among the encapsulated isolates. The majority of the encapsulated isolates were isolated from children under 14 years of age (76.7%), while NTHi isolates were identified in patients older than 15 years, particularly those ≥ 60 years old (40%). Ampicillin resistance was observed in 17.1% of cases, displaying ß-lactamase production as the principal resistance mechanism. MLST revealed a diverse NTHi population, whereas the encapsulated isolates presented a clonal structure. CONCLUSION: This study describes the prevalence of NTHi isolates circulating in Brazil after two decades of the Hib vaccine immunization program. Continuous universal surveillance is crucial for implementing prompt public health measures to prevent and control invasive Hi disease and monitor changes in antibiotic resistance profiles.

Molecular characterization and whole-genome sequencing of Corynebacterium diphtheriae causing skin lesion.

We present a case of skin lesion caused by nontoxigenic Corynebacterium diphtheriae. Genomic taxonomy analyses corroborated the preliminary identification provided by mass spectrometry. The strain showed a susceptible phenotype with increased exposure to penicillin, the first drug of choice for the treatment. An empty type 1 class integron carrying only the sul1 gene, which encodes sulfonamide resistance, was found flanked by transposases. Virulence factors involved in adherence and iron uptake, as well as the CRISPR-Cas system, were predicted. MLST analysis revealed the ST-681, previously reported in French Guiana, a European territory.

Molecular characterization and phylogenetic analysis of the first Corynebacterium rouxii strains isolated in Brazil: a recent member of Corynebacterium diphtheriae complex.

BACKGROUND: Corynebacterium diphtheriae complex was formed by the species C. diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis in the recent past. In addition to C. diphtheriae, C. ulcerans and C. pseudotuberculosis species can carry the tox gene, which encodes diphtheria toxin. Currently, three new species have been included in the complex: Corynebacterium rouxii, Corynebacterium silvaticum, and Corynebacterium belfantii. C. rouxii is derived from the ancient Belfanti biovar of C. diptheriae. We provide the complete genome sequences of two non-toxigenic strains C. rouxii isolated from a cat with a purulent infection in Brazil. The taxonomic status and sequence type, as well as the presence of resistance and virulence genes, and CRISPR-Cas system were additionally defined. RESULTS: The genomes showed an average size of 2.4 Mb and 53.2% GC content, similar to the type strain of the species deposited in Genbank/NCBI. Strains were identified as C. rouxii by the rMLST database, with 95% identity. ANI and DDH in silico were consistent with values above the proposed cut-off points for species limit, corroborating the identification of the strains as C. rouxii. MLST analyses revealed a new ST, which differs from ST-537 only by the fusA allele. No horizontal transfer resistance gene was predicted in both genomes and no mutation was detected in the constitutive genes gyrA and rpoB. Some mutations were found in the seven penicillin-binding proteins (PBPs) detected. The tox gene was not found, but its regulatory gene dtxR was present. Among the predicted virulence genes are those involved in iron uptake and adherence, in addition to the DIP0733 protein involved in epithelial cell adhesion and invasion. The CRISPR-Cas type I-E system was detected in both genomes, with 16 spacer sequences each. Of them, half are unknown according to the databases used, indicating that there is an unexplored reservoir of corynebacteriophages and plasmids. CONCLUSIONS: This is the first genomic study of C. rouxii reported in Brazil. Here we performed taxonomic analysis and the prediction of virulence factors. The genomic analyses performed in this study may help to understand the potential pathogenesis of non-toxigenic C. rouxii strains.

Phenotypic and molecular characterization and complete genome sequence of a Corynebacterium diphtheriae strain isolated from cutaneous infection in an immunized individual.

Diphtheria is an infectious disease potentially fatal that constitutes a threat to global health security, with possible local and systemic manifestations that result mainly from the production of diphtheria toxin (DT). In the present work, we report a case of infection by Corynebacterium diphtheriae in a cutaneous lesion of a fully immunized individual and provided an analysis of the complete genome of the isolate. The clinical isolate was first identified by MALDI-TOF Mass Spectrometry. The commercial strip system and mPCR performed phenotypic and genotypic characterization, respectively. The antimicrobial susceptibility profile was determined by the disk diffusion method. Additionally, genomic DNA was sequenced and analyzed for species confirmation and sequence type (ST) determination. Detection of resistance and virulence genes was performed by comparisons against ResFinder and VFDB databases. The isolate was identified as a nontoxigenic C. diphtheriae biovar Gravis strain. Its genome presented a size of 2.46 Mbp and a G + C content of 53.5%. Ribosomal Multilocus Sequence Typing (rMLST) allowed the confirmation of species as C. diphtheriae with 100% identity. DDH in silico corroborated this identification. Moreover, MLST analyses revealed that the isolate belongs to ST-536. No resistance genes were predicted or mutations detected in antimicrobial-related genes. On the other hand, virulence genes, mostly involved in iron uptake and adherence, were found. Presently, we provided sufficient clinical data regarding the C. diphtheriae cutaneous infection in addition to the phenotypic and genomic data of the isolate. Our results indicate a possible circulation of ST-536 in Brazil, causing cutaneous infection. Considering that cases of C. diphtheriae infections, as well as diphtheria outbreaks, have still been reported in several regions of the world, studies focusing on taxonomic analyzes and predictions of resistance genes may help to improve the diagnosis and to monitor the propagation of resistant clones. In addition, they can contribute to understanding the association between variation in genetic factors and resistance to antimicrobials.

Dynamics of antimicrobial resistance of Streptococcus pneumoniae following PCV10 introduction in Brazil: Nationwide surveillance from 2007 to 2019.

BACKGROUND: Brazil introduced 10-valent pneumococcal conjugate vaccine (PCV10) into its immunization program in 2010. We assessed antimicrobial susceptibility of Streptococcus pneumoniae (Spn) obtained from a national surveillance system for invasive pneumococcal diseases (IPD) before/after PCV10 introduction. METHODS: Antimicrobial non-susceptible isolates were defined as intermediate or resistant. Minimum inhibitory concentrations (MICs) to penicillin and ceftriaxone were analyzed by year. Antimicrobial susceptibility rates were assessed for each three-year-period using the pre-PCV10-period as reference. Susceptibility of vaccine-types was evaluated for 2017-2019. RESULTS: 11,380 isolates were studied. Spn with penicillin ≥ 0.125 mg/L and ceftriaxone ≥ 1.0 mg/L decreased in the three-years after PCV10 introduction (2011-2013: penicillin, 28.1-22.5%; ceftriaxone, 11.3%-7.6%) versus pre-PCV10-years (2007-2009: penicillin, 33.8-38.1%; ceftriaxone, 17.2%-15.6%). After 2013, the proportion of Spn with those MICs to penicillin and ceftriaxone increased to 39.4% and 19.7% in 2019, respectively. Non-susceptibility to penicillin and ceftriaxone increased in 2014-2016, and again in 2017-2019 especially among children < 5 years with meningitis (penicillin, 53.9%; ceftriaxone, 28.0%); multidrug-resistance reached 25% in 2017-2019. Serotypes 19A, 6C and 23A were most associated with antimicrobial non-susceptibility. CONCLUSIONS: Antimicrobial non-susceptible Spn decreased in the three-years after vaccination but subsequently increased and was associated with non-PCV10-types. Antimicrobial susceptibility surveillance is fundamental for guiding antibiotic therapy policies.

Cutaneous infection by non-diphtheria-toxin producing and penicillin-resistant Corynebacterium diphtheriae strain in a patient with diabetes mellitus.

Diphtheria is a potentially fatal infection, mostly caused by diphtheria toxin (DT)-producing Corynebacterium diphtheriae strains. During the last decades, the isolation of DT-producing C. diphtheriae strains has been decreasing worldwide. However, non-DT-producing C. diphtheriae strains emerged as causative agents of cutaneous and invasive infections. Although endemic in countries with warm climates, cutaneous diphtheria is rarely reported in Brazil. Presently, an unusual case of skin lesion in a Brazilian elderly diabetic patient infected by a penicillin-resistant non-DT-producing C. diphtheriae strain was reported. Laboratory diagnosis included mass spectrometry and multiplex PCR analyses. Since cutaneous diphtheria lesions are possible sources of secondary diphtheria cases and systemic diseases and considering that penicillin is the first line of antimicrobial agent for the treatment of these infections, the detection of penicillin-resistant strains of diphtheria bacilli should be a matter of concern. Thus, cases similar to the presently reported should be appropriately investigated and treated, particularly in patients with risk factor (s) for the development of C. diphtheriae invasive infections, such as diabetes. Moreover, health professionals must be aware of the presence of C. diphtheriae in cutaneous lesions of lower limbs, a common type of morbidity in diabetic patients, especially in tropical and subtropical countries.

Antimicrobial susceptibility of Streptococcus pneumoniae isolated from patients in the northeastern macroregion of São Paulo state, Brazil, 1998-2013 / Suscetibilidade antimicrobiana de Streptococcus pneumoniae isolados de pacientes na macrorregião nordeste do estado de São Paulo, Brasil, entre 1998 e 2013

ABSTRACT Introdution: There are reports worldwide about the increase in infections caused by Streptococcus pneumoniae resistant to antimicrobials. Objective: Evaluate the susceptibility profile of serotypes of Streptococcus pneumoniae associating them with pneumococcal invasive diseases (PID), as well as antimicrobial therapies. Method: This is a retrospective cross-sectional research involving secondary data from 1998 to 2013, in the northeastern macroregion of São Paulo state, Brazil, composed of Araraquara, Barretos, Franca and Ribeirão Preto regions, with 90 municipalities. At Instituto Adolfo Lutz (IAL), isolated strains from patients with PID were subjected to identification, serotyping and antimicrobial susceptibility testing. Results: From 796 strains analyzed, 14.8% (n = 118) were resistant to penicillin, being 3% (n = 24) with intermediate resistance and 11.8% (n = 94) with full resistance, especially in patients with meningitis. Moreover, resistance to ceftriaxone was 5.3%: 34 (4.3%) with intermediate resistance and 8 (1%) with full resistance. We point out that the greatest level of resistance profiles was observed against sulfamethoxazole/trimethoprim (SMT): 350 (49.4%). On the other hand, antimicrobial susceptibility was described above 90% to chloramphenicol: 99.6% (n = 696), erythromycin: 94.7% (n = 664), ceftriaxone: 94.7% (n = 754) and fully susceptible to vancomycin. Among the 18 most common serotypes, 9V and 14 showed less susceptibility to SMT, to penicillin and ceftriaxone; 19A to SMT and penicillin; 1 to SMT; 12F and 3 to chloramphenicol; 6A to SMT; 6B 23F to erythromycin and penicillin. Conclusion: Monitoring of Streptococcus pneumoniae antimicrobial resistance is essential to guide the appropriate empirical treatment of pneumococcal disease.

RESUMO Introdução: Em todo o mundo existem relatos de aumento das infecções causadas por Streptococcus pneumoniae resistentes aos antimicrobianos. Objetivo: Avaliar o perfil de suscetibilidade dos sorotipos de Streptococcus pneumoniae, associando-os com as doenças invasivas pneumocócicas (DIP), bem como com as terapias antimicrobianas. Método: Trata-se de um seguimento retrospectivo com enfoque em dados secundários de 1998 a 2013, na macrorregião nordeste do estado de São Paulo, Brasil, composta pelas regiões de Araraquara, Barretos, Franca e Ribeirão Preto, com 90 municípios. No Instituto Adolfo Lutz (IAL), as cepas isoladas de pacientes com DIP foram submetidas a identificação, sorotipagem e teste de suscetibilidade aos antimicrobianos. Resultados: Das 796 cepas analisadas, 14,8% (n = 118) apresentaram resistência a penicilina, sendo 3% (n = 24) com resistência intermediária e 11,8% (n = 94) com resistência plena, principalmente em pacientes com meningite. Para ceftriaxona, a resistência foi de 5,3%: 34 (4,3%) com resistência intermediária e 8 (1%) com resistência plena. Há de salientar que o maior nível de resistência das cepas foi observado para sulfametoxazol/trimetoprima (SMT): 350 (49,4%). Por outro lado, a suscetibilidade foi descrita acima de 90% para cloranfenicol: 99,6% (n = 696); eritromicina: 94,7% (n = 664); ceftriaxona: 94,7% (n = 754) e total para vancomicina. Entre os 18 sorotipos mais frequentes, 9V e 14 apresentaram menor suscetibilidade a SMT, penicilina e ceftriaxona; 19A a SMT e penicilina; 1 a SMT; 12F e 3 a cloranfenicol; 6A a SMT; 6B a eritromicina e 23F a penicilina. Conclusão: O monitoramento da resistência antimicrobiana do Streptococcus pneumoniae é fundamental para direcionar o tratamento empírico das doenças pneumocócicas.

Changes in serotype distribution of Haemophilus influenzae meningitis isolates identified through laboratory-based surveillance following routine childhood vaccination against H. influenzae type b in Brazil.

Following routine childhood vaccination against Haemophilus influenzae type b (Hib) disease in Brazil in 1999, passive laboratory surveillance reported increasing numbers of non-b serotypes and nontypeable H. influenzae (NTHi) from meningitis cases. To characterize this increase, we analyzed data on 3910 H. influenzae isolated from cerebrospinal fluid or blood from meningitis cases that were sent to the national reference laboratory for serotyping from 1990 to 2008. Hib accounted for 98% of H. influenzae meningitis isolates received during 1990-1999 versus 59% during 2000-2008, while non-b serotypes increased from 1% to 19% and NTHi increased from 2% to 22% of H. influenzae isolates received during the two periods. Higher proportions of non-b serotypes and NTHi than Hib were isolated from blood rather than cerebrospinal fluid. Estimated incidence rates for H. influenzae meningitis for Sao Paulo state remained below 1 case per million population during 2000-2008, although annual incidence of NTHi meningitis (mean, 0.03 cases per 100,000 population) increased in several age groups. Changes in surveillance for H. influenzae following introduction of Hib conjugate vaccine likely contributed to increased numbers of non-b and nontypeable H. influenzae meningitis isolates received at the national reference laboratory.

Re-avaliação da sorotipagem de Haemophilus influenzae: comparação da técnica de soroaglutinação em lâmina com a reação em cadeia da polimerase / Evaluation of the Haemophilus influenzae serotyping: comparison between slide agutination and polymerase chain reaction tests

Após a introdução da vacina conjugada para Haemophilus influenzae sorotipo b (Hib) nos Programas Nacionais de Imunização em diferentes países, casos de infecção grave causados pelo Hib tiveram um acentuado declínio. Logo, o valor preditivo do método tradicional de tipagem capsular, a soroaglutinação em lâmina (SAg) com antissoros específicos para os seis sorotipos capsulares de Hi, diminuiu. Para avaliar a magnitude de resultados discrepantes na realização da sorotipagem obtidos durante a vigilância epidemiológica de Hi, 258 cepas isoladas de doença invasiva e de portadores na nasofaringe foram estudadas por dois métodos de soroaglutinação: SAg método 1, no qual cepas de Hi são inicialmente triadas com antissoro tipo b, e SAg método 2, no qual as cepas são testadas contra todos os antissoros específicos em paralelo. Os resultados obtidos pelos dois métodos de SAg foram comparados, na forma de duplo-cego, com aqueles obtidos pela tipagem capsular por PCR. Os percentuais de freqüência dos tipos capsulares entre as três metodologias foram significantemente diferentes, envolvendo discrepâncias entre cepas do sorotipob e cepas não capsuladas (NC). Para cepas invasivas (n=131), os percentuais de concordância entre os SAg método 1 e SAg método 2 comparados com a PCR foram 68,0% e 88,3%, respectivamente, enquanto para cepas deportadores (n=127) os percentuais correspondentes foram 46,5% e 94,2%. A SAg método 2 permitiu maior acurácia na identificação dos sorotipos quando comparado com a SAg método 1, demonstrando boa correlação com a PCR. Ouso de antissoro polivalente utilizado como reagente de triagem para SAg mostrou poder discriminatório baixo com uma sensibilidade de 65,8% e especificidade de 91,7%. Com base nos resultados da primeira parte do estudo foi estabelecido um algoritimo de tipagem capsular de Hi para uso na rotina diagnóstica da vigilância epidemiológica. Este algoritimo pôde identificar com sucesso os tipos capsulares das 180 cepas de Hi, invasivas e...

Re-avaliação da sorotipagem de Haemophilus influenzae: comparação da técnica de soroaglutinação em lâmina com a reação em cadeia da polimerase

Após a introdução da vacina conjugada para Haemophilus influenzae sorotipo b (Hib) nos Programas Nacionais de Imunização em diferentes países, casos de infecção grave causados pelo Hib tiveram um acentuado declínio. Logo, o valor preditivo do método tradicional de tipagem capsular, a soroaglutinação em lâmina (SAg) com antissoros específicos para os seis sorotipos capsulares de Hi, diminuiu. Para avaliar a magnitude de resultados discrepantes na realização da sorotipagem obtidos durante a vigilância epidemiológica de Hi, 258 cepas isoladas de doença invasiva e de portadores na nasofaringe foram estudadas por dois métodos de soroaglutinação: SAg método 1, no qual cepas de Hi são inicialmente triadas com antissoro tipo b, e SAg método 2, no qual as cepas são testadas contra todos os antissoros específicos em paralelo. Os resultados obtidos pelos dois métodos de SAg foram comparados, na forma de duplo-cego, com aqueles obtidos pela tipagem capsular por PCR. Os percentuais de freqüência dos tipos capsulares entre as três metodologias foram significantemente diferentes, envolvendo discrepâncias entre cepas do sorotipob e cepas não capsuladas (NC). Para cepas invasivas (n=131), os percentuais de concordância entre os SAg método 1 e SAg método 2 comparados com a PCR foram 68,0% e 88,3%, respectivamente, enquanto para cepas deportadores (n=127) os percentuais correspondentes foram 46,5% e 94,2%. A SAg método 2 permitiu maior acurácia na identificação dos sorotipos quando comparado com a SAg método 1, demonstrando boa correlação com a PCR. Ouso de antissoro polivalente utilizado como reagente de triagem para SAg mostrou poder discriminatório baixo com uma sensibilidade de 65,8% e especificidade de 91,7%. Com base nos resultados da primeira parte do estudo foi estabelecido um algoritimo de tipagem capsular de Hi para uso na rotina ...

Evaluation of methodology for serotyping invasive and nasopharyngeal isolates of Haemophilus influenzae in the ongoing surveillance in Brazil.

To assess the magnitude of discrepant results obtained by routine Haemophilus influenzae serotyping, 258 isolates, collected by the epidemiological surveillance system in Brazil from individuals with invasive diseases or carriage, were evaluated by two slide agglutination (SlAg) methods: SlAg method 1, by which strains were initially screened with a serotype b-specific antiserum, and SlAg method 2, by which strains were tested against all serotype-specific antisera in parallel. Investigators comparing results of the two SlAg methods with those obtained by capsule type-specific PCR were blinded to the method used. The serotype prevalence rates found by the three methods were significantly different, involving discrepancies mainly between serotype b and noncapsulated (NC) isolates. For invasive isolates (n = 131), the overall agreement rate between SlAg method 1 or 2 and PCR was 68.0 or 88.3%, respectively, whereas for colonizing isolates (n = 127) the corresponding rate was 46.5 or 94.2%, respectively. SlAg method 2 improved the ascertainment of serotypes over that obtained with SlAg method 1, demonstrating good correlation with PCR. Use of the polyvalent antiserum as a screening reagent for SlAg for invasive and colonizing isolates showed poor discriminatory power, with a sensitivity of 65.8% and a specificity of 91.7%. We stress the importance of using a well-standardized SlAg methodology and suggest that reference laboratories should utilize PCR routinely to confirm SlAg results and to check all nonspecific SlAg reactions and apparent NC isolates by SlAg in order to provide reliable data on the prevalence of H. influenzae serotypes in the H. influenzae type b vaccine era.

Phenotypic and genotypic characterization of VanA Enterococcus isolated during the first nosocomial outbreak in Brazil.

We report the phenotypic and genotypic characterization of 50 VanA Enterococcus clinical isolates from infected patients and 97 isolates from colonized patients obtained during a nosocomial outbreak in a single hospital in São Paulo, Brazil during 1998. The identification of strains to the species level by conventional biochemical and phenotypic tests and by multiplex PCR assay had 100% agreement. Both E. faecalis and E. faecium were isolated from patients during this outbreak. The vanA genotype was confirmed by PCR. Antibiotic susceptibility testing showed that E. faecium isolates are generally less susceptible to antibiotics than E. faecalis. By PCR, 24 of 26 VRE strains tested carried the Tn1546 element. Pulsed-field gel electrophoresis identified five distinct patterns for E. faecalis (A, B, C, D, E) and three for E. faecium (M, N, and O). A single PFGE pattern was identified in the majority of strains of each species and does not discriminate between case and carrier isolates.

Catalase-negative, methicillin-resistant Staphylococcus aureus as a cause of septicemia

A catalase-negative methicillin-resistant Staphylococcus aureus (MRSA) was isolated from blood, venous catheter spike and bone marrow collected from an HIV-positive man with lobar pneumonia and sepsis after ten days of hospitalization. The isolate was resistant to oxacillin (positive for penicillin-binding protein 2'), ceftriaxone, clindamycin and clarithromycin, and susceptible to vancomycin. This is the first case of septicemia due to a catalase-negative S. aureus reported in Brazil, and, to our knowledge, it is the first case of catalase-negative MRSA reported in the literature. We believe that the patient acquired the S. aureus infection within the hospital environment since it was isolated ten days after hospitalization, it was isolated in a venous catheter spike, and the antibiotic resistance profile is similar to other S. aureus isolates recovered from infections in our hospital

Characterization of haemophilus influenzae isolated from invasive disease in Brazil from 1990 to 1999.

The Haemophilus influenzae serotype b (Hib) conjugate vaccine was introduced in the National Immunization Program in Brazil in the second half of 1999. A retrospective analysis on serotypes, biotypes, and antimicrobial resistance of Hi invasive strains obtained through Hi survey was conducted to document the characteristics of this pathogenic agent during a decade prior the use of Hib vaccine. A total 3,204 strains from 1990 to 1999 were studied, being 88.2% isolated from cerebrospinal fluid, 10.7% from blood, and 1.1% from pleural fluid. The rate of 90.9% of strains was obtained from children up to 4 years old, and the age group >6 months old to 1 year was the higher risk to Hi infection. Type b was, by far, the most common type (97.8%), followed in frequency by type a (0.5%); only 1.5% was a nontypable strain. Biotypes I and II accounted for 97.8% of isolates. Resistance to ampicillin (AM) and chloramphenicol (CO) was detected at rates of 18.1% and 19.1%, respectively, whereas simultaneous resistance to AM and CO was identified in 13.9% of strains. Total concordance was found between AM resistance and beta-lactamase production. No strain showed resistance to ceftriaxone and rifampicin. In conclusion, the data generated through this laboratory-based surveillance should serve as a reference for assessing the impact of Hib vaccination and to detect changes on the pattern of Hi diseases in the country.

Vancomycin-Resistant Enterococcus faecium: first case in Brazil

We report a fatal case of septicemia due to a vancomycin-resistant Enterococcus faecium in a 9 year-old girl with aplastic anemia. The isolate was also resistant to amplicillin, teicoplanin, gentamicin (high level), and streptomycin (high level). We believe that this is the first case of vancomycin-resistant Enterococcus (VRE) reported from a clinical specimen in Brazil.