IntroductionDiphtheria is a potentially fatal disease that still causes deaths mainly in unvaccinated children. Over the last decades, the incidence of this disease has reduced drastically in face to the increase in vaccination coverage. Diphtheria is mainly caused by toxigenic Corynebacterium diphtheriae, however, in more recent years, invasive infections due to nontoxigenic Corynebacterium diphtheriae have emerged. Given this epidemiological threat, the continued surveillance remains essential to guide prevention and control measures.MethodologyUsing whole genome sequencing, we characterized 299 Corynebacterium diphtheriae isolates, assessing their clinical origin, age distribution of patients, tox gene carriage, antimicrobial resistance markers, and phylogenetic relationships.ResultsThe tox gene was identified in 255 isolates (85.3%). Antimicrobial resistance genes aph(3’’)-Ib, aph(3’)-Ia, aph(6)Id, cmx, tet(33), tet(O), tet(W), and tet(Z) were detected in low (<10%) frequencies, but sul1 was found in 72 (24.1%) isolates. Phylogenetic analyses identified 11 main clades comprising 286 isolates, represented by 46 different sequence types (ST); the remaining 13 isolates were distributed in the other 12 ST. Twenty-one novel ST were described, comprising 51 isolates.DiscussionOur study represents the largest genomic survey of Corynebacterium diphtheriae in Latin America. These results enhance global understanding of diphtheria and reinforce the need for vigilance against reemergence in areas with suboptimal vaccination coverage.

Corynebacterium diphtheriae is the main etiological agent of diphtheria, a potentially fatal disease whose most severe signs and symptoms result from the action of an exotoxin, the diphtheria toxin (DT). Although non-toxigenic C. diphtheriae strains have been associated with several diseases, including cutaneous infections and endocarditis, they are not monitored in many countries, and their mechanisms of virulence and antimicrobial resistance remain underexplored. Therefore, this study aimed to provide a comprehensive characterization -through genomic, in vitro, and in vivo analyses - of a non-toxigenic C. diphtheriae strain (46855) isolated from a leg lesion, highlighting its pathogenic potential and resistance profile. The isolate was assigned to a novel sequence type (ST-925) and was found to be resistant to tetracycline and rifampin. Multiple antimicrobial resistance genes were predicted in the genome, such as tet(33), rbpA, and rpoB2, in addition to mutations in the rpoB gene. A diverse set of virulence-associated genes related to adhesion, iron uptake systems, gene regulation, and post-translational modification was also identified. The isolate was able to form biofilm in vitro and exhibited strong virulence in Galleria mellonella larvae and A549 human pneumocyte cells. Finally, the structural analysis of the rpoB gene, carried out for the first time in this study, linked the observed mutations to rifampin resistance in C. diphtheriae. In summary, the data revealed that C. diphtheriae 46855, although non-toxigenic, harbors multiple genes associated with antimicrobial resistance and virulence, emphasizing the need for greater surveillance and functional studies on non-toxigenic strains.

Although diphtheria is a vaccine-preventable disease, numerous cases are still reported around the world, as well as outbreaks in countries, including European ones. Species of the Corynebacterium diphtheriae complex are potentially toxigenic and, therefore, must be considered given the possible consequences, such as the circulation of clones and transmission of antimicrobial resistance and virulence genes. Recently, Corynebacterium rouxii was characterized and included among the valid species of the complex. Therefore, two cases of C. rouxii infection arising from infections in domestic animals are presented here. We provide molecular characterization, phylogenetic analyses, genome sequencing, and CRISPR-Cas analyses to contribute to a better understanding of the molecular bases, pathogenesis, and epidemiological monitoring of this species, which is still little studied. We confirmed its taxonomic position with genome sequencing and in silico analysis and identified the ST-918 for both strains. The clinical isolates were sensitive resistance to benzylpenicillin and rifampin. Antimicrobial resistance genes, including tetB, rpoB2, and rbpA genes, were predicted. The bla and ampC genes were not found. Several virulence factors were also detected, including adhesion, iron uptake systems, gene regulation (dtxR), and post-translational modification (MdbA). Finally, one prophage and the Type I-E CRISPR-Cas system were identified.

Cases of diphtheria, even in immunized individuals, are still reported in several parts of the world, including in Brazil. New outbreaks occur in Europe and other continents. In this context, studies on Corynebacterium diphtheriae infections are highly relevant, both for a better understanding of the pathogenesis of the disease and for controlling the circulation of clones and antimicrobial resistance genes. Here we present a case of cutaneous infection by multidrug-resistant Corynebacterium diphtheriae and provide its whole-genome sequencing. Genomic analysis revealed resistance genes, including tet(W), sul1, cmx, rpoB2, rbpA and mutation in rpoB. We performed phylogenetic analyzes and used the BRIG to compare the predicted resistance genes with those found in genomes from other significant isolates, including those associated with some outbreaks. Virulence factors such as spaD, srtBC, spaH, srtDE, surface-anchored pilus proteins (sapD), nonfimbrial adhesins (DIP0733, DIP1281, and DIP1621), embC and mptC (putatively involved in CdiLAM), sigA, dtxR and MdbA (putatively involved) in post-translational modification, were detected. We identified the CRISPR-Cas system in our isolate, which was classified as Type II-U based on the database and contains 15 spacers. This system functions as an adaptive immune mechanism. The strain was attributed to a new sequence type ST-928, and phylogenetic analysis confirmed that it was related to ST-634 of C. diphtheriae strains isolated in French Guiana and Brazil. In addition, since infections are not always reported, studies with the sequence data might be a way to complement and inform C. diphtheriae surveillance.

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. This study analyzed 1437 invasive Hi isolates in Brazil over 13 years, determining capsular serotypes, antimicrobial susceptibility, and genetic relatedness through multilocus sequence typing. The primary source of isolation for these invasive H. influenzae isolates was blood (54.4

The genus Corynebacterium is the largest genera among corynebacteria and has a range of species widely spread in ecological niches, some with epidemic potential and capable of causing fatal diseases. In recent years, due to the reclassifications and discoveries of new potentially toxin-producing species, microbiological identification and epidemiological control have been compromised, becoming possible only with sequencing techniques. Two bacterial strains isolated from a cat were identified by MALDI-TOF mass spectrometry as Corynebacterium diphtheriae and sent to the collaborating center of the Brazilian Ministry of Health for molecular identification and determination of toxigenicity potential, which were initially performed by multiplex PCR method. In addition, the antimicrobial susceptibility profile was determined according to BrCAST. Finally, for the final identification at the species level and effective epidemiological monitoring, the sequencing of the 16S rRNA and rpoB housekeeping genes was carried out. The isolates were identified as nontoxigenic C. diphtheriae strains by mPCR. Both strains were found susceptible to all antimicrobial agents. Although the identification at the species level was not possible through similarity analysis of S rRNA and rpoB housekeeping genes, the phylogenetic analysis showed that the isolates belonged to the species Corynebacterium rouxii with a high value of reliability. This is the first report of the isolation of C. rouxii in Latin America. Molecular identification, whether by the MALDI-TOF mass spectrometry or PCR techniques, does not discriminate C. rouxii from C. diphtheriae, requiring gene sequencing and phylogenetic analysis for correct identification at the species level.

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.

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.

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.

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 intro-duction. Methods: Antimicrobial non-susceptible isolates were defined as intermediate or resistant. Minimum inhibitory concentrations (MICs) to penicillin and ceftriaxone were analyzed by year. Antimicrobial sus-ceptibility 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%; ceftriax-one, 28.0%); multidrug-resistance reached 25% in 2017-2019. Serotypes 19A, 6C and 23A were most asso-ciated with antimicrobial non-susceptibility. Conclusions: Antimicrobial non-susceptible Spn decreased in the three-years after vaccination but subse-quently increased and was associated with non-PCV10-types. Antimicrobial susceptibility surveillance is fundamental for guiding antibiotic therapy policies. (c) 2021 Elsevier Ltd. All rights reserved.

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.

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 Sao Paulo state, Brazil, composed of Araraquara, Barretos, Franca and Ribeirao 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.

Background: The epidemiology and impact of multiple concurrent Human papillomavirus (HPV) infections on the natural history of cervical disease is uncertain, but could have significant implications for cervical cancer prevention and HPV vaccination strategies. Methods: A cross-sectional prevalence study was conducted to determine the overall prevalence of HPV and the rate of multiple concurrent HPV infections, in a cohort of sexually active HIV- uninfected South African adolescents. HPV genotyping was performed using the polymerase chain reaction. Results: Overall prevalence of HPV was 64.1%. Multiple concurrent HPV infections were found in 43.6% of participants and 68% of HPV-infected participants. Non-vaccine high-risk HPV (HR-HPV) genotypes were found much more often than vaccine types (HPV16 and HPV18). Conclusions: Our cohort of young South African females was found to have a high overall prevalence of HPV and multiple concurrent HPV infections. Most HR-HPV infections found were genotypes other than HPV16 or HPV18.

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. (C) 2011 Elsevier Ltd. All rights reserved.

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.

Em um paciente HIV-positivo, com pneumonia lobar e septicemia, foi isolada, após dez dias de internação, uma cepa de Staphylococcus aureus catalase-negativa, resistente a meticilina/ oxacilina (MRSA), de culturas de sangue, cateter venoso central e medula óssea. A cepa era resistente a oxacilina (PBP 2’ positivo), ceftriaxona, clindamicina e claritromicina, e sensível a vancomicina. Este é o primeiro caso, reportado no Brasil, de uma septicemia por S. aureus catalase-negativo e, em nosso conhecimento, o primeiro caso de um S. aureus catalase-negativo resistente a meticilina. Nós acreditamos que o paciente tenha adquirido a infecção no ambiente hospitalar, uma vez que esta cepa foi isolada aos dez dias de internação, foi isolada em cateter venoso central e o perfil de sensibilidade aos antimicrobianos é semelhante ao dos S. aureus de infecções nosocomiais que ocorrem em nosso hospital. 1. Doutora em Patologia, médica da Seção de Bacteriologia do Laboratório de Patologia Clínica do Instituto de Infectologia Emílio Ribas, São Paulo, Brasil. 2. Doutora em Ciências, pesquisadora do Departamento de Bacteriologia do Instituto Adolfo Lutz, São Paulo, Brasil. 3. Estagiária da Seção de Bacteriologia do Laboratório de Patologia Clínica do Instituto de Infectologia Emílio Ribas, São Paulo, Brasil. 4. Assistente técnico de pesquisa do Departamento de Bacteriologia do Instituto Adolfo Lutz, São Paulo, Brasil. 5. Assistente técnico de pesquisa do Departamento de Bacteriologia do Instituto Adolfo Lutz, São Paulo, Brasil. 6. Mestre em Microbiologia; biologista da Seção de Bacteriologia do Laboratório de Patologia Clínica do Instituto de Infectologia Emílio Ribas, São Paulo, Brasil. 7. Technical supervisor, University of Alberta Hospital, National Centre for Streptococcus, Alberta, Canada. Trabalho apresentado em forma de painel no XXI Congresso Brasileiro de Microbiologia (2001), sob o seguinte título: Staphylococcus aureus CatalaseNegative Septicemia: First Case Reported in Brazil. Catalase-negativo

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.

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.