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Staphylococcus spp. is a major nosocomial pathogen, particularly affecting immunocompromised patients and infants. It is associated with bacteremia, endocarditis, and co-infections. Methicillin-resistant Staphylococci (MRS) carry the mecA gene, encoding PBP2a, which confers resistance to beta-lactam antibiotics. The aim of this study is to investigate resistance profiles and develop a molecular method to identify nosocomial Staphylococcus spp. strains. A total of 64 strains from public hospitals in Rio de Janeiro were analyzed using phenotypic and molecular methods, with 17 classified as MDR. Different melting temperatures (Tm) were obtained through qPCR-HRM analysis, to identify S. aureus- (70.4 °C), S. haemolyticus- (79 °C), S. epidermidis- (74.1 °C) and mecA (70.5 °C)-positive strains (MRS). The mecA gene was detected in 51 strains, with 22 showing SCCmec type IV. The spread of MRSA and MDR Staphylococci, particularly MDR S. haemolyticus, is a growing concern. In our study, among 64 Staphylococci strains, only 11 were susceptible to methicillin, showing the continuous emergence of resistant strains. qPCR-HRM is a cost-effective, sensitive and fast method for rapid Staphylococcus spp. identification, aiding in nosocomial infection control.

The incidence of microcephaly in Brazil in 2015 was 20 times higher than in previous years. Congenital microcephaly is associated with genetic factors and several causative agents. Epidemiological data suggest that microcephaly cases in Brazil might be associated with the introduction of Zika virus. We aimed to detect and sequence the Zika virus genome in amniotic fluid samples of two pregnant women in Brazil whose fetuses were diagnosed with microcephaly. In this case study, amniotic fluid samples from two pregnant women from the state of Paraíba in Brazil whose fetuses had been diagnosed with microcephaly were obtained, on the recommendation of the Brazilian health authorities, by ultrasound-guided transabdominal amniocentesis at 28 weeks' gestation. The women had presented at 18 weeks' and 10 weeks' gestation, respectively, with clinical manifestations that could have been symptoms of Zika virus infection, including fever, myalgia, and rash. After the amniotic fluid samples were centrifuged, DNA and RNA were extracted from the purified virus particles before the viral genome was identified by quantitative reverse transcription PCR and viral metagenomic next-generation sequencing. Phylogenetic reconstruction and investigation of recombination events were done by comparing the Brazilian Zika virus genome with sequences from other Zika strains and from flaviviruses that occur in similar regions in Brazil. We detected the Zika virus genome in the amniotic fluid of both pregnant women. The virus was not detected in their urine or serum. Tests for dengue virus, chikungunya virus, Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus, HIV, Treponema pallidum, and parvovirus B19 were all negative. After sequencing of the complete genome of the Brazilian Zika virus isolated from patient 1, phylogenetic analyses showed that the virus shares 97–100% of its genomic identity with lineages isolated during an outbreak in French Polynesia in 2013, and that in both envelope and NS5 genomic regions, it clustered with sequences from North and South America, southeast Asia, and the Pacific. After assessing the possibility of recombination events between the Zika virus and other flaviviruses, we ruled out the hypothesis that the Brazilian Zika virus genome is a recombinant strain with other mosquito-borne flaviviruses. These findings strengthen the putative association between Zika virus and cases of microcephaly in neonates in Brazil. Moreover, our results suggest that the virus can cross the placental barrier. As a result, Zika virus should be considered as a potential infectious agent for human fetuses. Pathogenesis studies that confirm the tropism of Zika virus for neuronal cells are warranted. Consellho Nacional de Desenvolvimento e Pesquisa (CNPq), Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).

Background: Mozambique introduced the 10-valent pneumococcal conjugate vaccine (PCV10) in 2013 using a three-dose primary series with no booster dose (3p+0) and later switched to the PCV13 using a schedule of two primary doses with one booster (2p+1). We aimed to describe the burden and serotype distribution of pneumococcal meningitis in children under 5 years of age in Mozambique over an eleven-year period starting with the year of PCV10 introduction, and assess the impact of the PCV vaccine and schedule changes. Methods: We analysed meningitis surveillance data in Mozambique from March 2013 through to December 2023. Cerebrospinal fluid (CSF) samples were collected from eligible children in three referral hospitals (Maputo Central Hospital [south], Beira Central Hospital [central], and Nampula Central Hospital [north]). Culture and polymerase chain reaction assay (qPCR) were performed on each sample. S. pneumoniae-positive samples were subsequently serotyped using multiplex assay. We estimated annual incidence rates for pneumococcal meningitis in children under 5 years old following the PCVs’ introduction (2013–2023). The impact of the product switch and schedule change from PCV10/3p+0 to PCV13/2p+1 on the burden and serotype distribution of pneumococcal meningitis was assessed. Results: Of the 4075 CSF samples tested, 7.4% (301/4075) were positive for S. pneumoniae, 2.5% (103/4075) for H. influenzae, and 1.0% (42/4075) for N. meningitidis. Pneumococcal meningitis incidence in children under five reduced from 44.7 cases per 100,000 in 2013 to 4.6 cases per 100,000 in 2023, an 89.7% reduction. In the PCV13/2p+1 period (2020–2023), pneumococcal meningitis incidence was 51.2% lower than the PCV10/3p+0 period (2013–2017) (IRR 0.49, 95% CI 0.4–0.6; p < 0.001). PCV10-serotype pneumococcal meningitis incidence among children under five decreased by 65.6% in the PCV13/2p+1 period (IRR 0.34, 95% CI 0.2–0.6; p < 0.001). We detected zero cases of pneumococcal meningitis due to the PCV13-serotype in 2020–2023, whereas non-PCV10/13-serotypes increased by 76% (IRR 1.76, 95% CI 1.2–2.6; p = 0.004). The case–fatality proportion decreased by 71.9% (95% CI 62.9–84.8%) in the PCV13/2p+1 period. Conclusions: Since the introduction of PCVs in Mozambique, the burden of pneumococcal meningitis and deaths in children under 5 years of age has substantially decreased, as well as the prevalence of PCV13-serotypes. Higher valency PCVs are needed due to the increased prevalence of non-PCV10/13-serotypes. Funding: Gavi, The Vaccine Alliance, reference number: MOZ-HSS-2-INS; WHO Reference: 2014405143-0, creation DFC to support HIB & Surveillance System.

Abstract The aim of this study was to determine the prevalence Cronobacter from 30 samples of oats and 30 of linseeds commercially available in Brazil. The detection of Cronobacter was as according to the ISO 22964:2017. The isolates were characterized according to their phenotypically using Vitek 2.0 and antibiotic susceptibility profile. Molecular characterization was accomplished by real-time PCR targeting dnaG gene, PCR targeting rpoB gene, multiplex-PCR targeting cgcA gene and fusA allele sequencing. A total of 34 samples (56.7%) contained Cronobacter; 19 (63.3%) of linseeds and 15 (50.0%) of oats. The isolates were identified as C. sakazakii (n = 18, 52.9%), C. dublinensis (n = 7, 20.6%), C. turicensis (n = 6, 17.7%) and C. malonaticus (n = 3, 8.8%). Thirty-four Cronobacter isolates were assigned to 11 different fusA alleles of which 3 were new (169, 170 and 171). The PCR targeting rpoB gene and cgcA gene failed to identify 19 isolates. Seven (20.6%) strains showed resistance or intermediate/resistance to tetracycline, and one (2.9%) strain had intermediate resistance to piperacilin-tazobactam. The presence of Cronobacter in oats and linseeds indicate that these foods can be a potential threat to human health, particularly when preparing food for elderly or immunosuppressed persons. The incorrect use of this foods for feeding of neonates (<6 months) by careers should also be avoided. This study shows that oats and linseeds offered in markets, presenting products contaminated by Cronobacter, can be a potential threat to human health, particularly for elderly, immunosuppressed and neonates.

Mycoplasma is the smallest self-replicating bacteria, figuring as common contaminant of eukaryotic cell cultures. Production inputs and operator’s manipulation seem to be the main sources of such contamination. Many analytical approaches have been applied for mycoplasma detection in cell cultures and also in biological products. However, unless they were validated, only indicator cell culture and bacteriological culture are considered as compendial methods for quality control of biological products. Nano-flow cytometry has been pointed out as an alternative technique for addressing prokaryotic and eukaryotic cell viability being a substantial tool for reference material production. In this study, a viability-flow-cytometry assay was standardized for M. gallisepticum and then applied to other cell-culture-contaminant mycoplasmas. For this, M. galliseticum’s growth rate was observed and different treatments were evaluated to establish low viability cultures (cell death-induced control). Distinct viability markers and their ideal concentrations (titration) were appraised. Ethanol treatment showed to be the best death-inducing control. CFDA and TOPRO markers revealed to be the best choice for detecting live and dead mycoplasma frequencies, respectively. The standardized methodology was applied to Mycoplasma arginini, M. hyorhinis, M. orale, Spiroplasma citri and Acholeplasma laidlawii. Significant statistical difference was observed in the percentage of viable cells in comparison to ethanol treatment for A. laidlawii in CFDA and in both markers for M. gallisepticum, M. hyorhinis and S. citri. In summary, we standardized a flow cytometry assay for assessing M. gallisepticum − and potentially other species – viability and ultimately applied for reference material production improving the quality control of biological products.

In sub Saharan Africa, the epidemiology, including the distribution of serogroups of strains of N. meningitidis is poorly investigated in countries outside \"the meningitis belt\". This study was conducted with the aim to determine the distribution of serogroups of strains of N. meningitidis causing meningococcal meningitis in children and adults in Mozambique. A total of 106 PCR confirmed Neisseria meningitidis Cerebrospinal Fluid (CSF) samples or isolates were obtained from the biobank of acute bacterial meningitis (ABM) surveillance being implemented by the National Institute of Health, at three central hospitals in Mozambique, from January to December 2014. Serogroups of N. meningitidis were determined using conventional PCR, targeting siaD gene for Neisseria meningitidis. Outer Membrane Proteins (OMP) Genotyping was performed by amplifying porA gene in nine samples. Of the 106 PCR confirmed Neisseria meningitidis samples, the most frequent serotype was A (50.0%, 53/106), followed by W/Y (18.9%, 20/106), C (8.5%, 9/106), X (7.5%, 8/106) and B (0.9%, 1/106). We found non-groupable strains in a total of 15 (14.2%) samples. PorA genotypes from nine strains showed expected patterns with the exception of two serogroup C strains with P1.19,15,36 and P1.19-36,15 and one serogroup X with P1.19,15,36, variants frequently associated to serogroup B. Our data shows that the number of cases of meningococcal meningitis routinely reported in central hospitals in Mozambique is significant and the most dominant serogroup is A. In conclusion, although serogroup A has almost been eliminated from the \"meningitis belt\", this serogroup remains a major concern in countries outside the belt such as Mozambique.

Neisseria meningitidis infections are a major issue for global health. The invasive MenC ST-103 clonal complex (CC103) has been the most prevalent in meningococcal outbreaks in Brazil, occurring also in several countries worldwide. Here we have analysed the population structure and accessory genome of MenC CC103 strains from a global perspective. An in-depth phylogenomic analysis revealed a lineage of N. meningitidis causing meningitis in Brazil and the United Kingdom. This lineage was also characterized as harbouring a particular accessory genome composed of CRISPR/Cas and restriction modification systems. This lineage was also characterized by a genomic island resembling an integrative and conjugative element. This island carried genes potentially associated with virulence and fitness. We propose this accessory gene repertoire could be contributing to the spatial-temporal persistence of the invasive MenC CC103 lineage.

Neisseria meningitidis serogroup B has been predominant in Brazil, but no broadly effective vaccine is available to prevent endemic meningococcal disease. To understand genetic diversity among serogroup B strains in Brazil, we selected a nationally representative sample of clinical disease isolates from 2004, and a temporally representative sample for the state of São Paulo (1988-2006) for study (n = 372). We performed multi-locus sequence typing (MLST) and sequence analysis of five outer membrane protein (OMP) genes, including novel vaccine targets fHbp and nadA. In 2004, strain B:4:P1.15,19 clonal complex ST-32/ET-5 (cc32) predominated throughout Brazil; regional variation in MLST sequence type (ST), fetA, and porB was significant but diversity was limited for nadA and fHbp. Between 1988 and 1996, the São Paulo isolates shifted from clonal complex ST-41/44/Lineage 3 (cc41/44) to cc32. OMP variation was associated with but not predicted by cc or ST. Overall, fHbp variant 1/subfamily B was present in 80% of isolates and showed little diversity. The majority of nadA were similar to reference allele 1. A predominant serogroup B lineage has circulated in Brazil for over a decade with significant regional and temporal diversity in ST, fetA, and porB, but not in nadA and fHbp.

Biocides play an important role in healthcare-associated infection control by either minimizing or preventing microorganism dissemination. This study evaluated the susceptibility of Pseudomonas aeruginosa clinical isolates to a quaternary ammonium (QAC) disinfectant and antibiotics, and verified the presence of qacE Delta 1, a determinant of resistance to QAC. The disinfectant test was the Association of Official Analytical Chemists Use-Dilution Test, and polymerase chain reaction was used to examine for qacE Delta 1. The qacE Delta 1 gene was detected in 48% of the isolates. Eighty-eight percent of the multiresistant isolates carried qacE Delta 1 gene, while 35% of the non-multiresistant isolates was positive to this gene, and multiresistance well correlated with its presence. Among isolates tested for the disinfectant, 46% showed a reduced susceptibility to the disinfectant. qacE Delta 1 gene was present in 70% of the susceptible isolates to the biocide, whereas 90% of the less susceptible strains harbored this gene. Reduced susceptibility to the disinfectant was independent of presence of qacE Delta 1 suggesting that it does not play an important role in biocide resistance in P. aeruginosa. As far as we know, it is the first report confirming this fact and testing with disinfectant at its in-use concentration. The evidence of less susceptible strains than the reference bacterium used in disinfectant testing, and the high percentage of qacE Delta 1 gene detected are of special concern and suggests continued investigation in laboratory and in situ, not only in healthcare settings, but also in all areas of biocide usage, including different micro-organisms and biocides.

Biocides play an important role in healthcare-associated infection control by either minimizing or preventing microorganism dissemination. This study evaluated the susceptibility of Pseudomonas aeruginosa clinical isolates to a quaternary ammonium (QAC) disinfectant and antibiotics, and verified the presence of qacEΔ1 , a determinant of resistance to QAC. The disinfectant test was the Association of Official Analytical Chemists Use-Dilution Test, and polymerase chain reaction was used to examine for qacEΔ1 . The qacEΔ1 gene was detected in 48% of the isolates. Eighty-eight percent of the multiresistant isolates carried qacEΔ1 gene, while 35% of the non-multiresistant isolates was positive to this gene, and multiresistance well correlated with its presence. Among isolates tested for the disinfectant, 46% showed a reduced susceptibility to the disinfectant. qacEΔ1 gene was present in 70% of the susceptible isolates to the biocide, whereas 90% of the less susceptible strains harbored this gene. Reduced susceptibility to the disinfectant was independent of presence of qacE Δ1 suggesting that it does not play an important role in biocide resistance in P. aeruginosa . As far as we know, it is the first report confirming this fact and testing with disinfectant at its in-use concentration. The evidence of less susceptible strains than the reference bacterium used in disinfectant testing, and the high percentage of qacEΔ1 gene detected are of special concern and suggests continued investigation in laboratory and in situ, not only in healthcare settings, but also in all areas of biocide usage, including different micro-organisms and biocides.